**Immunomodulatory E**ff**ects of the** *Meretrix Meretrix* **Oligopeptide (QLNWD) on Immune-Deficient Mice**

**Wen Zhang 1,**† **, Lei Ye 1,**† **, Fenglei Wang <sup>2</sup> , Jiawen Zheng <sup>1</sup> , Xiaoxiao Tian <sup>1</sup> , Yan Chen 1,\* , Guofang Ding <sup>1</sup> and Zuisu Yang <sup>1</sup>**


Received: 4 November 2019; Accepted: 3 December 2019; Published: 5 December 2019

**Abstract:** The aim of this study was to explore the immunomodulatory effects of the *Meretrix meretrix* oligopeptide (MMO, QLNWD) in cyclophosphamide (CTX)-induced immune-deficient mice. Compared to untreated, CTX-induced immune-deficient mice, the spleen and thymus indexes of mice given moderate (100 mg/kg) and high (200 mg/kg) doses of MMO were significantly higher (*p* < 0.05), and body weight loss was alleviated. Hematoxylin-eosin (H&E) staining revealed that MMO reduced spleen injury, thymus injury, and liver injury induced by CTX in mice. Furthermore, MMO boosted the production of immunoglobulin G (IgG) and hemolysin in the serum and promoted the proliferation and differentiation of spleen T-lymphocytes. Taken together, our findings suggest that MMO plays a vital role in protection against immunosuppression in CTX-induced immune-deficient mice and could be a potential immunomodulatory candidate for use in functional foods or immunologic adjuvants.

**Keywords:** *Meretrix meretrix* oligopeptides; cyclophosphamide; immunomodulatory; immune-deficient mice

#### **1. Introduction**

Immunoregulation can be broadly divided into positive regulation and negative regulation, both of which are the result of complex regulation of the immune system. Sometimes regulation in only one direction is triggered, but most immune regulation is bidirectional in order to maintain a stable steady-state. Immunomodulators can be classified into three general types: immunopotentiators, immunosuppressants, and two-way immunomodulators [1–3]. When the body experiences diseases or immune abnormalities, the application of immunomodulators can restore immune function to normal. There are many types of immunomodulators, such as bacterial preparations (e.g., lipopolysaccharide (LPS)), chemical preparations (e.g., cyclophosphamide (CTX)), and biochemical preparations (e.g., thymosin) [4]. However, some chemical immunomodulators have serious side effects, which not only have a specific inhibitory effect on the cause of immune diseases, but also have general inhibitory effects on normal tissue cells [5]. Inflammation, infection, tumors, organ bleeding, and loss of pregnancy have all been reported as being induced after the administration of chemical immunomodulators [6]. Untreated chronic inflammation, however, inhibits natural killer (NK) cells and T cells, which are key participants in the immune system, and limits the success of immunotherapy [7]. More recently, immunomodulators from natural extracts have attracted much attention in the field due to their lesser side-effects when used in humans [8,9]. For example, Hong et al. [10] showed that *Cervus nippon mantchuricus* extract (NGE) has immuno-enhancing effects

on RAW264.7 macrophage cells in immunosuppressed mice. Purified leaf extracts of *Melia azedarach* L. (CDM) exerted anti-herpetic activity, inhibited NFκB translocation to the nucleus, and modulated both interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) responses in macrophages in one recent study [11]. Therefore, further exploration of natural and effective immunomodulators with lesser side effects seems to be a very worthwhile research pursuit.

Bioactive peptides are small proteins, composed of amino acids, which often have unique physiological functions not possessed by large proteins or their constituent amino acids, such as antibacterial, antiviral, anti-oxidant, antifungal, calcium-binding, or anti-tumor properties [12–14]. Moreover, many bioactive peptides can be absorbed and digested even more quickly than free amino acids, and thus have become popular research topics and promising functional factors in the international food industry [15]. As the most common kind of bioactive peptide, immunologically active peptides stimulate the proliferation of lymphocytes, enhance the phagocytic abilities of macrophages, improve the body's resistance to external pathogens, and generally enhance the body's immunity to infection. Recently, such immunoregulatory peptides have attracted much research attention. For example, Yang et al. [16] reported that a marine oligopeptide from chum salmon could significantly enhance the capacity of lymphocyte proliferation in mice. Gao et al. [17] reported that collagen hydrolysates from yak bones exhibited immunomodulatory effects on CTX-induced immunosuppressed mice by increasing both innate and adaptive immunity. Li et al. [18] reported that a novel pentapeptide (RVAPEEHPVEGRYLV) from *Cyclina sinensis* could stimulate macrophage activity to activate the NFκB signaling pathway, and further in vivo studies revealed that this novel pentapeptide has immunomodulatory effects on CTX-induced immunosuppression in mice [19].

In a previous study of ours, an oligopeptide (QLNWD) was purified from the hydrolysate of *Meretrix meretrix* oligopeptide (MMO), and was shown to have the ability to aid in reversing the effects of nonalcoholic fatty liver disease (NAFLD) in mice [20]. We also investigated the immunomodulatory effect of this oligopeptide in vitro [21], and our results indicated that MMO has the effect of promoting the activation of RAW264.7 cells and the potential to enhance the non-specific immunity. However, the immunoregulatory activity of this oligopeptide in vivo is unknown. The aim of the present study was to explore the immunomodulatory effects of MMO on mice with CTX-induced immunosuppression in vivo. The effects of MMO on the thymus and spleen indexes of the mice were investigated, as well as morphological changes to their spleens, thymuses, and livers as observed microscopically using hematoxylin-eosin (H&E) staining. The stimulation index change in spleen T-lymphocytes was also determined in the present study. This study will provide a foundation for the further development of MMO as an immunopotentiator.

#### **2. Results and Discussion**

#### *2.1. Comparison of Body Weight*

The body weight of mice is a direct indicator of their physical condition. Previous evidence has shown that weight recovery can effectively increase the number of T-cell subsets and macrophages, which are vital components of the murine immune system [22–24]. As shown in Figure 1, it was observed that the weights of the mice in the positive drug or MMO-treated groups were significantly reduced when compared to the control group in the five days prior to the commencement of the study. Over the next 10 days, the mice in both the positive control group and the MMO-treated group saw marked increases in their body weights. However, the body weight of mice in the disease model became stable after 7 days, as it seems that CTX can cause immunodeficiency in mice, resulting in reduced appetite. This result indicated that MMO had an effect on alleviating the degree of immunosuppression induced by CTX on the mice.

**Figure 1.** The body weight changes of immunosuppressed study mice. Negative control: saline; positive control: 25 mg/kg of levamisole; disease model: 80 mg/kg of CTX; low-dose MMO: 50 mg/kg of MMO; medium-dose MMO: 100 mg/kg of MMO; and high-dose MMO: 200 mg/kg of MMO.

#### *2.2. Thymus and Spleen Indexes*

The thymus and spleen are representative immune organs. The thymus is one of the primary lymphoid organs [25], and the innate and adaptive immune responses to antigens and pathogens are initiated by the spleen, which is considered to be an important organ for assessing immune system function [26]. The thymus and spleen indexes can thus be used to roughly estimate the strength of immune function, which is a superficial and lagging indicator [27]. Our results showed that the spleen and thymus indexes of the model group were visibly reduced (*p* < 0.05) when compared to the negative control group. The spleen indexes of the middle (100 mg/kg) and high (200 mg/kg) MMO groups were significantly higher than those of the model group (*p* < 0.05), which was similar for their thymus indexes as well (Figure 2). These results indicate that MMO may be able to effectively alleviate the atrophy of both the spleen and the thymus caused by CTX.

**Figure 2.** MMO-induced changes to the immune organ indexes of mice. \*, a significant difference when compared to the negative control (*p* < 0.05); # , a significant difference when compared to the disease model, *p* < 0.05 values were considered to be statistically significant.

#### *2.3. Morphological Observations of Mouse Organs*

To gain further insight into the effectiveness of MMO on CTX-induced immunosuppression in the mice, H&E staining was used to observe subtle morphological changes to the spleens, thymus glands, and livers of the mice in each group (Figures 3–5).

The spleen is the largest immune organ of the body, accounting for 25% of the total lymphoid tissue, and also contains a large number of lymphocytes, dendritic cells, neutrophils, natural killer cells, and macrophages [28]. As shown in Figure 3, there was a clear dividing line between the red and white medulla in the normal group (Figure 3A). The splenic corpuscle in the white medulla was nearly round, the lymphatic sheath structure around the artery was complete, the splenic cord in the red medulla was connected, and the splenic sinus was obvious. In the model group, the boundary between the white pulp and the red pulp was blurred and the splenic corpuscle in the white pulp was scattered (Figure 3B). Compared to the normal group, the lymphatic sheath around the artery was thinner and the area of the red pulp was smaller, which suggested that CTX may have damaged the T and B cells in the spleen, significantly reduced the lymphoid tissue, and led to overall atrophy of the spleen. In the positive control group, multiple intact splenic corpuscles were seen, with an enlarged white medullary margin and thickened lymphatic sheath around the central artery (Figure 3C). The general structure of the splenic corpuscle was observed in the low-dose MMO group, but the boundary between the red and white medulla was still not obvious (Figure 3D). In the medium-dose MMO group, the marginal area of white pulp could be observed (Figure 3E). Red and white medulla were clearly observed in the high-dose MMO group and the white medulla margins were widened (Figure 3F). Overall, MMO gradually returned the spleen structure to an organizational form similar to that of the normal group. The white pulp part, for example, became clearer and, for the high-dose MMO group in particular, presented with a shape very similar to that of the positive control group. These results suggest that MMO can restore lymphocyte white marrow, increase T and B cells in marginal regions, and reduce CTX-induced spleen cell apoptosis in mice [29].

**Figure 3.** H&E staining of mouse spleen (×400). R: red medulla; W: white medulla; M1: marginal area; C1: central artery. (**A**) Negative control; (**B**) disease model; (**C**) positive control; (**D**) low-dose (50 mg/kg) MMO; (**E**) medium-dose (100 mg/kg) MMO; and (**F**) high-dose (200 mg/kg) MMO.

Thymus atrophy also showed a similar trend in all of the mice. The cortex of the thymus contains thymocytes, which produce thymosin that can stimulate the proliferation and differentiation of T-lymphocytes, activate the major histocompatibility complex (MHC) colony factor transmitting signal [30], and accelerate the presentation of antigens. In the normal group, cortical and medullary structures were clear and distinct and obvious thymus bodies were observed in the medulla (Figure 4A). In the model group, the cortex and medulla were intercalated, the thymus corpuscle was shrunken and unclear in the visual field, the cortical area was smaller, and the number of T-lymphocytes was significantly reduced (Figure 4B), which all indicate significant immunosuppression when compared

to the normal group. Cells and T-lymphocytes in the thymus of the positive control group were significantly increased when compared to the negative control group and multiple thymus corpuscles were observed in the visual field (Figure 4C). The cortex and medulla of the low-dose MMO group still could not be distinguished, but there was an increase in T cells in the cortex (Figure 4D). In the medium-dose MMO group, the cortex and medulla could be distinguished only roughly (Figure 4E). In the high-dose MMO group, however, the cortex and medulla were distinct and T-lymphocytes were significantly increased (Figure 4F)—a similar morphology to that of the positive control group. With increasing doses of MMO, cortical thymocytes increased, which demonstrated that MMO could activate the immune response and reduce the thymus injury induced by CTX [30].

**Figure 4.** H&E staining of mice thymus (×200). C2: cortical; M2: medulla; T: thymus corpuscle. (**A**) Negative control; (**B**) disease model; (**C**) positive control; (**D**) low-dose (50 mg/kg) MMO; (**E**) medium-dose (100 mg/kg) MMO; and (**F**) high-dose (200 mg/kg) MMO.

**Figure 5.** H&E staining of mice livers (×400). C3: central vein; H: hepatic cord; L: liver sinusoidal. (**A**) Negative control; (**B**) disease model group; (**C**) positive control; (**D**) low-dose (50 mg/kg) MMO; (**E**) medium-dose (100 mg/kg) MMO; and (**F**) high-dose (200 mg/kg) MMO.

The liver is the central hub of the body's metabolism, with functions such as detoxification and hematopoiesis [31]. To investigate whether there was an effect on the liver after using immunosuppressive agents and MMO, the histological structure of the mouse liver was observed. The hepatic lobule structure of the negative control group was clear and complete, with radial hepatic cords that radiated out in all directions and were arranged neatly around the central vein. Morphology of the hepatocytes was also regular and liver sinusoidal structures were observed (Figure 5A). In the disease model group, the hepatic cord was ruptured and disordered and degeneration and necrosis of hepatocytes was evidenced by a reduction in vacuoles and even absence of part of the nucleus. The structure of the liver sinusoids was not obvious, indicating that the cytotoxicity of CTX caused damage to the mouse liver (Figure 5B). In the positive control group, the hepatic cord around the

central vein recovered to the radial structure and the hepatocyte nucleus also showed a round shape (Figure 5C). The liver morphologies of the medium-dose and the high-dose MMO groups both bore a close resemblance to that of the positive control group (Figure 5E,F). Our results showed that MMO can effectively reduce the cytotoxicity of CTX-induced liver injury in mice.

#### *2.4. Serum Immunoglobulin G (IgG) Levels*

IgG is one of the most abundant proteins in human serum, accounting for about 10–20% of plasma protein [32]. Detection of IgG levels can help to indirectly judge the immune function of the body [33]. Vikas et al. [34] explored the changes in IgG levels in mice treated with galactose. The results showed that the IgG concentration in the galactose-treated mice was higher than that of the normal group, indicating that galactose had the potential to upregulate IgG production. The effect of MMO on IgG content in mice serum is shown in Figure 6. The IgG level in the model group was markedly reduced when compared to the negative control group (*p* < 0.05). From the doses of 50 mg/kg to 200 mg/kg, the MMO-treated groups seemed to have significant dose-dependent increases in IgG levels (*p* < 0.05), when compared to the disease model group. Moreover, the IgG levels in the high-dose MMO group were higher than the negative control and close to the positive control group, which suggests that high doses of MMO have a very beneficial effect on the restoration of serum immunoglobulins in immunocompromised mice.

**Figure 6.** The effects of MMO on immunoglobulin G (IgG) content in mouse serum. \*, a significant difference when compared to the negative control, *p* < 0.05. # , a significant difference when compared to the disease model, *p* < 0.05. *p* < 0.05 values were considered to be statistically significant.

#### *2.5. Serum Hemolysin*

− Hemolysin reflects the proliferation and differentiation of hemolytic B cells and is one of the main nonspecific indexes used to measure the immune function of the body [35,36]. The half hemolysis value (HC50) and the hemolysin proliferation rate are routinely used to evaluate the effects of natural extract products on humoral immunity in mice [36]. As shown in Table 1, in contrast to the negative control group, the HC<sup>50</sup> of the model group dropped by 0.68 ± 0.05. However, the HC<sup>50</sup> levels of the MMO-treated groups (50, 100, 200 mg/kg) were raised by 0.26 ± 0.05, 1.69 ± 0.02, and 3.20 ± 0.05, respectively. The proliferation rate of hemolysin in the disease model group was −0.78% ± 0.05 when compared to the negative control group, which indicated that serum hemolysin was inhibited by CTX. However, the proliferation rates of the MMO-treated groups exceeded that of the negative control group across the board, suggesting a supra-accelerating effect of MMO on CTX-damaged mice. Similarly, Pan et al. [35] reported that milk protein hydrolysate (MPH) increased immunological function by triggering hemolysin formation in mice. Liu et al. [37] showed that cottonseed meal oligopeptide (PFC) significantly increased the HC<sup>50</sup> levels in mice by 1.39 ± 0.45, 2.59 ± 0.20, and 2.46 ± 0.41 when given

doses of 5 mg/mL, 10 mg/mL, and 20 mg/mL, respectively. Our results were consistent with these findings and indicated that MMO has the effect of alleviating immunosuppression induced by CTX in mice.


**Table 1.** The effect of MMO on HC<sup>50</sup> and the hemolysin proliferation rate.

Note: \* indicates a significant difference over the Negative Control group.

#### *2.6. T Lymphocyte Assessment*

As the main cells in both the thymus and the spleen, T-lymphocytes can assist B cells to produce antibodies, kill target cells, and promote mitogen responses [38,39]. Relevant studies have reported that mitogens, such as Concanavalin A (ConA) and phytohemagglutinin (PHA), can stimulate lymphocytes to release a wide variety of cytokines in vitro as well as induce the simultaneous stimulatory and inhibitory activities of different T cell populations [40]. Evidence has shown that one effect of ConA stimulation of T-lymphocytes may be to enhance endocytosis of the cell membrane and studies have speculated that cell density and cell contact area are associated with the stimulation of ConA, reaching a peak of growth at 24 h [41,42]. In this study, we used ConA to stimulate spleen T-lymphocytes extracted from the spleens of each group of mice and observed the stimulation effects after 24 h. We observed that the stimulation of T-lymphocytes in the model group was lower than in the negative control, which indicated that CTX inhibited the responses of T cells in the lymphatic systems of those mice. The net proliferation of T-lymphocytes in the low-dose (50 mg/kg) MMO group was similar to that of the model group. However, the net proliferation of T-lymphocytes in the high-dose MMO-treated group was higher than that of the negative control group, indicating that MMO stimulated the proliferation of T-lymphocytes and reduced the inhibitory effects of CTX on T-lymphocytes in those mice (Figure 7). Combined with the H&E staining results, we speculated that spleen atrophy was reduced by MMO and that the activity of T-lymphocytes was increased in CTX-immunocompromised mice.

**Figure 7.** The stimulation index change induced by MMO in spleen T-lymphocytes from mice. \*, a significant difference when compared to the negative control (*p* < 0.05); # , a significant difference when compared to the disease model (*p* < 0.05). *p* < 0.05 values were considered to be statistically significant.

#### **3. Materials and Methods**

#### *3.1. Animals*

Sixty male ICR mice (20–23 g) were provided by the Experiment Animal Center of Zhejiang Province (certificate no SCXK 2014-0001). All the mice were kept under conventional and uniform conditions at 22 ◦C. The study proceeded after the mice were given seven days to acclimatize to their new environment.

#### *3.2. Materials and Chemical Reagents*

The MMO (QLNWD) [13] used for the experiments was synthesized by China Peptides Co., Ltd. (Shanghai, China). The CTX and levamisole were provided by Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). An H&E staining kit was supplied by Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Sheep red blood cells (SRBC) and guinea pig serum were obtained from Zhengzhou Baiji Biological Engineering Co. Ltd. (Henan, China). A mouse IgG enzyme linked immunosorbent assay (ELISA) kit was purchased from Shanghai Fusheng Industrial Co. Ltd. (Shanghai, China). Hanks' balanced salt solution (HBSS) and ConA were purchased from Solarbio (Beijing, China). NLRP3 rabbit monoclonal antibody was purchased from Cell Signaling Technology (Massachusetts, USA). A 3,3′ -diaminobenzidine (DAB) immunohistochemistry color development kit was purchased from BBI Life Science Corporation Co., Ltd. (Shanghai, China). Ammonium-chloride-potassium (ACK) lysis buffer was offered by Beyotime Biotechnology (Shanghai, China).

#### *3.3. Animal Groupings and Treatments*

Animal groupings and procedures were performed according to the methods in Zhang et al. [43] with some slight modifications. The mice were randomly divided into 6 groups, each of which contained 10 mice. Each mouse had its body weight recorded, received an intraperitoneal injection of 0.2 mL, and was fed the same weight of feed every day at the same time. The experimental groups were administered three dosage concentrations of MMO: 50 mg/kg body weight (BW) (low dose), 100 mg/kg BW (medium dose), and 200 mg/kg BW (high dose). The negative control group and the disease model group were given normal saline (NS, 0.9% NaCl) injections. The positive control group was protected from the effects of CTX by 2.5 mg/kg BW of levamisole given over 10 days prior to the experiment [44]. On day 1 of the experiment, all groups except the negative control group were injected with 80 mg/kg BW CTX (Table 2, after having fasted without water deprivation for 24 h beforehand [45].



#### *3.4. Body Weight and Immune Organ Index Changes*

The body weights of all mice were recorded once every other day for 15 days total. Before being sacrificed by cervical dislocation, each mouse was weighed a final time. The immune organs and the spleen and thymus glands were harvested, rinsed using NS, blotted by gauze immediately, and weighed in order to calculate each mouse's immune organ index (IOI) using Equation (1), before finally proceeding to dissection:

$$\text{ROI} = \frac{\text{immume organ weight}}{\text{bodyweight}} \times 100\%. \tag{1}$$

#### *3.5. Histomorphological Observation*

Following dissection, the tissues were fixed in 4% paraformaldehyde for 24 h to 48 h, embedded in paraffin, sliced to 5 µm sections, stained using an H&E staining kit, and sealed with neutral gum. The histomorphological changes of the organ tissues of each group were observed under an optical microscope (CX31, Olympus) and photographed with a CCD-NC 6051 photographic system.

#### *3.6. Determination of IgG Serum Content*

Blood was sampled from the eyes 24 h after each mouse's last intraperitoneal injection. The serum and plasma were separated using a refrigerated centrifuge (4 ◦C, 5000 rpm, 5 min). The amount of IgG in the serum was measured by a mouse IgG ELISA kit from Shanghai Fusheng Industrial Co. Ltd.

#### *3.7. Detection of Serum Hemolysin*

The mice serum samples were diluted 100-fold in 96-well plates at 100 µL per well. The sample wells were mixed with 5% sheep red blood cells (SRBC) (50 µL) and 10% guinea pig serum (50 µL), while control wells had just 5% SRBC (50 µL) added to them. The 96-well plates were placed in a 37 ◦C water bath for 30 min, after which the reaction was stopped in ice water and the supernatants were collected and analyzed at 540 nm in a microplate reader (SpectraMax M2, Molecular Devices, San Jose, CA, USA). The half hemolysis value (HC50) and the hemolysin content change showed the change of hemolysin in the serum samples of the mice (Equations (2) and (3)).

$$\text{HC}\_{50} = \frac{\text{OD value of sample} \times \text{dilution ratio}}{\text{OD value of SRBC}},\tag{2}$$

$$\text{Proification rate} = \frac{(\text{HC}\_{50}\text{ of sample} - \text{HC}\_{50}\text{ of control})}{\text{HC}\_{50}\text{ of control}}.\tag{3}$$

#### *3.8. Proliferation of Spleen T-Lymphocytes*

Mice spleen T-lymphocytes were extracted by the method described by Cai et al. [46]. The spleens of the mice were carefully dissected on a sterile bench, washed with HBSS, cleaned of blood and unrelated tissues, and ground on a 200-mesh stainless steel mesh, after which the cells were collected in a clean centrifuge tube. After being centrifuged at 1000 rpm for 5 min, the supernatant was discarded and the pellet was mixed with 2 mL of ammonium-chloride-potassium (ACK) lysis buffer for 5 min, washing three times with HBSS in between each step. After centrifugation under uniform conditions, the remaining cells were resuspended in RPMI-1640 complete medium, cultured in a cell culture incubator for 12 h, and stored for subsequent multiplex reaction experiments.

The T lymphocyte multiplication experiment method described by Ye et al. [47] was adopted for this study as well. The pre-preparation cell suspension was then put into a 96-well plate and the number of cells was 1 × 10<sup>6</sup> cells/mL. Each concentration was set into four complex wells (200 µL). Then, 10 µL of ConA (5 µg/mL) was added among 2 wells and 10 µL NS added as control. The plate was incubated in an incubator (37 ◦C, 5% CO2) for 24 h, to which was added 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-*H*-tetrazolium bromide (MTT) during the 20 h. Finally, it was treated with 150 µL DMSO for 10 min and the absorbance was detected under 490 nm. The T lymphocyte-multiplication extent was represented by the stimulation index (SI), calculated as shown below Equation (4):

$$\text{SI} = \frac{\text{OD value of sample well (average)}}{\text{OD value of control (average)}} \tag{4}$$

## *3.9. Statistical Analysis*

The experimental data were analyzed and processed by SPSS 19.0 statistical software. The figures were expressed as mean ± standard deviation (SD), analyzed using a one-way analysis of variance (ANOVA) test, and *p* < 0.05 values were considered to be statistically significant.

## **4. Conclusions**

In general, we have conclusively shown that MMO has immunomodulatory effects on CTX-immunocompromised mice. Compared to the disease model group, 100 mg/kg and 200 mg/kg doses of MMO were shown to significantly increase the spleen and thymus indexes (*p* < 0.05) and alleviate CTX-induced body weight loss in our experimental mice. The spleen immune injuries and thymus injuries induced by CTX were also alleviated in the MMO-treated groups. Furthermore, MMO may increase the levels of IgG and hemolysin in mouse serum and promote the proliferation of spleen T-lymphocytes. Our findings suggest that MMO plays a vital role in protection against immunosuppression in CTX-treated mice. Transcriptomics and proteomics will be used to further reveal its immune regulatory mechanism in our future studies in vitro and in vivo. We hope that our findings will provide a foundation for further study of MMO as an immunoregulatory adjuvant or functional food additive.

**Author Contributions:** Y.C. conceived and designed the experiments. W.Z., L.Y., F.W., J.Z., X.T., Z.Y., and G.D. performed the statistical analysis of the data. W.Z. and Y.L. wrote the manuscript.

**Funding:** This work was financially supported by Zhejiang Provincial Natural Science Foundation of China (grant No. LQ18B060004), the National Natural Science Foundation of China (grant No. 21808208) and the Scientific Research Start-up Funds of Zhejiang Ocean University (grant No. 11135090118).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Marine Collagen Peptides Promote Cell Proliferation of NIH-3T3 Fibroblasts via NF-**κ**B Signaling Pathway**

**Fei Yang <sup>1</sup> , Shujie Jin <sup>2</sup> and Yunping Tang 2,\***


Received: 30 September 2019; Accepted: 18 November 2019; Published: 19 November 2019 -

**Abstract:** Marine collagen peptides (MCPs) with the ability to promote cell proliferation and migration were obtained from the skin of *Nibea japonica*. The purpose of MCPs isolation was an attempt to convert the by-products of the marine product processing industry to high value-added items. MCPs were observed to contain many polypeptides with molecular weights ≤ 10 kDa and most amino acid residues were hydrophilic. MCPs (0.25–10 mg/mL) also exhibited 2, 2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, superoxide anion, and 2′ -azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging activities. Furthermore, MCPs promoted the proliferation of NIH-3T3 cells. In vitro scratch assays indicated that MCPs significantly enhanced the scratch closure rate and promoted the migration of NIH-3T3 cells. To further determine the signaling mechanism of MCPs, western blotting was used to study the expression levels of nuclear factor kappa-B (NF-κB) p65, IκB kinase α (IKKα), and IκB kinase β (IKKβ) proteins of the NF-κB signaling pathway. Our results indicated protein levels of NF-κB p65, IKKα and IKKβ increased in MCPs-treated NIH-3T3 cells. In addition, MCPs increased the expression of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF-β) in NIH-3T3 cells. Therefore, MCPs, a by-product of *N. japonica,* exhibited potential wound healing abilities in vitro.

**Keywords:** *Nibea japonica*; marine collagen peptides; proliferation; wound healing; processing by-products

#### **1. Introduction**

Owing to the boom in marine product processing industry, there is a huge production of by-products that are either discarded or simply used as animal feed or fertilizer [1,2]. Hence, there is an urgent requirement to explore methods for using such by-products to yield high value-added items. Bioactive peptides from marine resources possess several physiological functions, including antioxidative [3], anticancer [4], antibacterial [5], angiotensin-converting-enzyme (ACE) inhibitory [6], immunomodulatory [7], hepatoprotective [8], and wound healing activities [9]. Therefore, extraction of bioactive peptides from marine wastes and by-products might offer new avenues for their utilization, consequently preventing environmental pollution and creating enormous economic benefits [6,10].

Collagen extracted from marine by-products is in demand due to the absence of religious restrictions on its use, low immunogenicity, and non-cytotoxicity [11,12]. Marine collagen undergoes enzymatic and chemical hydrolysis to generate marine collagen peptides (MCPs). [13]. Compared to collagen, MCPs possess several advantages, such as ease of absorption for its lower molecular weight and unique physiological functions (including antioxidation) [3,14], high affinity to calcium [15], antihypertensive [16,17], and wound healing activities [18,19].

Currently, the effects of collagen peptides or their combinations with other functional ingredients on wound healing have been the focus of many studies because of their outstanding antioxidant and antimicrobial properties [13,18]. Wound healing is a complex process involving cell-matrix interactions, inflammation, new tissue formation and tissue remodeling [19,20]. Fibroblasts are responsible for regeneration and remodeling of connective tissue in healthy skin [18,20]. So far, there are only few studies dedicated to explore the mechanism of wound healing induced by MCPs in vitro or in vivo. Previous researches have shown a close association between wound healing and nuclear factor kappa enhancer binding protein (NF-κB) signaling pathway [5,21]. NF-kB is related to cell proliferation, cell adhesion, inflammation and elimination of reactive oxygen species (ROS) [20]. In addition, the NF-kB signaling pathway has been reported to be involved with cutaneous [7,22] and corneal epithelial wound healing [21]. As NF-kB signaling and MCPs are both connected to wound healing, we hypothesized that this pathway might play an important role in wound healing induced by MCPs.

Previously, marine collagen was successfully obtained from the skin of *Nibea japonica,* and the physicochemical properties and biocompatibility were determined [11,23]. In an attempt to identify functional MCPs, in the present study, MCPs were extracted from *N. japonica* skins for their functional assessment. We also analyzed the molecular weight, amino acid content and antioxidant activities of the extracted MCPs. Our study showed that MCPs can promote cell proliferation and migration of NIH-3T3 fibroblasts via the NF-kB signaling pathway.

#### **2. Results and Discussions**

#### *2.1. Determination of Molecular Weight Distribution of MCPs*

The HPLC spectrum of the standard molecular weight samples is shown in Figure 1A and the regression equation obtained is as follows:

$$\text{lg}\text{Mw} = -0.2753\text{Rt} + 7.3148\tag{1}$$

**Figure 1.** The HPLC spectra of the standard molecular weight samples (**A**) and marine collagen peptides (MCPs) from skin of *Nibea japonica* (**B**).

The coefficient of regression (R<sup>2</sup> ) was 0.9652 indicating good linear relationship and the molecular weight distribution of MCPs could be determined based on the above equation. The HPLC spectrum of MCPs from the *N. japonica* skins is shown in Figure 1B. Components less than 1, 3, 5 and 10 kDa accounted for 55.25%, 79.29%, 85.71% and 90.31% of the spectrum respectively indicating these MCPs primarily contained a large number of low molecular weight polypeptides. Furthermore, *N. japonica* MCPs had better water solubility than the marine collagen [11] essentially because their low molecular weight structures possess many water-exposed polar amino acid residues, leading to the formation of more hydrogen bonds [24,25].

#### *2.2. Amino Acid Content of MCPs*

The amino acid content of MCPs from *N. japonica* skins is shown in Figure 2. The studied MCPs comprised seven essential amino acids (11.49%) and ten non-essential amino acids (70.48%). Glycine was the principal amino acid in MCPs, accounting for approximately 21.22% of the total amino acid composition, followed by proline (10.55%), alanine (9.79%), hydroxyproline (9.28%), arginine (7.47%) and glutamic acid (4.48%). Furthermore, no cysteine was detected in the concerned MCPs. In our previous studies, we confirmed that the collagen from *N. japonica* skins is a type I collagen [11,23]. Cysteine being exclusively present in type III collagen [12] our results confirm our previous observation. MCPs usually contain a high concentration of Gly-Xaa-Yaa triplets, where Xaa is usually proline and Yaa is most likely hydroxyproline [11,12]. The high content of glycine, proline and hydroxyproline in MCPs was consistent with the high frequency of occurrence of the Gly-Pro-Hyp sequence in the collagen. Furthermore, the glycine (21.22%), proline (10.55%), hydroxyproline (9.28%) and arginine (7.47%) contents in MCPs from *N. japonica* skins was similar to that in MCPs from tilapia skin (where the percentage of glycine, proline, hydroxyproline, and arginine were 20.92%, 11.32%, 10.28% and 7.96%, respectively) [13]. The majority of the amino acid residues were hydrophilic, such as hydroxyproline, arginine, glutamic acid, and aspartic acid. This was consistent with the good water solubility of MCPs from *N. japonica* skins.

**Figure 2.** Amino acids content of MCPs extracted from skin of *Nibea japonica*. Note: \* essential amino acid. All assays were performed in triplicate.

#### *2.3. Antioxidant Activity of MCPs*

NADPH oxidase stimulates inflammatory cells to produce large amounts of ROS during the inflammatory phase of wound healing [26,27]. Normally, ROS are scavenged by antioxidants, and there is a balance between ROS production and neutralization [26]. On the contrary, this balance is disturbed in a wound, where excessive ROS are produced. Excessive ROS induction is associated with activation of pro-apoptotic proteins resulting in cell death and necrosis and can be harmful for wound healing. MCPs are widely used in the skin care industry to promote wound healing due to their antioxidant properties and other beneficial properties [28]. Therefore, the antioxidant activities of MCPs (0.25–10 mg/mL) from the skin of *N. japonica* were evaluated using four different radical scavenging assays.

As illustrated in Figure 3, MCPs (0.25–10 mg/mL) obtained from *N. japonica* skins could scavenge DPPH, hydroxyl, superoxide anion and ABTS radicals. The concentration of MCPs was related to the scavenging activities of these four free radicals. The scavenging activities of these four free radicals also increased in proportion to MCPs concentration. However, as shown in Figure 3, the antioxidant activities of MCPs were relatively lower than that of ascorbic acid (approximately 0–60% at concentrations between 0.25 and 10 mg/mL), and should be improved for use in wound healing. Recently, several functional ingredients, such as chitosan, chemically modified chitosan or nicotinamide were used to improve the antioxidant activity of peptides to promote wound healing. For example, the *N*-succinyl chitosan-collagen peptide copolymer manufactured with transglutaminase possessed better antioxidant activity and could be used as a wound healing biomaterial [28]. Nicotinyl-isoleucine-valine-histidine (NA-IVH), manufactured by combining nicotinamide and jellyfish peptides (IVH), showed significant enhancement of radical scavenging function and can promote wound healing under hyperglycemic condition [26]. Therefore, MCPs have to be modified to improve their wound healing properties for subsequent application in wound healing.

**Figure 3.** 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (**A**), Hydroxyl (**B**), superoxide anion (**C**) and ABTS (**D**) radical scavenging activities of MCP s from *Nibea japonica* skins. All assays were performed in triplicate.

#### *2.4. Cell Proliferation of NIH-3T3*

Various types of cells are known to undergo migration and proliferation during wound healing. Fibroblasts are the key components of normal wound healing and play an important role from late inflammation to complete epithelialization [29]. The present study demonstrated that MCPs have the potential to promote the growth of NIH-3T3 cells. As shown in Figure 4, the viability rate of NIH-3T3 cells treated with varied concentrations of MCPs increased significantly post 72 h of incubation. The viability of cells treated with 25 µg/mL MCPs was 37% more than that of the negative control (NC) group, but was lower than that of the positive control (PC) group. Our observation is in agreement with the results obtained using MCPs from tilapia, which promoted L929 fibroblast proliferation [18]. Thus, MCPs showed significant proliferation in vitro and have potential to be used for wound healing or cosmetic application.

**Figure 4.** Relative cell viability as affected by 72 h treatment of different concentrations of MCPs from *Nibea japonica* skin. Negative control (NC): adding 0.4% serum DMEM to cells; Experimental group: MCP was dissolved by 0.4% serum DMEM in concentrations of 6.25, 12.5, 25, 50, and 100 µg/mL and then added to the prepared cells; Positive control (PC): adding 10% serum DMEM to cells. \* *p* < 0.05 and \*\* *p* < 0.001 vs. NC. The data were expressed as the mean ± standard deviation (*x* ± *s*, *n* = 6).

#### *2.5. E*ff*ect of MCPs on the Scratch Wound Closure In Vitro*

Fibroblast migration can accelerate the process of wound re-epithelialization and promote wound closure during healing [30]. Previously, in vitro scratch test has often been used to simulate wound healing [18,20]. Therefore, we used the above assay on NIH-3T3 cells to evaluate the effect of MCPs from *N. japonica* skins on the wound healing process. As shown in Figure 5, the migration of cells to the scratched area was evident after 12 h and 24 h. In addition, in the presence of MCPs, the wound area was significantly reduced in a dose-dependent manner compared to the control group without MCPs. Significant scratch closure mediated by MCPs was observed after 24 h. In particular, the effect of 50 µg/mL MCPs on in vitro wound healing was highly statistically significant (Figure 5B) and the scratch was almost completely sealed (Figure 5A). Our results indicated that MCPs was capable of inducing NIH-3T3 cell migration and potentially promote wound healing. This may be because abundant amino acids residues in MCPs provide a suitable environment for NIH-3T3 cells to proliferate and migrate (although the mechanism is not clearly delineated).

**Figure 5.** Effect of MCPs from *Nibea japonica* skins on the scratch closure in vitro. (**A**) Representative optical images showed the cells migrated toward wound gap after 12 h and 24 h incubation; (**B**) Wound closure rate (%) that affected by MCPs for 12 h and 24 h. The data was obtained by using Image J 1.38 software and were expressed as the mean ± standard deviation (*x* ± *s*, *n* = 6). \* *p* < 0.05 and \*\* *p* < 0.001 vs. control.

#### *2.6. MCPs Activated the NF-*κ*B Signaling Pathway in NIH-3T3 Fibroblasts*

NF-κB is a transcription factor that regulates the expression of multiple genes involved in a variety of cellular functions including cell migration, proliferation, adhesion and survival [20,31]. Therefore, for further confirmation of role of MCPs towards activation of the signaling pathway through NF-κB the protein expression levels of some related proteins were evaluated using western blotting. As shown in Figure 6, NF-κB p65, IκB kinase α (IKKα), and IκB kinase β (IKKβ) levels increased significantly after treatment of different concentrations of MCPs in a dose-dependent manner. These results indicated that MCPs can promote NIH-3T3 cell migration and proliferation via the NF-κB signaling pathway.

**Figure 6.** MCPs activated the NF-κB signaling pathway and increased expression of its target pathways in NIH-3T3 fibroblast cells. (**A**) Western blot analysis of the NF-κB p65, IKKα, and IKKβ in the NIH-3T3 cells treated with different concentrations MCPs overnight. (**B**) The expression levels of NF-κB p65, IKKα, and IKKβ analyzed by western blotting. The data was obtained by using Image J 1.38 software and were expressed as the mean ± standard deviation (*x* ± *s*, *n* = 6). \* *p* < 0.05 and \*\* *p* < 0.001 vs. control.

#### *2.7. Western Blot Analysis of Growth Factors*

Wound healing is a complex process regulated by different signaling pathways, various cytokines and certain growth factors [19,32]. Epidermal growth factor (EGF) can enhance the migration and proliferation of fibroblasts. In addition, EGF promotes angiogenesis and epithelization and triggers growth factor secretion by fibroblasts, which ultimately leads to accelerated wound healing [33]. Fibroblast growth factor (FGF) can enhance angiogenesis, cell migration and proliferation to promote wound healing [34]. Vascular endothelial growth factor (VEGF) is the main growth factor that triggers angiogenesis and stimulates wound healing [20,32]. Transforming growth factor (TGF-β) can also induce various processes such as secretion of extracellular matrix proteins, proliferation, migration, and angiogenesis [35]. Therefore, we further assessed the effects of MCPs from *N. japonica* skin on the expression of EGF, FGF, VEGF, and TGF-β in this study. As shown in Figure 7, the protein levels of EGF, FGF, VEGF, and TGF-β increased significantly after treatment with various concentrations of MCPs. These results support the notion that MCPs can be applied for promoting wound healing. In addition, a continuous over-expression of such growth factors, without a turning-back point towards their initial levels after wound healing, may be linked to other non-beneficial proliferation-related manifestations such as cancerous neoangiogenesis because of an unresolved inflammatory process [36]. So, further in vivo experiments should be applied to the wound surface of the skin to demonstrate the effect of MCPs, and the role of NF-κB signaling pathway or growth factors in promoting wound healing.

**Figure 7.** Effect of MCPs on the protein expression levels of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF-β) in NIH-3T3 fibroblast cells. (**A**) Western blot analysis of the EGF, FGF, VEGF, and TGF in the NIH-3T3 cells treated with different concentrations MCPs overnight (**B**) Protein expression levels of EGF, FGF, VEGF, and TGF analyzed by western blotting. The data was obtained by using Image J 1.38 software and were expressed as the mean ± standard deviation (*x* ± *s*, *n* = 6). \* *p* < 0.05 and \*\* *p* < 0.001 vs. control.

#### **3. Materials and Methods**

#### *3.1. Materials*

*N. japonica* skins available in our laboratory [11], and the NIH-3T3 fibroblasts were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). Antibodies raised against NF-κB p65 (cat. No. AF0246), IKKα (cat. No. AF0198), IKKβ (cat. No. AI137), VEGF (cat. no. AF1309), and TGF-β (cat. No. AF0198) were purchased from Beyotime Biotechnology (Shanghai, China). β-actin (cat. no. K200058M) was purchased from Solarbio (Beijing, China). Detection antibodies for EGF (cat. no. 184265) and FGF (cat. no. ab171941) were procured from Abcam (Cambridge, England). MTT cell proliferation and cytotoxicity assay kit (AR1156) was purchased from Boster Biological Technology co. Itd (Wuhan, China). All other reagents were of analytical grade.

#### *3.2. Preparation of MCPs from N. japonica skin*

The non-collagenous proteins and fat were removed from the *N. japonica* skins following protocol described by Tang et al. [11]. Following it, the fish skins were heated at 100 ◦C for 10 min, and hydrolyzed in presence of neutral protease (1500 U/g). We adjusted the initial pH of the solution to 7.0 and the enzymatic hydrolysis was performed at 45 ◦C for 3 h. The above step was followed by enzyme deactivation at 100 ◦C for 10 min. After centrifugation, the supernatant of MCPs was collected and lyophilized for further study.

#### *3.3. Determination of the Molecular Weight Distribution of MCPs*

The molecular weight distribution of MCPs was analyzed using high pressure liquid chromatography (HPLC) (Agilent 1200, CA, USA). We used a TSK gel G2000 SWXL analytical column (4.6 × 250 mm, 5 µm) at UV 220 nm at 25 ◦C, and a mobile phase of acetonitrile/water/trifluoroacetic acid (45:55:0.1) at the flow rate of 0.5 mL/min. The standard samples comprised of peroxidase (40,000 Da), aprotinin (6500 Da), Arg-Val-Ala-Pro-Glu-Glu-His-Pro-Val-Glu-Gly-Arg-Tyr-Leu-Val (1750 Da) [7], and Tyr-Val-Pro-Gly-Pro (530 Da) [4] which were loaded into the column by turn. The standard curve of retention time and absorbance was plotted. The MCPs solution was then filtered using 0.22 µm micropore film and injected under the same conditions. Finally, the molecular weight distribution of MCPs was calculated according to the standard curve equation.

#### *3.4. Amino Acid Content*

The amino acid content was determined according to the Chinese national standard (GB5009124-2016). MCPs were first dissolved in 6 M HCl solution and hydrolyzed at 110 ◦C for 24 h. The hydrolysate was further diluted with citric acid buffer and analyzed using an amino acid analyzer L-8900 (Hitachi, Tokyo, Japan). The hydroxyproline content was analyzed as per procedure described by Tang et al. [11].

#### *3.5. Antioxidant Activity of MCPs*

The hydroxyl, 2, 2-diphenyl-1-picrylhydrazyl (DPPH), 2, 2′ -azino-bis-3-ethylbenzothiazoline-6 sulfonic acid (ABTS), and superoxide anion radical scavenging activity of MCPs was analyzed according to Zhao et al. [37].

#### *3.6. Proliferation of NIH-3T3 Fibroblasts in Presence of MCPs*

The MTT assay was employed to assess proliferation of MCPs [11]. Briefly, NIH-3T3 cells were seeded in a 96-well plate at a density of 2 × 10<sup>5</sup> cells/mL and cultured in complete medium (Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin) at 37 ◦C in a 5% CO<sup>2</sup> incubator. Upon 80–90% cellular confluence the culture medium was substituted with fresh maintenance medium (DMEM contains 0.4% FBS), and the cells were further grown for duration of 24 h at 37 ◦C. Further the cells were exposed to different concentrations of MCPs (0, 6.25, 12.5, 25, 50 and 100 µg/mL) in the maintenance medium, and cultured for 24, 48 and 72 h respectively. Cells grown in the same volume of complete medium served as the positive control group. The optical density (OD) at 490 nm was determined using a microplate reader (SpectraMa, Molecular Devices Co., San Jose, CA, USA), and relative cell viability (%) was calculated using the following formula:

$$\text{Relative cell viability (\%)} = \left[1 - \text{(OD treated/OD untreated)}\right] \times 100\% \tag{2}$$

#### *3.7. In Vitro Scratch Wound Assay*

NTH-3T3 cells were seeded in a 6-well plate at a density of 2 × 10<sup>5</sup> cells/mL and incubated in complete medium until cell confluence reached about 80–90%. The cells were further grown for next 24 h at 37 ◦C in 5% CO<sup>2</sup> incubator. A uniform scratch wound was created using a 200 µL sterile pipette tip, and the wound debris was removed through phosphate buffer saline (PBS) wash. The scratched cells were then treated with different concentrations of MCPs (0, 12.5, 25 and 50 µg/mL) and cultured for 12 or 24 h. Scratch closure was evaluated using an inverted microscope (Olympus, Tokyo, Japan) and the scratch area was analyzed using the Image J 1.38 software (NIH, Bethesda, MD, USA). We enumerated the scratch closure rate (%) based on the following formula:

$$\text{Scracth closure rate (\%)} = (\text{A}\_0 - \text{A}\_t) / \text{A}\_0 \times 100\% \tag{3}$$

where A<sup>0</sup> represents the scratch area at 0 h and A<sup>t</sup> represents the same at the designated time point.

#### *3.8. Western Blot Analysis*

Western blotting of target proteins helped to confirm the proliferation of MCPs on NIH-3T3 cells., We used the technique according to Jiang et al. [38] with certain modifications. We used a seeding density of 2 × 10<sup>5</sup> cells/mL for culturing NTH-3T3 cells and treated with varied concentrations of MCPs (0, 12.5, 25 and 50 µg/mL) for 24 h. Subsequently, the cells were collected and lysed in radioimmunoprecipitation assay (RIPA) lysis solution. Protein concentration of cellular lysates was obtained using the bicinchoninic acid (BCA) protein assay. Further, an equivalent amount of denatured protein sample (30 µg) was resolved using 12% sodium dodecyl sulfate (SDS)-polacrylamide gel. After electrophoresis, the gel was transferred onto a polyvinylidene difluoride (PVDF) membrane. Non-specific binding was prevented through incubation with 5% skimmed milk for 1 h followed

by overnight incubation with diluted (1:1000) primary antibodies (NF-κB p65, IKKα, IKKβ, VEGF, EGF, FGF, and TGF-β) at 4 ◦C. We finally incubated the membranes in diluted (1:1000) secondary antibodies for 1 h at room temperature. The target protein bands were visualized using enhanced chemiluminescence and the density was enumerated using the Image J 1.38 software (NIH, Bethesda, MD, USA). We used β-Actin as an internal control.

#### *3.9. Statistical Analysis*

We represented all experimental data as the mean ± standard deviation (*x* ± *s*, *n* = 6) and analyzed using the SPSS software version 24.0 (SPSS Inc., Chicago, IL, USA). Statistical significance of the data was determined using one-way analysis of variance (ANOVA).

#### **4. Conclusions**

In the present study, MCPs prepared from the skin of *N. japonica* exhibited potential cell proliferation and migration activities. Our results indicated that MCPs are rich in polypeptides with molecular weights ≤ 10 kDa. MCPs could scavenge DPPH, hydroxyl, superoxide anion, and ABTS radical as well as promoted the proliferation and migration of NIH-3T3 cells. In vitro scratch assays also reflected that MCPs significantly affect the scratch closure rate. MCPs further increased the protein levels of NF-κB p65, IKKα, and IKKβ, which are prominent members of the NF-κB signaling pathway, as well as those of certain growth factors such as EGF, FGF, VEGF, and TGF-β in NIH-3T3 cells (as revealed through western blotting). In conclusion, our results indicated that MCPs from the skin of *N. japonica* possess potential to promote wound healing. The findings may provide guidance for high value-added utilization by-products of marine processing industry. In the future, in vivo experiments are needed to apply to the wound surface of the skin to demonstrate role of MCPs in promoting wound healing.

**Author Contributions:** Y.T. conceived and designed the experiments. F.Y. and S.J. performed the experiments and carried out statistical analysis of the data. F.Y. and S.J. wrote the manuscript.

**Funding:** This work was financially supported by the National Natural Science Foundation of China (grant No. 41806153).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Sample Availability:** Samples are available from the first or corresponding author.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **An Updated Review on Pharmaceutical Properties of Gamma-Aminobutyric Acid**

**Dai-Hung Ngo <sup>1</sup> and Thanh Sang Vo 2,\***


Academic Editors: María Dolores Torres and Elena Falqué López Received: 27 May 2019; Accepted: 19 July 2019; Published: 24 July 2019

**Abstract:** Gamma-aminobutyric acid (Gaba) is a non-proteinogenic amino acid that is widely present in microorganisms, plants, and vertebrates. So far, Gaba is well known as a main inhibitory neurotransmitter in the central nervous system. Its physiological roles are related to the modulation of synaptic transmission, the promotion of neuronal development and relaxation, and the prevention of sleeplessness and depression. Besides, various pharmaceutical properties of Gaba on non-neuronal peripheral tissues and organs were also reported due to anti-hypertension, anti-diabetes, anti-cancer, antioxidant, anti-inflammation, anti-microbial, anti-allergy, hepato-protection, reno-protection, and intestinal protection. Therefore, Gaba may be considered as potential alternative therapeutics for prevention and treatment of various diseases. Accordingly, this updated review was mainly focused to describe the pharmaceutical properties of Gaba as well as emphasize its important role regarding human health.

**Keywords:** anti-hypertension; bioactivity; Gaba; Gaba-rich product; health benefit

#### **1. Introduction**

Gamma-aminobutyric acid (Gaba) is a non-protein amino acid that is widely distributed in nature. Especially, Gaba is present in high concentrations in different brain regions [1]. Besides, it was also found in various foods such as green tea, soybean, germinated brown rice, kimchi, cabbage pickles, yogurt, etc. Generally, Gaba was produced by l-glutamic acid under the catalyzation of glutamic acid decarboxylase [2]. In the nervous system, newly synthesized Gaba is packaged into synaptic vesicles and then released into the synaptic cleft to diffuse to the target receptors on the postsynaptic surface [3]. Numerous studies have identified two distinct classes of Gaba receptor including Gaba<sup>A</sup> and Gaba<sup>B</sup> [4]. These receptors are different due to their pharmacological, electrophysiological, and biochemical properties. Gaba<sup>A</sup> receptor is Gaba-gated chloride channels located on the postsynaptic membrane, while Gaba<sup>B</sup> receptor is G protein-coupled receptors located both pre- and postsynaptic.

Gaba is well known as the major inhibitory neurotransmitter in the mammalian central nervous system. It was reported to play vital roles in modulating synaptic transmission, promoting neuronal development and relaxation, and preventing sleeplessness and depression [5–9]. Notably, various biological activities of Gaba were documented due to anti-hypertension, anti-diabetes, anti-cancer, antioxidant, anti-inflammation, anti-microbial, and anti-allergy. Moreover, Gaba was also reported as a protective agent of liver, kidney, and intestine against toxin-induced damages [10]. In this contribution, the pharmaceutical properties of Gaba on non-neuronal peripheral tissues and organs were mainly focused to emphasize its beneficial role in prevention and treatment of various diseases.

#### **2. Pharmaceutical Properties of Gaba**

#### *2.1. Neuroprotective E*ff*ect*

It has been reported that the damage of nervous tissue triggers inflammatory response, causing the release of various inflammatory mediators such as reactive oxygen species (ROS), nitric oxide, and cytokines. These mediators can cause several neuronal degenerations in the central nervous system such as Alzheimer's, Parkinson's, and multiple sclerosis [11,12]. So far, numerous studies have been reported regarding the important roles of Gaba on neuro-protection against the degeneration induced by toxin or injury (Figure 1 and Table 1). According to Cho et al. (2007), Gaba produced by the kimchi-derived *Lactobacillus buchneri* exhibited a protective effect against neurotoxic-induced cell death [13]. Moreover, Gaba-enriched chickpea milk can protect neuroendocrine PC-12 cells from MnCl2-induced injury, improve cell viability, and reduce lactate dehydrogenase release [14]. On the other hand, Zhou and colleagues have determined that Gaba receptor agonists also possessed neuroprotective effect against brain ischemic injury. Both Gaba<sup>A</sup> and Gaba<sup>B</sup> receptor agonist (muscimol and baclofen) could significantly protect neurons from the death induced by ischemia through increasing nNOS (Ser847) phosphorylation [15]. Likewise, the administration of Gaba<sup>B</sup> receptor agonist baclofen significantly alleviated neuronal damage and suppressed cytodestructive autophagy via up-regulating the ratio of Bcl-2/Bax and increasing the activation of Akt, GSK-3β, and ERK [16]. Additionally, co-activation of Gaba receptor agonists (muscimol and baclofen) resulted in the attenuation of Fas/FasL apoptotic signaling pathway, inhibition of the kainic acid-induced increase of thioredoxin reductase activity, the suppression of procaspase-3 activation, and the decrease in caspase-3 cleavage. It indicates that co-activation of Gaba receptor agonists results in neuroprotection by preventing caspase-3 denitrosylation in kainic acid-induced seizure of rats [17].



β

**Figure 1.** Therapeutic targets for neuroprotective activity of Gaba.

#### *2.2. Neurological Disorder Prevention*

Neurologic disorder is associated to dysfunction in part of the brain or nervous system, resulting in physical or psychological symptoms. It includes epilepsy, Alzheimer's disease, cerebrovascular diseases, multiple sclerosis, Parkinson's disease, neuroinfections, and insomnia [18]. It was evidenced that Gaba can suppress neurodegeneration and improve memory as well as cognitive functions of the brain (Figure 2 and Table 2). According to Okada et al. (2000), the usefulness of Gaba-enriched rice germ on sleeplessness, depression, and autonomic disorder was examined [19]. Twenty female patients were administered by Gaba-rich rice germ for three times per day. It was observed that the most common mental symptoms during the menopausal and pre-senile period such as sleeplessness, somnipathy, and depression were remarkedly improved in more than 65% of the patients with such symptoms. Likewise, oral administration of Gaba-rich Monascus-fermented product exhibited the protective effect against depression in the forced swimming rat model. Its antidepressant effect was suggested due to recovering the level of monoamines norepinephrine, dopamine, and 5-hydroxytryptamine in the hippocampus [20]. Meanwhile, Yamatsu et al. (2016) reported that Gaba administration significantly shortened sleep latency and increased the total non-rapid eye movement sleep time, indicating the essential role of Gaba in the prevention of a sleep disorder [21]. Moreover, the mixture of Gaba and l-theanine could decrease sleep latency, increase sleep duration, and up-regulate the expression of Gaba and glutamate GluN1 receptor subunit [22]. On the other hand, the electroencephalogram assay has revealed the significantly roles of Gaba in increasing alpha waves, decreasing beta waves, and enhancing IgA levels under stressful conditions. It indicates that Gaba is able to induce relaxation, diminish anxiety, and enhance immunity under stressful conditions [23]. The administration of Gaba-enriched product fermented by kimchi-derived lactic acid bacteria also improved long-term memory loss recovery in the cognitive function-decreased mice and increased the proliferation of neuroendocrine PC-12 cells in vitro [24]. Moreover, the Gaba-enriched fermented *Laminaria japonica* (GFL) provided a protective effect against cognitive impairment associated with dementia in the elderly [25]. In addition, Reid and colleagues have shown that GFL could improve cognitive impairment and neuroplasticity in scopolamineand ethanol-induced dementia model mice [26]. Especially, GFL was effective in increasing serum brain-derived neurotrophic factor level that associated with lower risk for dementia and Alzheimer's disease in middle-aged women [27]. These results indicate that the use of Gaba-enriched functional foods may improve depression, sleeplessness, cognitive impairment, and memory loss.

**Figure 2.** Preventive action of Gaba on neurological disorders.


**Table 2.** Neurological disorder prevention of Gaba.

#### *2.3. Anti-Hypertensive E*ff*ect*

Hypertension is known to relate to a high blood pressure condition, causing various cardiovascular diseases such as ischemic and hemorrhagic stroke, myocardial infarction, and heart and kidney failure [28]. Particularly, angiotensin-I converting enzyme (ACE) was revealed to play an important role in the regulation of blood pressure via converting angiotensin I into the potent vasoconstrictor angiotensin II [29]. Hence, ACE is one of the among therapeutic targets for the control of hypertension. According to Nejati et al. [30], the milk fermented by *Lactococcus lactis* DIBCA2 and *Lactobacillus plantarum* PU11 exhibited an ACE inhibitory activity up to an IC<sup>50</sup> value of 0.70 ± 0.07 mg/mL. Similarly, high ACE inhibitory activity was also observed by Gaba, which was achieved from *L. plantarum* NTU 102-fermented milk [31]. Moreover, *L. brevis*-fermented soybean containing approximately 1.9 g/kg Gaba was found to possess higher ACE inhibitory activity than the traditional soybean product [32]. Besides, the fermentation of a soybean solution by kimchi-derived lactic acid bacteria in the optimized condition has achieved a Gaba content of up to 1.3 mg/g soybean seeds, and its ACE inhibitory activity was observed up to 43% as compared to the control [33]. Notably, high Gaba content (10.42 mg/g extract) and significant ACE inhibitory activity (92% inhibition) was also determined by the fermented lentils [34].

On the other hand, the anti-hypertensive activity of Gaba was also reported in numerous studies using different experimental models (Table 3). Kimura et al. [35] have investigated the effect of Gaba on blood pressure in spontaneously hypertensive rats. It was observed that the intraduodenal administration of Gaba (0.3 to 300 mg/kg) caused a dose-related decrease in the blood pressure in 30 to 50 min. The hypotensive effect of Gaba was suggested due to attenuating a sympathetic transmission through the activation of the Gaba<sup>B</sup> receptor at presynaptic or ganglionic sites. Moreover, the lowering effect of Gaba-enriched dairy product on the blood pressure of spontaneously hypertensive and normotensive Wistar-Kyoto rats was also determined [36]. Notably, the clinical trial has confirmed that daily supplementation of 80 mg of Gaba was effective in the reduction of blood pressure in adults with mild hypertension [37]. Therefore, the consumption of Gaba-enriched dairy product would be beneficial for the down-regulation of hypertension. Indeed, the administration of Gaba-enriched rice grains brings about 20 mmHg decrease in blood pressure in spontaneously hypertensive rats, while there was no significant hypotensive effect in normotensive rats [38]. Likewise, the significant anti-hypertensive activity and the serum cholesterol-lowering effect of Gaba-rich brown rice were shown in spontaneously hypertensive rats as compared to the control [39,40]. In the clinical trial, the effects of Gaba-enriched white rice on blood pressure in 39 mildly hypertensive adults has been examined in a randomized, double blind, placebo-controlled study [41]. It was revealed that the consumption of the Gaba rice could improve the morning blood pressure as compared with the placebo rice after the 1st week and during the 6th and 8th weeks. In the same trend, Tsai and colleagues have determined that Gaba-enriched Chingshey purple sweet potato-fermented milk by lactic acid bacteria (*L. acidophilus* BCRC 14065, *L. delbrueckii* ssp. lactis BCRC 12256, and *L. gasseri* BCRC 14619) was able to reduce both systolic blood pressure and diastolic blood pressure in spontaneously hypertensive rats [42]. The alleviative effect of probiotic-fermented purple sweet potato yogurt on cardiac hypertrophy in spontaneously hypertensive rat hearts was also further determined by Lin and colleagues [43].

In addition, the other Gaba-rich products from bean, tomato, and bread were also reported to be effective in the attenuation of hypertension in vivo. Definite decreases in systolic and diastolic blood pressure values and blood urea nitrogen level were achieved in spontaneously hypertensive rats fed with Gaba-enriched beans [44,45]. Likewise, the anti-hypertensive activity of a Gaba-rich tomato was evidenced to decrease blood pressure in spontaneously hypertensive rats significantly [46]. Moreover, the blood pressure of patients with pre- or mild- to moderate hypertension was significantly decreased during the consumption of 120 g/day of Gaba-rich bread [47]. Accordingly, Gaba-enriched dairy foods may be preferred to use for anti-hypertensive therapeutics.


#### **Table 3.** Anti-hypertensive effect of Gaba.

#### *2.4. Anti-Diabetic E*ff*ect*

Diabetes is an endocrine disorder that is associated with dysregulation of carbohydrate metabolism and deficiency of insulin secretion or insulin action, causing chronic hyperglycemia [48]. So far, diabetic diseases can be managed by pharmacologic interventions [49]. However, the lowering blood glucose effect of pharmacological drugs is accompanied with various disadvantages such as drug resistance, side effects, and even toxicity [50]. Therefore, the proper diet and exercise have been recommended and preferred as alternative therapeutics for the regulation of diabetic diseases. Notably, Gaba and Gaba-enriched natural products have been evidenced as effective agents in lowering blood glucose, attenuating insulin resistance, stimulating insulin release, and preventing pancreatic damage (Figure 3 and Table 4). Soltani and colleagues have shown that Gaba enhanced islet cell function via producing

membrane depolarization and Ca(2+) influx, activating PI3-K/Akt-dependent growth and survival pathways, and restoring the β-cell mass [51]. Moreover, Gaba preferentially up-regulated pathways linked to β-cell proliferation and rose to a distinct subpopulation of β cells with a unique transcriptional signature, including urocortin3, wnt4, and hepacam2 [52]. Especially, the combined use of Gaba and sitagliptin was superior in increasing β-cell proliferation, reducing cell apoptosis, and suppressing α-cell mass [53]. On the other hand, Gaba was found to enhance insulin secretion in pancreatic INS-1 β-cells [54]. In the pre-clinical trial model, Gaba administration could decrease the ambient blood glucose level and improve the glucose excursion rate in streptozotocin-induced diabetic mice [53]. Furthermore, oral treatment with Gaba significantly reduced the concentrations of fasting blood glucose, improved glucose tolerance and insulin sensitivity, and inhibited the body weight gain in the high fat diet-fed mice [55]. Notably, Gaba potentially inhibited the diabetic complication related to the nervous system via suppressing the Fas-dependent and mitochondrial-dependent apoptotic pathway in the cerebral cortex [56].

**Figure 3.** Therapeutic targets for anti-diabetic activity of Gaba.

β The fact that the germination of rice and the fermentation of foods are accompanied with the increase in Gaba content [57,58], therefore, the pre- and germinated rice and fermented foods were highly appreciated for their roles in positive regulation of diabetes and its complication. According to Hagiwara and colleagues, the feeding of pre-germinated brown rice diet to diabetic rats significantly decreased blood glucose, adipocytokine PAI-1 concentration, and plasma lipid peroxide [59]. Moreover, pre-germinated brown rice lowered HbA(1c) and adipocytokine (TNF-α and PAI-1) concentration and increased the adiponectin level in type-2 diabetic rats, leading to the prevention of potential diabetic complications [60]. In addition, high fat diet-induced diabetic pregnant rats fed with the germinated brown rice lead to the increase in adiponectin levels and the reduction of insulin, homeostasis model assessment of insulin resistance, leptin, and oxidative stress in their offspring [61]. On the other hand, blackish purple pigmented rice with a giant embryo significantly decreased blood glucose and plasma insulin levels, adipokine concentrations, and hepatic glucose-regulating enzyme activities in ovariectomized rats [62]. Meanwhile, glucose homeostasis was greatly improved through the intervention of Gaba-enriched wheat bran in the context of a high-fat diet rat [63]. The supplement of Gaba-enriched rice bran to obese rats also exhibited an efficient effect on lowering serum sphingolipids, a marker of insulin resistance [64]. In clinical trials, Ito and colleagues have suggested that the intake of pre-germinated brown rice was effective in lowering postprandial blood glucose concentration without insulin secretion increase [65]. Likewise, Hsu et al. [66] and Suzuki et al. [67] have confirmed that pre-germinated brown rice decreased blood glucose and hypercholesterolemia in type 2 diabetes patients.

Beside germinated rice, fermented foods are also known to contain a significant amount of Gaba and possess potential anti-diabetic activity. The oral administration of hot water extract of the fermented tea obtained by tea-rolling processing of loquat (*Eriobotrya japonica*) significantly decreased the blood glucose level and serum insulin secretion in maltose-loaded Sprague–Dawley rats [68]. Similarly, anti-diabetic effects of green tea fermented by cheonggukjang was observed via decreasing water intake and lowering blood glucose and HbA1c levels in diabetic mice [69]. In addition, mung bean fermented by *Rhizopus* sp. [70], yogurt fermented by *Streptococcus salivarius* subsp. thermophiles fmb5 [71], and soybean extract fermented by *Bacillus subtilis* MORI [72] could enhance their anti-hyperglycemic effect via reducing blood glucose, HbA1c, cholesterol, triglyceride, and low-density lipoprotein levels in diabetic mice. In the same trend, the milk fermented by commercial strain YF-L812 (*S. thermophilus*, *L. delbrueckii* subsp. *bulgaricus*), standard strains. *B. breve* KCTC 3419, and *L. sakei* LJ011. Fermented milk was effective in decreasing fasting blood glucose, serum insulin, leptin, glucose and insulin tolerance, total cholesterol, triglycerides, and low density lipoprotein cholesterol [73]. Especially, the consumption of probiotic-fermented milk (kefir) by type 2 diabetic patents lowered HbA1C level, homeostatic model assessment of insulin resistance, and homocysteine amount [74,75]. Accordingly, the germinated rice and fermented foods, which contain a high amount of Gaba, could be used as anti-diabetic functional food for maintaining health and preventing complications in type 2 diabetes.


**Table 4.** Anti-diabetic effect of Gaba.


**Table 4.** *Cont*.

#### *2.5. Anti-Cancer E*ff*ect*

Cancer is involved in the unregulated cell proliferation, apoptosis suppression, invasion, and metastasis [76]. Current cancer therapies are related to surgery, radiation treatment, and chemotherapy treatment, which are widely applied for treatment of all kinds of cancers. However, these therapies possess major disadvantages including cancer recurrence, drug resistance, and side effects. Hence, the discovery of alternative medicines with desirable properties is always necessary. In this regard, Gaba was emerged as a promising compound that is able to regulate cancer due to the induction of apoptosis and inhibition of proliferation and metastasis (Table 5). Gaba-enriched brown rice extract significantly retarded the proliferation rates of L1210 and Molt4 leukemia cells and enhanced apoptosis of the cultured L1210 cells [77]. Moreover, Schuller et al. [78] suggested that Gaba had a tumor suppressor function in small airway epithelia and pulmonary adenocarcinoma, providing the approach for the prevention of pulmonary adenocarcinoma in smokers. According to Huang and colleagues, Gaba was determined to inhibit the activity and expression of MMP-2 and MMP-9 in cholangiocarcinoma QBC939 cells, suggesting its role in prevention of invasion and metastasis in cancer [79]. Song and

colleagues also found the inhibitory effects of Gaba on the proliferation and metastasis of colon cancer cells (SW480 and SW620 cells) due to the up-pressing cell cycle progression (G2/M or G1/S phase), attenuating mRNA expression of EGR1-NR4A1 and EGR1-Fos axis, and disrupting MEK-EGR1 signaling pathway [80]. Especially, the co-treatment of Gaba and Celecoxib significantly inhibited systemic and tumor VEGF, PGE2, and cAMP molecules and down-regulated COX-2 and p-5-LOX protein in pancreatic cancer cells [81]. Moreover, the prolonged administration of Gaba at 1000 mg/kg body weight significantly decreased the number of gastric cancers of the glandular stomach in Wk 52 rats. In parallel, the histological method also revealed the role of Gaba on decreasing the labeling index of the antral mucosa and increasing the serum gastrin level [82]. Likewise, the pre-treatment of Gaba also significantly reduced intrahepatic liver metastasis and primary tumor formation in mice and inhibited human liver cancer cell migration and invasion via the induction of liver cancer cell cytoskeletal reorganization [83]. Meanwhile, the increase in the activity of Gaba<sup>A</sup> receptor contributed to the down-regulation of alpha-fetoprotein mRNA expression and cell proliferation in malignant hepatocyte cell line [84].



#### *2.6. Antioxidant E*ff*ect*

The free radicals contain one or more unpaired electrons that are generated from the living organisms and external sources. The high level of free radicals could cause the damage of the body's tissues and cells, leading to human aging and various diseases [85,86]. Thus, consumption of natural products with high anti-oxidant effect is useful for the prevention of free radical-caused diseases [86]. Herein, the antioxidant property of Gaba has been evidenced in numerous studies (Figure 4). It was shown that Gaba was able to trap the reactive intermediates during lipid peroxidation and react readily with malondialdehyde under physiological conditions [87]. Moreover, the administration of Gaba significantly decreased malondialdehyde concentration and increased the activity of superoxide dismutase and glutathione peroxidase in the cerebral cortex and hippocampus of acute epileptic state rats [88]. In other studies, the protective effect of Gaba against H2O2-induced oxidative stress in pancreatic cells [89] and human umbilical vein endothelial cells [90] was observed via reducing cell death, inhibiting reactive oxygen species (ROS) production, and enhancing antioxidant defense systems. Similarly, gamma rays-induced oxidative stress in the small intestine of rats was significantly

ameliorated via decreasing malondialdehyde and advanced oxidation protein productions, increasing catalase and glutathione peroxidase activities, preventing mucosal damage and hemorrhage, and inducing the regeneration of the small intestinal cells [91]. Gaba also attenuated brain oxidative damage associated with insulin alteration in streptozotocin-treated rats [92]. On the other hand, Gaba from *L. brevis*-fermented sea tangle solution was observed to exhibit stronger antioxidant activity than positive control BHA in scavenging DPPH and superoxide radicals and inhibiting xanthine oxidase [93]. Meanwhile, the Gaba-rich germinated brown rice extract considerably scavenged hydroxyl radical and thiobarbituric acid-reactive substances in both cell-free medium and post-treatment culture media, indicating its radical scavenging capacity in both direct and indirect action [94]. Recently, brew-germinated pigmented rice vinegar was also suggested as a new product with high antioxidant activity [95].

**Figure 4.** Modulatory activity of Gaba for antioxidant promotion.

#### *2.7. Anti-Inflammatory E*ff*ect*

β α β α β Inflammation response is triggered by the stimulation of various factors such as physical damage, ultra violet irradiation, microbial invasion, and immune reactions [96]. It is associated with the production of a large range of pro-inflammatory mediators such cytokine, NO, and PGE<sup>2</sup> [97]. Notably, Gaba was indicated as an inhibitor of inflammation via decreasing pro-inflammatory mediator production and ameliorating inflammatory symptom (Figure 5). At the early time, Han et al. [98] have determined the anti-inflammatory activity of Gaba via inhibiting the production and expression of iNOS, IL-1β, and TNF-α in LPS-stimulated RAW 264.7 cells. As the result, it contributed to the reduction of the whole healing period and enhancement of wound healing at the early stage. Likewise, Gaba suppressed inflammatory cytokine production and NF-kB inhibition in both lymphocytes and pancreatic islet beta cells [99]. Recently, Gaba-enriched sea tangle *L. japonica*, Gaba-rich germinated brown rice, and Gaba-rich red microalgae *Rhodosorus marinus* were reported for their inhibitory capacities on inflammatory response. Gaba-enriched sea tangle *L. japonica* extract suppressed nitric oxide production and inducible nitric oxide synthase expression in LPS-induced mouse macrophage RAW 264.7 cells [100]. Gab-rich germinated brown rice inhibited IL-8 and MCP-1 secretion and ROS production from Caco-2 human intestinal cells activated by H2O<sup>2</sup> and IL-1β [101]. Gaba-rich red microalgae*Rhodosorus marinus* extract negatively modulated expression and release of pro-inflammatory IL-1α in phorbol myristate acetate-stimulated normal human keratinocytes, therefore indicating the potential treatment of sensitive skins, atopia, and dermatitis [102]. Besides, the roles of Gaba in the attenuation of gut inflammation and improvement of gut epithelial barrier were suggested via inhibiting IL-8 production and stimulating the expression of tight junction proteins as well as the expression of TGF-β cytokine in Caco-2 cells [103].

**Figure 5.** Therapeutic targets for anti-inflammatory activity of Gaba.

#### *2.8. Anti-Microbial E*ff*ect*

Gaba tea is a kind of Gaba-enriched tea by the repeating treatments of alternative anaerobic and aerobic conditions. The Gaba tea extract exhibited inhibitory activity against *Vibrio parahaemolyticus, Staphylococcus aureus, Bacillus cereus, Salmonella typhimurium,* and *Escherichia coli* [104]. Gaba could increase *Pseudomonas aeruginosa* virulence due to stimulation of cyanogenesis, reduction in oxygen accessibility, and overexpression of oxygen-scavenging proteins. Gaba also promotes specific changes in the expression of thermostable and unstable elongation factors involved in the interaction of the bacterium with the host proteins [105]. Recently, the role of Gaba in anti-microbial host defenses was elucidated by Kim and colleagues [106]. Treatment of macrophages with Gaba enhanced phagosomal maturation and anti-microbial responses against mycobacterial infection. This study identified the role of Gabaergic signaling in linking anti-bacterial autophagy to enhance host innate defense against intracellular bacterial infection including *Mycobacteria, Salmonella,* and *Listeria*.

#### *2.9. Anti-Allergic E*ff*ect*

γ Allergy is a disorder of the immune system associating with an exaggerated reaction of the immune system to harmless environmental substances. Allergic reaction is characterized by the excessive activation of mast cells and basophils, leading to release various mediators such as histamine and an array of cytokines [107]. Among them, histamine is considered as the major target for potential anti-allergic therapeutics. Herein, the inhibitory activity of Gaba on histamine release from the activated mast cells was investigated in vitro [108,109]. Rat basophilic leukemia cells and rat peritoneal exudate cells sensitized with anti-dinitrophenyl (DNP) IgE and challenged with DNP-conjugated bovine serum albumin resulted in the release of histamine in a cell culture medium. However, IgE-mediated histamine release was inhibited by Gaba treatment in both cells. Conversely, the inhibitory activities of Gaba were lowered by the addition of CGP35348, a Gaba<sup>B</sup> receptor antagonist. It indicated that Gaba inhibited degranulation from basophils and mast cells via Gaba<sup>B</sup> receptor on the cell surface. On the other hand, Hokazono et al. [110] have evaluated the protective effect of Gaba against the development of atopic dermatitis (AD)-like skin lesions in NC/Nga mice. It was observed that Gaba could prevent the development of AD-like skin lesions in mice via alleviating serum immunoglobulin E (IgE) and splenocyte IL-4 production. The combined administration of Gaba and the fermented barley extract remarkedly increased splenic cell interferon-γ production, indicating the domination of Th1/Th2 balance to Th1 response. Hence, the simultaneous intake of Gaba and the fermented barley extract was encouraged to ameliorate allergic symptoms such as atopic dermatitis (Figure 6).

**Figure 6.** Therapeutic targets for anti-allergic activity of Gaba.

#### *2.10. Hepatoprotective E*ff*ect*

The long-term use of ethanol can cause liver damage and unfavorable lipid profiles in humans. The toxic acetaldehyde is formed from alcohol under catalysis of alcohol dehydrogenase, causing various adverse effects such as thirst, vomiting, fatigue, headache, and abdominal pain [111]. For the first time, Oh and colleagues have evaluated the protective effect of Gaba-rich germinated brown rice against the toxic consequences of chronic ethanol use [112]. Interestingly, serum low-density lipoprotein cholesterol, liver aspartate aminotransferase, and liver alanine aminotransferase levels were decreased in mice fed both ethanol and brown rice extract for 30 days. Furthermore, the brown rice extract significantly increased serum and liver high-density lipoprotein cholesterol concentrations and reduced liver triglyceride and total cholesterol concentrations. In the same trend, Lee et al. [113] have reported that Gaba-rich fermented sea tangle (GFST) could prevent ethanol and carbon tetrachloride-induced hepatotoxicity in rats. The oral administration of GFST decreased the serum levels of glutamic pyruvate transaminase, gamma glutamyl transpeptidase, and malondialdehyde levels and increased antioxidant enzyme such as superoxide dismutase, catalase, and glutathione peroxidase [113]. Moreover, GFST increased the activities and transcript levels of major alcohol-metabolizing enzymes, such as alcohol dehydrogenase and aldehyde dehydrogenase, and reduced blood concentrations of alcohol and acetaldehyde [114]. In an in vitro study, the protective effects of GFST against alcohol hepatotoxicity in ethanol-exposed HepG<sup>2</sup> cells were revealed by preventing intracellular glutathione depletion, decreasing gamma-glutamyl transpeptidase activity, and suppressing cytochrome P450 2E1 enzyme expression [115]. These results indicated that Gaba-rich foods might have a pharmaceutical role in the prevention of chronic alcohol-related diseases (Figure 7).

**Figure 7.** Mechanism of the action of Gaba for hepatoprotection.

#### *2.11. Renoprotective E*ff*ect*

Acute kidney injury is involved in kidney damage and cell death, causing high morbidity and mortality worldwide [116]. The renoprotective agents derived from natural products may be essential for the prevention or treatment of kidney injury-related diseases. Indeed, numerous studies have evidenced the protective effect of Gaba against acute kidney injury (Figure 8). According to Kim et al. (2004), the physiological changes caused by acute renal failure such as body weight and kidney weight gain, urea nitrogen and creatinine elevation, creatinine clearance reduction, sodium FE(Na) secretion, and urine osmolarity decrease in rats were significantly improved by oral administration of Gaba [117]. Moreover, the status of serum albumin decrease, urinary protein increase, and serum lipid profile was completely improved by Gaba. In addition, Gaba alleviated nephrectomy-induced oxidative stress by increasing superoxide dismutase and catalase, and decreasing lipid peroxidation in rats [118]. Furthermore, Gaba reduced tubular fibrosis, tubular atrophy, and the transforming growth factor-beta1 and fibronectin expression [119]. The acute tubular necrosis was also apparently reduced to normal proximal condition by Gaba treatment [120]. In another study, Talebi and colleagues have shown the protective effect of Gaba on kidney injury induced by renal ischemia-reperfusion in ovariectomized rats via decreasing serum levels of creatinine and blood urea nitrogen, kidney weight, and kidney tissue damage [121]. Meanwhile, the increases in alanine amino transferase and aspartate amino transferase activities, urea and creatinine levels, malondialdehyde and advanced oxidation protein levels, and oxidative damage to the kidney tissues induced by γ-irradiated- and streptozotocin-treated rats were markedly attenuated by Gaba administration in rats [122]. Especially, Gaba was observed to ameliorate kidney injury induced by renal ischemia/reperfusion injury in a gender dependent manner [123]. These results emphasized the protective effect of Gaba against the renal damage involving in renal failure. γ

**Figure 8.** Mechanism of the action of Gaba for renoprotection.

#### *2.12. Intestinal Protective E*ff*ect*

⁺ ⁺ ⁺ ⁺ Chen and colleagues have examined the beneficial roles of Gaba on intestinal mucosa in vivo [124,125]. It was shown that heat stress-induced chicken decreased the activity of Na+-K+-ATPase, maltase, sucrase, and alkaline phosphatase enzymes in intestinal mucosa [124]. Moreover, heat stress caused the marked decline in villus length, mucosa thickness, intestinal wall thickness, and crypt depth in the duodenum and ileum [125]. However, the treatment of Gaba administration markedly increased the activity of maltase, sucrase, alkaline phosphatase, and Na+-K+-ATPase [124]. Furthermore, Gaba enhanced villus length, mucosa thickness, intestinal wall thickness, and crypt depth in the duodenum and ileum [125]. It indicated that Gaba could effectively alleviate heat stress-induced damages of the intestinal mucosa. In a further study, they investigated the effect of Gaba supplementation on the growth performance, intestinal immunity, and gut microflora of the weaned piglets [126]. Notably, Gaba supplementation improved the growth performance, inhibited

proinflammatory cytokines (IL-1 and IL-18) expression, promoted anti-inflammatory cytokines (IFN-γ, IL-4, and IL-10) expression, and increased the dominant microbial populations, the community richness, and diversity of the ileal microbiota. On the other hand, Xie and colleagues also investigated the effect of Gaba on colon health in mice [127]. It was observed that the female Kunming mice administrated with Gaba at doses of 40 mg/kg/d for 14 days could increase the concentrations of acetate, propionate, butyrate, and total short chain fatty acids, and decreased pH value in colonic and cecal contents. Recently, Kubota and colleagues have revealed that Gaba attenuated ischemia reperfusion-induced alterations in intestinal immunity via increasing IgA secretion, alpha-defensin-5 expression, and superoxide dismutase activity in the rat small intestine [128]. Besides, Jiang and colleagues also showed the protective effect of Gaba against intestinal mucosal barrier injury of colitis induced by 2,4,6-trinitrobenzene sulfonic acid and alcohol [129]. These results have evidenced the physiological function of Gaba in improvement and promotion of intestinal health.

#### *2.13. Other Pharmaceutical Properties*

Yang et al. [130] have examined the modulatory effects of Gaba on cholesterol-metabolism-associated molecules in human monocyte-derived macrophages (HMDMs). It was found that Gaba was effective in the reduction of cholesterol ester in lipid-laden HMDMs via suppressing the expression of scavenger receptor class A, lectin-like oxidized low-density lipoprotein receptor-1, and CD36, and promoting the expression of ATP-binding cassette transporter 1, ATP-binding cassette sub-family G member 1, and scavenger receptor class B type I. Moreover, the production of TNF-α was decreased and the activation of signaling pathways (p38MAPK and NF-κB) was repressed in the presence of Gaba. The inhibitory effect of Gaba on the formation of human macrophage-derived foam cells suggests its role in the prevention of atherosclerotic lesions.

Yang et al. [131] have investigated whether Gaba ameliorate fluoride-induced a thyroid injury in vivo. The model of hypothyroidism was conducted by exposing NaF (50 mg/kg) to adult male mice for 30 days. Thereafter, thyroid hormone production, oxidative stress, thyroid function-associated genes, and side effects during therapy were measured. Interestingly, Gaba supplementation remarkedly promoted the expression of thyroid thyroglobulin, thyroid peroxidase, and sodium/iodide symporter. Moreover, it improved the thyroid redox state, the expression of thyroid function-associated genes, and liver metabolic protection. These findings indicate that Gaba has a therapeutic potential in hypothyroidism.

In regarding to the growth hormone, the oral administration of Gaba was reported to elevate the resting and post-exercise immunoreactive growth hormone and immunofunctional growth hormone concentrations in humans [132]. Moreover, the administration of Gaba is likely to increase the concentrations of plasma growth hormone and the rate of protein synthesis in the rat brain [133,134]. Recently, the role of Gaba in the enhancement of muscular hypertrophy in men after progressive resistance training was also evaluated by Sakashita and colleagues [135]. They found that the combination of Gaba and whey protein was effective in increasing whole body fat-free mass, thus enhancing exercise-induced muscle hypertrophy.

Indeed, the excessive production of free radicals and oxidants causes oxidative stress that damages cell membranes and other structures such as DNA, lipids, and proteins [136]. Particularly, the damage of cell membranes and lipoproteins by hydroxyl and peroxynitrite radicals causes lipid peroxidation and formation of cytotoxic and mutagenic agents such as malondialdehyde and conjugated diene compounds [137]. Moreover, the free radicals and oxidants can change protein structure and lose enzyme activity. Various mutations may also result from oxidants-induced DNA damages. Therefore, oxidative stress can induce a variety of chronic and degenerative diseases such as cancer, cardiovascular disease, neurological disease, pulmonary disease, rheumatoid arthritis, nephropathy, and ocular disease [138]. In this sense, antioxidants play an important role in the neutralization of free radicals, protection of the cells from toxic effects, and prevention of disease pathogenesis [139]. As a result, the antioxidant activity of Gaba may partly contribute to its biological effects such as anti-hypertension, anti-diabetes, anti-cancer, antioxidant, anti-inflammation, anti-microbial, anti-allergy, hepato-protection, reno-protection, and intestinal protection.

#### **3. Conclusions**

The fact that consumers have paid much attention to natural products in order to promote and maintain their health. Simultaneously, various functional foods derived from natural products have been developed along with the tendency of consumers. Herein, Gaba has been evidenced as a powerful bioactive compound with numerous health beneficial effects. Thus, the functional foods produced from Gaba are believed to be able to prevent and/or treat different diseases, especially hypertension, diabetes, and neurological disorders. Whereby, the researches into large-scale production, biotechnological techniques, and high Gaba-producing strains will be remarkedly increased in food industry. However, the further testing and validation due to the safety and efficacy of Gaba consumption are necessary in clinical trials.

**Author Contributions:** We declare that this review was done by the authors named in this article. The review was conceived and designed by D.-H.N. The data were collected and analyzed by D.-H.N. and T.S.V. The manuscript was written by T.S.V. All authors read and approved the manuscript for publication.

**Funding:** This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.02-2018.304.

**Acknowledgments:** This review is also supported by Nguyen Tat Thanh University, Ho Chi Minh city, Vietnam and Thu Dau Mot University, Binh Duong province, Vietnam.

**Conflicts of Interest:** There are no conflicts to declare.

#### **References**


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