*Article* **Curcumin and Nano-Curcumin Mitigate Copper Neurotoxicity by Modulating Oxidative Stress, Inflammation, and Akt/GSK-3**β **Signaling**

**Wedad S. Sarawi <sup>1</sup> , Ahlam M. Alhusaini <sup>1</sup> , Laila M. Fadda <sup>1</sup> , Hatun A. Alomar <sup>1</sup> , Awatif B. Albaker <sup>1</sup> , Amjad S. Aljrboa <sup>1</sup> , Areej M. Alotaibi 1,2, Iman H. Hasan <sup>1</sup> and Ayman M. Mahmoud 3,\***


**Citation:** Sarawi, W.S.; Alhusaini, A.M.; Fadda, L.M.; Alomar, H.A.; Albaker, A.B.; Aljrboa, A.S.; Alotaibi, A.M.; Hasan, I.H.; Mahmoud, A.M. Curcumin and Nano-Curcumin Mitigate Copper Neurotoxicity by Modulating Oxidative Stress, Inflammation, and Akt/GSK-3β Signaling. *Molecules* **2021**, *26*, 5591. https://doi.org/10.3390/ molecules26185591

Academic Editors: Stefano Castellani, Massimo Conese and William Blalock

Received: 30 August 2021 Accepted: 11 September 2021 Published: 15 September 2021

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**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** Copper (Cu) is essential for multiple biochemical processes, and copper sulphate (CuSO<sup>4</sup> ) is a pesticide used for repelling pests. Accidental or intentional intoxication can induce multiorgan toxicity and could be fatal. Curcumin (CUR) is a potent antioxidant, but its poor systemic bioavailability is the main drawback in its therapeutic uses. This study investigated the protective effect of CUR and N-CUR on CuSO<sup>4</sup> -induced cerebral oxidative stress, inflammation, and apoptosis in rats, pointing to the possible involvement of Akt/GSK-3β. Rats received 100 mg/kg CuSO<sup>4</sup> and were concurrently treated with CUR or N-CUR for 7 days. Cu-administered rats exhibited a remarkable increase in cerebral malondialdehyde (MDA), NF-κB p65, TNF-α, and IL-6 associated with decreased GSH, SOD, and catalase. Cu provoked DNA fragmentation, upregulated BAX, caspase-3, and p53, and decreased BCL-2 in the brain of rats. N-CUR and CUR ameliorated MDA, NF-κB p65, and pro-inflammatory cytokines, downregulated pro-apoptotic genes, upregulated BCL-2, and enhanced antioxidants and DNA integrity. In addition, both N-CUR and CUR increased AKT Ser473 and GSK-3β Ser9 phosphorylation in the brain of Cu-administered rats. In conclusion, N-CUR and CUR prevent Cu neurotoxicity by attenuating oxidative injury, inflammatory response, and apoptosis and upregulating AKT/GSK-3β signaling. The neuroprotective effect of N-CUR was more potent than CUR.

**Keywords:** curcumin; GSK-3β; inflammation; DNA damage; oxidative stress

### **1. Introduction**

Copper (Cu) is a redox-active metal found in many organs and tissues. It is essential for a plethora of biochemical processes such as blood clotting, iron absorption, protein homeostasis, energy production, and cellular metabolism [1]. It acts as a cofactor necessary for many redox-regulating proteins [2]. Cu homeostasis is maintained within the normal level by precise regulatory mechanisms that regulate its absorption, excretion, and blood level [3]. Genetic alteration in Cu-regulating ATPases, *ATP7A*, and *ATP7B* can cause Menkes disease (MD) and Wilson disease (WD), respectively [2,4,5]. MD is associated with a defect in Cu absorption and severe Cu deficiency, while WD results in Cu toxicity and affects several organs, including the liver, brain, and eye [2]. Chronic exposure to Cu has been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease [6], Parkinson's disease [7], and familial amyotrophic lateral sclerosis (ALS) [2,8].

Copper sulphate (CuSO4) is a well-known pesticide used for repelling pests that decreases the crop yield in agriculture. It is commonly used in tissue culture incubators to minimise the contamination risk as it has bactericidal and fungicidal properties. Accidental

or intentional CuSO<sup>4</sup> intoxication can induce multiorgan dysfunctions that could be fatal. The systemic absorption of Cu occurs through the gastrointestinal tract, lungs, and skin [9]. The clinical manifestations of Cu toxicity are erosive gastropathy, acute liver and kidney injuries, intravascular hemolysis, arrhythmia, rhabdomyolysis, and seizures [10]. Although the mechanisms of CuSO<sup>4</sup> toxicity are not fully addressed, they represent a combination of significant oxidative stress and endocrine perturbation in the vulnerable organs of the body [11]. Animal studies showed that the chronic oral administration of CuSO<sup>4</sup> causes liver and kidney functional impairment due to increased Cu levels in the respective organs [12]. The toxic effects of Cu on the liver and kidney have been studied extensively, while the toxicities of other vital organs of the body are less documented. Similar to other metals, the management of Cu toxicity includes the use of chelating agents such as D-penicillamine, tetrathiomolybdate, and trientine [13]. Other chelators such as deferoxamine (DFO) have an affinity for Cu binding [14]. Despite the effectiveness of these chelators, they often associated with some serious adverse effects on cardiovascular, gastrointestinal, respiratory, and nervous systems, which necessitates the use of safer alternatives. In addition, the limited or moderate effectiveness of these chelators has been found in some cases.

Curcumin (CUR) is a hydrophobic polyphenolic compound found natively in turmeric [15]. It exhibits antioxidant [16], antimicrobial [17], anti-inflammatory, pulmoprotective [18], anti-diabetic [19], hepatoprotective [20–22], nephroprotective [23], and antitumor actions [24]. In addition to these pharmacological effects, CUR possesses neuroprotective activity where it protected the brain against oxidative injury induced by heavy metals [25]. CUR–cyclodextrin/cellulose nanocrystals (CNCx) exerted more potent antiproliferative effect on prostate and colorectal cancer cell lines than CUR [26]. In addition, CNCx mitigated oxidative stress and improved myelination, and the cellular, electrophysiological, and functional characteristics of Charcot–Marie–Tooth-1A transgenic rats [27]. Recently, Iurciuc-Tincu et al. have immobilized CUR into polysaccharide particles and reported increased stability and bioavailability [28,29]. CUR loading to polysaccharides facilitated overcoming the gastric juice barrier and efficient absorption in the intestine [28,29]. CUR has shown a modulatory effect on glycogen synthase kinase-3 (GSK-3) activity [30], and we have recently reported the involvement of GSK-3β inhibition in mediating its protective efficacy against lead hepatotoxicity [20]. GSK-3β is implicated in neuronal survival; however, the exact mechanism is not clear-cut [31]. Studies have demonstrated increased neuronal death following the overexpression of GSK-3β [32], whereas its knockdown prevents apoptosis [33]. Despite the potent pharmacological effects of CUR, its poor systemic bioavailability and rapid metabolism represent the main drawbacks in its therapeutic uses, which is a problem that was amended by nanoparticle encapsulation [34]. In comparison to the native form, nano-CUR (N-CUR) has a higher solubility and stability but similar activity [15]. Therefore, this nanoformulation can significantly enhance the cell permeability and show more protective effects in vitro and in vivo. This study was conducted to investigate the involvement of the Akt/GSK-3β pathway in CuSO4-induced cerebral oxidative stress, inflammation, and apoptosis in rats and the ameliorative effect of CUR and N-CUR.

#### **2. Results**

#### *2.1. N-CUR and CUR Attenuate Cu-Induced Cerebral Oxidative Stress*

The ameliorative effect of CUR and N-CUR on oxidative stress in the brain of Cuexposed rats was evaluated through the assessment of malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). Cerebral MDA was significantly elevated in Cu-administered rats when compared with the control group (*p* < 0.001; Figure 1A). In contrast, cerebral GSH content (Figure 1B), SOD activity (Figure 1C), and CAT activity (Figure 1D) were decreased in Cu-administered rats (*p* < 0.001). Treatment with DFO, CUR, and N-CUR decreased MDA and increased GSH, SOD, and CAT in the brain of Cu-administered rats. The effect of both CUR and N-CUR on cerebral MDA was significant compared to DFO (*p* < 0.01).

glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT). Cerebral MDA was significantly elevated in Cu-administered rats when compared with the control group (*p*  < 0.001; Figure 1A). In contrast, cerebral GSH content (Figure 1B), SOD activity (Figure 1C), and CAT activity (Figure 1D) were decreased in Cu-administered rats (*p* < 0.001). Treatment with DFO, CUR, and N-CUR decreased MDA and increased GSH, SOD, and CAT in the brain of Cu-administered rats. The effect of both CUR and N-CUR on cerebral

MDA was significant compared to DFO (*p* < 0.01).

**Figure 1.** N-CUR and CUR attenuate Cu-induced cerebral oxidative stress. Treatment with N-CUR, CUR, and DFO decreased MDA (**A**) and increased GSH (**B**), SOD (**C**), and CAT (**D**) in the brain of Cu-administered rats. Data are mean ± SEM, (*n* = 8). \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001. **Figure 1.** N-CUR and CUR attenuate Cu-induced cerebral oxidative stress. Treatment with N-CUR, CUR, and DFO decreased MDA (**A**) and increased GSH (**B**), SOD (**C**), and CAT (**D**) in the brain of Cu-administered rats. Data are mean ± SEM, (*n* = 8). \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001.

#### *2.2. N-CUR and CUR Suppress Cerebral Inflammation in Cu-Administered Rats 2.2. N-CUR and CUR Suppress Cerebral Inflammation in Cu-Administered Rats*

Cerebral levels of NF-κB p65, TNF-α, and IL-6 were assayed to determine the ameliorative effect of CUR and N-CUR on inflammation induced by Cu ingestion (Figure 2). Cu administration increased NF-κB p65 (Figure 2A), TNF-α (Figure 2B), and IL-6 (Figure 2C) in the cerebrum of rats (*p* < 0.001). All treatments (DFO, CUR, and N-CUR) decreased the levels of cerebral NF-κB p65, TNF-α, and IL-6 significantly (*p* < 0.001). N-CUR was more effective in decreasing cerebral NF-κB p65 (*p* < 0.05) than DFO, and TNF-α, and IL-6 as compared to either DFO or CUR. Cerebral levels of NF-κB p65, TNF-α, and IL-6 were assayed to determine the ameliorative effect of CUR and N-CUR on inflammation induced by Cu ingestion (Figure 2). Cu administration increased NF-κB p65 (Figure 2A), TNF-α (Figure 2B), and IL-6 (Figure 2C) in the cerebrum of rats (*p* < 0.001). All treatments (DFO, CUR, and N-CUR) decreased the levels of cerebral NF-κB p65, TNF-α, and IL-6 significantly (*p* < 0.001). N-CUR was more effective in decreasing cerebral NF-κB p65 (*p* < 0.05) than DFO, and TNF-α, and IL-6 as compared to either DFO or CUR. *Molecules* **2021**, *26*, x FOR PEER REVIEW 4 of 14

**Figure 2.** N-CUR and CUR suppress inflammation in Cu-administered rats. Treatment with N-CUR, CUR, and DFO decreased cerebral (**A**) NFκB p65, (**B**) TNF-α, and (**C**) IL-6. Data are mean ± SEM, (*n* = 8). \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001. **Figure 2.** N-CUR and CUR suppress inflammation in Cu-administered rats. Treatment with N-CUR, CUR, and DFO decreased cerebral (**A**) NFκB p65, (**B**) TNF-α, and (**C**) IL-6. Data are mean ± SEM, (*n* = 8). \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

cerebrum of rats exposed to Cu as compared to the control group, as depicted in Figure 3. In contrast, rats administered with Cu exhibited a remarkable downregulation of cerebral BCL-2 expression. DFO, CUR, and N-CUR significantly downregulated BAX, p53, and caspase-3 and upregulated BCL-2 in the cerebrum of Cu-administered rats. The effect of N-CUR on BAX and BCL-2 was significant when compared with CUR, whereas its effect

was more potent on BAX, caspase-3, and p53 than the effect of DFO.

#### *2.3. N-CUR and CUR Prevent Apoptosis in Cu-Administered Rats*

The expression levels of BAX, caspase-3, and p53 were significantly increased in the cerebrum of rats exposed to Cu as compared to the control group, as depicted in Figure 3. In contrast, rats administered with Cu exhibited a remarkable downregulation of cerebral BCL-2 expression. DFO, CUR, and N-CUR significantly downregulated BAX, p53, and caspase-3 and upregulated BCL-2 in the cerebrum of Cu-administered rats. The effect of N-CUR on BAX and BCL-2 was significant when compared with CUR, whereas its effect was more potent on BAX, caspase-3, and p53 than the effect of DFO. *Molecules* **2021**, *26*, x FOR PEER REVIEW 5 of 14

**Figure 3.** N-CUR and CUR prevent apoptosis in Cu-administered rats. (**A**) Representative blots showing changes in the expression of BAX, BCL-2, and caspase-3. (**B**–**E**) N-CUR, CUR, and DFO decreased (**B**) BAX, increased (**C**) BCL-2, and downregulated (**D**) caspase-3, and (**E**) p53 expression in the brain of Cu-administered rats. Data are mean ± SEM, (*n* = 8). \* *p* < 0.05, \*\* *p* < 0.01 and **Figure 3.** N-CUR and CUR prevent apoptosis in Cu-administered rats. (**A**) Representative blots showing changes in the expression of BAX, BCL-2, and caspase-3. (**B**–**E**) N-CUR, CUR, and DFO decreased (**B**) BAX, increased (**C**) BCL-2, and downregulated (**D**) caspase-3, and (**E**) p53 expression in the brain of Cu-administered rats. Data are mean ± SEM, (*n* = 8). \* *p* < 0.05, \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

\*\*\* *p* < 0.001. The beneficial effects of CUR and N-CUR against Cu-induced cerebral cell death were further confirmed via assessment of DNA fragmentation (Figure 4). Cu-administered rats showed an increase in DNA fragmentation levels as compared to the control group (*p* < 0.001). All treatments (DFO, CUR and N-CUR) prevented the deleterious effect The beneficial effects of CUR and N-CUR against Cu-induced cerebral cell death were further confirmed via assessment of DNA fragmentation (Figure 4). Cu-administered rats showed an increase in DNA fragmentation levels as compared to the control group (*p* < 0.001). All treatments (DFO, CUR and N-CUR) prevented the deleterious effect of Cu on DNA integrity.

#### of Cu on DNA integrity. *2.4. N-CUR and CUR Upregulate AKT/GSK-3β Signaling in Cu-Administered Rats*

To investigate the effect of Cu and the ameliorative effect of DFO, CUR, and N-CUR on cerebral AKT/GSK3β signaling, the phosphorylation levels of AKT and GSK3β were determined using Western blotting (Figure 5). Cu-treated rats exhibited a significant decrease in pAKT Ser473 and pGSK-3β Ser9 as compared to the normal rats (*p* < 0.001). Treatment with DFO, CUR, or N-CUR increased cerebral AKT and GSK-3β phosphorylation levels. N-CUR exerted a stronger effect on AKT/GSK-3β signaling than DFO and CUR.

\*\*\* *p* < 0.001.

of Cu on DNA integrity.

**Figure 3.** N-CUR and CUR prevent apoptosis in Cu-administered rats. (**A**) Representative blots showing changes in the expression of BAX, BCL-2, and caspase-3. (**B**–**E**) N-CUR, CUR, and DFO decreased (**B**) BAX, increased (**C**) BCL-2, and downregulated (**D**) caspase-3, and (**E**) p53 expression in the brain of Cu-administered rats. Data are mean ± SEM, (*n* = 8). \* *p* < 0.05, \*\* *p* < 0.01 and

The beneficial effects of CUR and N-CUR against Cu-induced cerebral cell death were further confirmed via assessment of DNA fragmentation (Figure 4). Cu-administered rats showed an increase in DNA fragmentation levels as compared to the control group (*p* < 0.001). All treatments (DFO, CUR and N-CUR) prevented the deleterious effect

**Figure 4.** N-CUR, CUR, and DFO prevent DNA fragmentation in the brain of Cu-administered rats. DNA fragmentation was assessed by (**A**) agarose gel electrophoresis and (**B**) colorimetric methods. Data are mean ± SEM, (*n* = 8). \*\*\* *p* < 0.001. crease in pAKT Ser473 and pGSK-3β Ser9 as compared to the normal rats (*p* < 0.001). Treatment with DFO, CUR, or N-CUR increased cerebral AKT and GSK-3β phosphorylation levels. N-CUR exerted a stronger effect on AKT/GSK-3β signaling than DFO and CUR.

**Figure 5.** N-CUR and CUR upregulate AKT/GSK-3β signaling in Cu-administered rats. (**A**) Representative blots of pAKT, AKT, pGSK-3β, and GSK-3β. (**B**,**C**) N-CUR, CUR, and DFO increased AKT Ser473 (**B**) and GSK-3β Ser9 (**C**) phosphorylation in the brain of Cu-administered rats. Data are mean ± SEM, (*n* = 8). \*\*\**p* < 0.001. **Figure 5.** N-CUR and CUR upregulate AKT/GSK-3β signaling in Cu-administered rats. (**A**) Representative blots of pAKT, AKT, pGSK-3β, and GSK-3β. (**B**,**C**) N-CUR, CUR, and DFO increased AKT Ser473 (**B**) and GSK-3β Ser9 (**C**) phosphorylation in the brain of Cu-administered rats. Data are mean ± SEM, (*n* = 8). \*\*\* *p* < 0.001.

#### *2.5. N-CUR and CUR Upregulate Brain-Derived Neurotrophic Factor (BDNF) in Cu-2.5. N-CUR and CUR Upregulate Brain-Derived Neurotrophic Factor (BDNF) in Cu-Administered Rats*

*Administered Rats*  The administration of Cu resulted in a significant downregulation of BDNF expression in the cerebrum of rats, as shown in Figure 6. Treatment of the Cu-administered rats with DFO, CUR, or N-CUR increased the levels of cerebral BDNF. While the effect of CUR on BDNF was significant as compared to DFO, the effect of N-CUR was more potent when The administration of Cu resulted in a significant downregulation of BDNF expression in the cerebrum of rats, as shown in Figure 6. Treatment of the Cu-administered rats with DFO, CUR, or N-CUR increased the levels of cerebral BDNF. While the effect of CUR on BDNF was significant as compared to DFO, the effect of N-CUR was more potent when compared to both treatments.

compared to both treatments.

**Figure 6.** N-CUR and CUR upregulate BDNF in Cu-administered rats. Data are mean ± SEM, (*n* = 8). \*\* *p* < 0.01 and \*\*\* *p* < 0.001. **Figure 6.** N-CUR and CUR upregulate BDNF in Cu-administered rats. Data are mean ± SEM, (*n* = 8). \*\* *p* < 0.01 and \*\*\* *p* < 0.001.

#### **3. Discussion**

**3. Discussion**  Cu is the third most abundant essential transition metal in humans, and the brain is the second organ containing the highest content after the liver [35]. It is essential for antioxidant defenses, energy homeostasis, and many other physiological processes [1]. However, it may cause neurotoxicity and contribute to the pathogenesis of neurodegenerative diseases [1], where oxidative stress represents the main underlying mechanism [36]. The present results revealed the development of cerebral oxidative stress manifested by ele-Cu is the third most abundant essential transition metal in humans, and the brain is the second organ containing the highest content after the liver [35]. It is essential for antioxidant defenses, energy homeostasis, and many other physiological processes [1]. However, it may cause neurotoxicity and contribute to the pathogenesis of neurodegenerative diseases [1], where oxidative stress represents the main underlying mechanism [36]. The present results revealed the development of cerebral oxidative stress manifested by elevated MDA and decreased GSH, SOD, and CAT in Cu-administered rats.

vated MDA and decreased GSH, SOD, and CAT in Cu-administered rats. Cu cycles easily between stable oxidised and unstable reduced states to coordinate ligands and enzymes and facilitate redox reactions, thereby acting as a cofactor for many enzymes [37]. Although the redox nature makes Cu essential for many biological processes, it renders it toxic because of the generation of highly reactive hydroxyl radicals [36]. In addition, Cu can increase mitochondrial reactive oxygen species (ROS) generation and alter the activity of respiratory chain enzymes [38]. The generated ROS are potent oxidising agents that provoke the oxidative damage of lipids, proteins, and DNA [39], leading to lipid peroxidation (LPO), DNA breaks, and other deleterious effects [40]. Accordingly, LPO was elevated and GSH, SOD, and CAT were declined in the brain of Cuadministered rats in the present study. Given the role of oxidative stress in mediating Cu toxicity, CUR can suppress neurotoxicity via its radical-scavenging and antioxidant properties. Here, rats that received CUR and N-CUR exhibited a remarkable reduction in cerebral MDA levels and enhanced GSH, SOD, and CAT. The antioxidant efficacy of CUR has been reported in numerous studies that employed animal models of neurotoxicity induced by D-galactosamine, fluoride, formaldehyde, rotenone, vincristine, tetrachlorobenzoquinone, pentylenetetrazole, acrylamide, and other agents (reviewed in [41]). In addition, CUR reduced cerebellar LPO in lead-intoxicated rats [25]. These beneficial effects were attributed to the potent radical-scavenging activity of CUR. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a redox-sensitive factor that regulates antioxidant genes and suppresses oxidative stress [42], might also have a role in the neuroprotective activity of CUR. In this context, CUR enhanced Nrf2 and antioxidant defenses in Cu cycles easily between stable oxidised and unstable reduced states to coordinate ligands and enzymes and facilitate redox reactions, thereby acting as a cofactor for many enzymes [37]. Although the redox nature makes Cu essential for many biological processes, it renders it toxic because of the generation of highly reactive hydroxyl radicals [36]. In addition, Cu can increase mitochondrial reactive oxygen species (ROS) generation and alter the activity of respiratory chain enzymes [38]. The generated ROS are potent oxidising agents that provoke the oxidative damage of lipids, proteins, and DNA [39], leading to lipid peroxidation (LPO), DNA breaks, and other deleterious effects [40]. Accordingly, LPO was elevated and GSH, SOD, and CAT were declined in the brain of Cu-administered rats in the present study. Given the role of oxidative stress in mediating Cu toxicity, CUR can suppress neurotoxicity via its radical-scavenging and antioxidant properties. Here, rats that received CUR and N-CUR exhibited a remarkable reduction in cerebral MDA levels and enhanced GSH, SOD, and CAT. The antioxidant efficacy of CUR has been reported in numerous studies that employed animal models of neurotoxicity induced by D-galactosamine, fluoride, formaldehyde, rotenone, vincristine, tetrachlorobenzoquinone, pentylenetetrazole, acrylamide, and other agents (reviewed in [41]). In addition, CUR reduced cerebellar LPO in lead-intoxicated rats [25]. These beneficial effects were attributed to the potent radical-scavenging activity of CUR. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a redox-sensitive factor that regulates antioxidant genes and suppresses oxidative stress [42], might also have a role in the neuroprotective activity of CUR. In this context, CUR enhanced Nrf2 and antioxidant defenses in rat cerebellar granule neurons challenged with hemin [43] and quinolinic acid-induced neurotoxicity [44].

rat cerebellar granule neurons challenged with hemin [43] and quinolinic acid-induced neurotoxicity [44]. The upregulation of BDNF in the brain of Cu-administered rats treated with CUR and N-CUR might have a role in boosting the antioxidant defenses through Nrf2 activation. In accordance, a recent study demonstrated that CUR increased BDNF in the brain of quinolinic acid-intoxicated rats, and this activated ERK1/2 and consequently enhanced Nrf2 expression and GSH levels [44]. BDNF belongs to the neurotrophin family and is The upregulation of BDNF in the brain of Cu-administered rats treated with CUR and N-CUR might have a role in boosting the antioxidant defenses through Nrf2 activation. In accordance, a recent study demonstrated that CUR increased BDNF in the brain of quinolinic acid-intoxicated rats, and this activated ERK1/2 and consequently enhanced Nrf2 expression and GSH levels [44]. BDNF belongs to the neurotrophin family and is involved in the maintenance of adult neuronal function [45]. In astrocytes, BDNF has been proposed to play a role in regulating Nrf2 and their metabolic cooperation between

neurons [46]. In a model of traumatic brain injury with transplanted neuronal stem cells, BNDF induced Nrf2-mediated antioxidant response [47]. Therefore, this study introduced new information that the upregulation of BDNF plays a role, at least in part, in the protective effect of CUR against Cu neurotoxicity and that N-CUR has a stronger effect on modulating BDNF expression. However, the lack of data showing changes in Nrf2 expression could be considered a limitation of this study.

In addition to the attenuation of oxidative stress, CUR and N-CUR suppressed NF-κB and pro-inflammatory cytokines in the brain of Cu-administered rats. The inflammatory response observed in Cu-administered rats is a direct consequence of excessive ROS generation. The activation of NF-κB and subsequent release of many inflammatory mediators occur as a result of increased cellular ROS [48]. The pro-inflammatory action of Cu is driven by its potential to catalyse ROS generation and decreasing GSH [36], which is an effect reported in the present study. CUR and N-CUR effectively ameliorated cerebral inflammation in Cu-administered rats. N-CUR decreased the levels of TNF-α and IL-6 significantly when compared with CUR, demonstrating enhanced anti-inflammatory activity of the nano form. The ability of CUR to suppress inflammation has been reported in several studies. In a rat model of acrylamide neurotoxicity, Guo et al. [49] showed that CUR attenuated neuroinflammation by decreasing TNF-α and IL-1β levels. In addition, CUR decreased circulating TNF-α in an animal model of lead neurotoxicity [50].

Apoptotic cell death was observed in the brain of Cu-administered rats in the present study. BAX, caspase-3, and p53 were upregulated, whereas the anti-apoptotic BCL-2 was declined in the brain of rats as a result of Cu ingestion. Cu-mediated ROS generation induces mitochondrial permeability transition in astrocytes [51] and hepatocytes [52], leading to cell death via apoptosis. Excess ROS can activate the pro-apoptotic protein BAX, which increases cytochrome *c* release by promoting the loss of membrane potential via mitochondrial voltage-dependent anion channel [53]. Oxidative stress can also provoke p53 nuclear accumulation and its binding to specific DNA sequences, leading to the transcription of genes involved in cell death [54] and the release of mitochondrial cytochrome *c* and the activation of caspases [55]. In contrast, BCL-2 suppresses the release of cytochrome *c* and prevents apoptosis [56]. CUR downregulated the pro-apoptotic factors and increased BCL-2 expression, demonstrating an anti-apoptotic effect that is a direct consequence of its antioxidant and anti-inflammatory properties. The effect of N-CUR on BAX and BCL-2 expression was more potent than CUR. The cytoprotective efficacy of CUR has been reported in a *Drosophila* model of Huntington's disease [57]. In this model, CUR competently ameliorated neurodegeneration, cytotoxicity, and the compromised neuronal function [57].

To further explore the mechanism underlying the neuroprotective effect of CUR in Cu-administered rats and whether N-CUR is more potent, we determined their effect on AKT/GSK-3β signaling. The phosphorylation of AKT Ser473 and GSK-3β Ser9 was decreased in the brain of Cu-administered rats. While CUR ameliorated the altered phosphorylation levels of these proteins, N-CUR remarkably activated AKT/GSK-3β signaling. Activated AKT mediates the regulation of different processes, including cell growth and proliferation through the phosphorylation of GSK-3, mTOR, NF-κB, and other proteins [58]. AKT controls the activity of GSK-3β, which is active in resting cells, through phosphorylation at Ser9 [59]. Increased GSK-3β activity provoked liver injury [60], whereas its inhibition accelerated the generation of hepatocytes and protected against acetaminophen [61] and lead toxicity [62]. In neuronal cells, the overexpression of GSK-3β induced apoptosis [32,63], demonstrating its crucial role in cell death. BAX phosphorylation has been suggested to be stimulated through GSK-3, and the mutation of GSK-3 inhibited BAX mitochondrial translocation [64]. Moreover, GSK-3 can work in concert with JNK to orchestrate neuronal apoptosis [65]. In the current study, Cu ingestion decreased AKT Ser473 and GSK-3β Ser9 phosphorylation. Reduced inhibition of GSK-3β through its phosphorylation at Ser9 due to suppressed AKT coincides with the observed upregulation of BAX and other mediators of apoptosis. Therefore, the neuroprotective effect of CUR could be directly connected to its ability to activate AKT, which then inhibits GSK-3β-mediated apoptosis. Accordingly,

activation of the AKT/GSK-3β signaling by CUR conferred protection against liver injury induced by heavy metals [20]. In support of our findings, computational approaches have demonstrated that CUR inhibits GSK-3β by fitting into its binding pocket [66,67]. This inhibitory effect has been confirmed by an in vitro study showing that the IC<sup>50</sup> of CUR's inhibitory activity was 66.3 nM [66]. Furthermore, studies demonstrating the effect of CUR on GSK-3 activity in several diseases have been reviewed by McCubrey et al. [30].

In addition to the findings of this study, Balasubramanian [68,69] presented important quantum chemical insights into the neuroprotective mechanism of CUR and its efficacy to prevent Alzheimer0 s disease. The dual property of CUR to be nonpolar in parts and polar in other parts is due to the presence of both phenolic and enolic protons combined with an aliphatic hydrophobic bridge. This property enables CUR to cross the blood–brain barrier (BBB) and bind to and prevent the polymerisation of amyloid-β (Aβ) oligomers [69]. By employing quantum chemical computations, Balasubramanian [68] studied the chelate complexes of CUR with Cu(II) and other transition metal ions that provoke the polymerisation of Aβ and formation of neurotoxic conformations, reporting that the β-diketone bridge, through the loss of an enolic proton of CUR, is the primary site of chelation. CUR can form stable chelate complexes at the β-diketone bridge, thereby scavenging neurotoxic metal ions and inhibiting Aβ polymerisation and the subsequent generation of neurotoxic conformations [68]. Moreover, the ability of piperine, an alkaloid present in black pepper, to enhance the bioavailability and neuroprotective efficacy of CUR is noteworthy of mention. Through the use of quantum chemical and molecular docking, Patil et al. [70] demonstrated that piperine increased the bioavailability of CUR (20-fold) by inhibiting the enzymes mediating CUR glucuronosylation and by intercalating into CUR layers through intermolecular hydrogen bonding [70]. These processes enhance the metabolic transport and consequently the bioavailability of CUR [70]. In support of these findings, Singh et al. [71] reported the protective effect of CUR with piperine, a bioavailability enhancer, against neurotoxicity induced by 3-nitropropionic acid (3-NP) in rats. When supplemented with piperine, CUR improved motor function, attenuated oxidative stress and inflammatory cytokines, and modulated catecholamines and dopamine turnover in the striatum of 3-NP-admninstered rats [71].

#### **4. Materials and Methods**

#### *4.1. Chemicals and Reagents*

CuSO4, CUR, carboxymethylcellulose (CMC), thiobarbituric acid, agarose, reduced glutathione (GSH), and pyrogallol were obtained from Sigma (St. Louis, MO, USA). Liposomal N-CUR was obtained from Lipolife (Essex, UK), and DFO was purchased from Novartis Pharma AG (Rotkreuz, Switzerland). TNF-α and IL-6 ELISA kits were supplied by R&D Systems (Minneapolis, MN, USA), and the NF-κB p65 ELISA kit was obtained from MyBiosource (San Diego, CA, USA). Antibodies against pAKT Ser473, AKT, pGSK-3β Ser9, GSK-3β, BDNF, and β-actin were supplied by Novus Biologicals (Centennial, CO, USA). Primers were obtained from Sigma (St. Louis, MO, USA). Other chemicals were supplied by standard manufacturers.

#### *4.2. Animals and Treatments*

Forty male Wistar rats, weighing 180–200 g, were obtained from the Animals Care Centre at King Saud University. The animals were given free access to food and water and acclimatised for one week under standard conditions and 12 h light/dark cycle and free access to food and water. All experimental procedures were conducted in accordance with the requirements of the research ethics Committee at King Saud University (Ethical reference no. SE-19-129). After acclimatisation, the rats were randomly allocated into five groups (*n* = 8) as follows:


Twenty-four h after the last treatment, the rats were sacrificed under ketamine/xylazine anesthesia. Blood was collected via cardiac puncture and serum was separated by centrifugation. The rats were dissected, and the brain was removed and kept frozen in liquid nitrogen. Other parts from the cerebrum were homogenised in cold PBS (10% *w*/*v*), centrifuged at 5000 rpm for 15 min at 4 ◦C, and the supernatant was used for assessment of MDA, GSH, SOD, CAT, TNF-α, IL-6, and NF-κB p65.

#### *4.3. Determination of MDA and Antioxidants*

MDA was determined as previously described [73]. GSH, SOD, and CAT were assayed according to the methods of Ellman [74], Marklund and Marklund [75], and Cohen et al. [76], respectively.

#### *4.4. Determination of NF-κB p65, TNF-α, IL-6, and p53*

NF-κB p65 was assayed using a specific ELISA kit (MyBioSource, San Diego, CA, USA), and TNF-α and IL-6 were assayed using R&D Systems (Minneapolis, MN, USA) ELISA kits. p53 was determined using ELISA kit supplied by Novus Biologicals (Centennial, CO, USA).

#### *4.5. Determination of DNA Fragmentation*

Agarose electrophoresis and the colorimetric methods [77] were used to assess DNA fragmentation. The results were presented as a fold change of the control.

#### *4.6. Gene Expression*

Changes in the expression of BAX, BCL-2, and caspase-3 were determined by RT-PCR as previously described [78]. Briefly, RNA was isolated from the frozen brain samples using TRIzol (ThermoFisher Scientific, Waltham, MA, USA). Following treatment with RNase-free DNase (Qiagen, Hilden, Germany), RNA was quantified using a nanodrop. RNA samples with OD260/OD280 nm ratio of ≥ 1.8 were reverse transcribed into cDNA. The produced cDNA was amplified using PCR master mix (Qiagen, Hilden, Germany) and the primer pairs listed in Table 1. The PCR products were loaded in 1.5% agarose gel, electrophoresed, and the bands were visualised using UV transilluminator. The images were analysed by ImageJ (version 1.32j, NIH, USA), and the values were normalised to β-actin.


### **Table 1.** Primers used for RT-PCR.

#### *4.7. Western Blotting*

The samples were homogenized in RIPA buffer supplemented with proteinase/phosphatase inhibitors, centrifuged, and protein concentration was determined in the supernatant using Bradford protein assay kit (BioBasic, Markham, Canada). Forty µg protein from each sample was subjected to 10% SDS/PAGE and electrotransferred to nitrocellulose membranes. The membranes were subjected to blocking in 5% milk in TBST followed by incubation overnight at 4 ◦C with primary antibodies against pAKT Ser473, AKT, pGSK-3β Ser9, GSK-3β, BDNF, and β-actin. The probed membranes were washed, and secondary antibodies were added. After washing, the membranes were washed with TBST, developed using Clarity™ Western ECL Substrate from BIO-RAD (Hercules, CA, USA), and then visualised in ImageQuant LAS 4000. The band intensity was quantified using ImageJ (version 1.32j, NIH, USA).

#### *4.8. Statistical Analysis*

The obtained data are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed by one-way ANOVA and Tukey0 s post hoc test using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA). A *p* value < 0.05 was considered significant.

#### **5. Conclusions**

These results confer new information on the protective effect of N-CUR on Cu neurotoxicity. N-CUR and CUR attenuated oxidative stress, inflammation, cell death, and oxidative DNA damage in the brain of Cu-administered rats. The modulatory effect of N-CUR and CUR on AKT/GSK-3β signaling was involved, at least in part, in their protective activity against Cu neurotoxicity. The neuroprotective effect of N-CUR was stronger when compared to the native form, which is an effect that could be attributed to the improved properties of CUR.

**Author Contributions:** Conceptualisation, W.S.S.; A.M.A. (Ahlam M. Alhusaini) and A.M.M.; methodology, W.S.S.; A.M.A. (Ahlam M. Alhusaini); L.M.F.; H.A.A.; A.B.A.; A.S.A.; I.H.H. and A.M.M.; validation, W.S.S.; A.M.A. (Ahlam M. Alhusaini) and A.M.M.; formal analysis, W.S.S.; A.M.A. (Ahlam M. Alhusaini) and A.M.M.; investigation, W.S.S.; A.M.A. (Ahlam M. Alhusaini); L.M.F.; H.A.A.; A.B.A.; I.H.H. and A.M.M.; resources, W.S.S.; A.M.A. (Ahlam M. Alhusaini); A.M.A. (Areej M. Alotaibi) and A.M.M.; data curation, W.S.S. and A.M.M.; writing—original draft preparation, A.M.M.; writing—review and editing, A.M.M.; visualisation, W.S.S.; A.M.A. (Ahlam M. Alhusaini); I.H.H. and A.M.M.; supervision, A.M.A. (Ahlam M. Alhusaini) and A.M.M.; project administration, A.M.A. (Ahlam M. Alhusaini); I.H.H. and W.S.S.; funding acquisition, A.M.A. (Ahlam M. Alhusaini) and W.S.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** Please add: This research was funded by the Deanship of Scientific Research at King Saud University, grant number RG-1441-546.

**Institutional Review Board Statement:** The experiment was conducted according to the guidelines of the National Institutes of Health (NIH publication No. 85–23, revised 2011) and was approved by the research ethics Committee at King Saud University (Ethical reference no. SE-19-129).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data analysed or generated during this study are included in this manuscript.

**Acknowledgments:** The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group number RG-1441-546.

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

**Sample Availability:** Samples of the compounds are not available from the authors.

### **References**


## *Review* **Caffeic Acid on Metabolic Syndrome: A Review**

**Nellysha Namela Muhammad Abdul Kadar 1,2, Fairus Ahmad <sup>1</sup> , Seong Lin Teoh <sup>1</sup> and Mohamad Fairuz Yahaya 1,\***


**Abstract:** Metabolic syndrome (MetS) is a constellation of risk factors that may lead to a more sinister disease. Raised blood pressure, dyslipidemia in the form of elevated triglycerides and lowered highdensity lipoprotein cholesterol, raised fasting glucose, and central obesity are the risk factors that could lead to full-blown diabetes, heart disease, and many others. With increasing sedentary lifestyles, coupled with the current COVID-19 pandemic, the numbers of people affected with MetS will be expected to grow in the coming years. While keeping these factors checked with the polypharmacy available currently, there is no single strategy that can halt or minimize the effect of MetS to patients. This opens the door for a more natural way of controlling the disease. Caffeic acid (CA) is a phytonutrient belonging to the flavonoids that can be found in abundance in plants, fruits, and vegetables. CA possesses a wide range of beneficial properties from antioxidant, immunomodulatory, antimicrobial, neuroprotective, antianxiolytic, antiproliferative, and anti-inflammatory activities. This review discusses the current discovery of the effect of CA against MetS.

**Keywords:** caffeic acid; metabolic syndrome; phenolic compound; obesity; dyslipidemia; hyperglycemia; hypertension

#### **1. Introduction**

Metabolic syndrome (MetS) has affected almost one fifth of the adult population and increases the risk of cardiovascular disease, type-2 diabetes, and all-cause mortality compared to a healthy person [1]. In Asia, Malaysia is recognized as one of the countries that has a high MetS prevalence [2]. MetS is a complication of the modern lifestyle that includes overeating and underactivity [3]. With the current COVID-19 pandemic situation and increasing state of sedentary lifestyle, the numbers are bound to be more than the expected figures in the coming years [4].

The current definition of MetS still uses the Harmonized Criteria that state that abnormal findings of 3 out of 5 of the following risk factors would qualify a person of having MetS: raised blood pressure, dyslipidemia (raised triglycerides (TG) and lowered high-density lipoprotein cholesterol), raised fasting glucose, and central obesity [5,6]. These components have the ability to precede into cardiac dysfunction, but together, they can also cause an additional risk to morbidity and mortality [7]. Although MetS has been collectively accepted as an alarming condition, the clinical world has yet to mutually agree on a uniform terminology and diagnostic criteria. This is mainly due to the adversity of genetic predisposition, diet history, and physical, geographical, and endocrinal attributes that together take part in forming this intricate syndrome [8]. One of the causes of MetS is the increase in oxidative stress and chronic inflammation. In many instances, it has been shown that an antioxidant imbalance may play a role in its development where there is an overproduction of reactive oxygen species (ROS) and nitrogen (RNS) species that can react

**Citation:** Muhammad Abdul Kadar, N.N.; Ahmad, F.; Teoh, S.L.; Yahaya, M.F. Caffeic Acid on Metabolic Syndrome: A Review. *Molecules* **2021**, *26*, 5490. https://doi.org/10.3390/ molecules26185490

Academic Editors: Stefano Castellani and Massimo Conese

Received: 20 August 2021 Accepted: 6 September 2021 Published: 9 September 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 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 (https:// creativecommons.org/licenses/by/ 4.0/).

with virtually all biomolecules, causing oxidative damage [9,10]. Similarly, human studies have also shown that MetS is associated with oxidative stress and a proinflammatory state that comes with a high antioxidant defense in the peripheral blood mononuclear cells assumed to be derived from a pre-activation state of human cells [11].

Although obesity and insulin resistance remain at the root of MetS pathogenesis, other factors such as chronic stress and dysregulation of the hypothalamic–pituitary–adrenal axis and autonomic nervous system, increased cellular oxidative stress, renin–angiotensin– aldosterone activity, and intrinsic tissue glucocorticoid reaction, as well as the newly discovered miRNAs, have been identified to play roles in this condition [12,13].

At the core of many pathological diseases, including MetS, an increase in ROS has played a crucial element that can be tipped over with the aid of a longstanding diet comprising antioxidants [14]. Reactive species are essential signaling molecules that are involved in nearly every physiological activity, from cell division to metabolic regulation. They modulate the activity of biomolecules, and redox-sensitive transcription factors activate a cell's adaptive endogenous response, including antioxidant defense. The degree of reactive species production and neutralization that are tightly associated with oxidative metabolism determines the redox homeostasis of cells and their surroundings. Setting the redox states of cells is critical in both health and disorders such as MetS [15]. The question is, which antioxidant and at what aliquot would be the optimum elixir to shorten the period in combating the specific diseases.

The research world has, for many years, focused on a more natural approach toward combatting human diseases. Synthetic medications have slowly proven its downside over years of pharmacological use. Polypharmacy in the treatment of MetS has become a substantial healthcare burden due to adverse drug reactions, morbidity, and cost [16]. One of the phytonutrient compounds that caught the attention of researchers were the flavonoids. These are a very diverse group of polyphenolic compounds that consists of a benzo-γ-pyrone and can be found in several parts of a plant. They are classified as plant secondary metabolites having a polyphenolic structure [17,18]. These compounds, which can be found in abundance in the Mediterranean diet, has increasingly shown a beneficial effect in maintaining cardiometabolic and cardiovascular health, which, in turn, reduces the risks of MetS development. This positive impression may be due to the diets that are high in polyphenolic antioxidant content derived from vegetables, grapes, and olive oils [19]. Similarly, treatment with naringin, a type of glycoside flavonoid, has been reported to reverse MetS by reducing visceral obesity, blood glucose, blood pressure, and lipid profile [20].

In this review, we discuss a phenolic compound found in many herbs, caffeic acid (CA), or its chemical name 3,4-dihydroxycinnamic acid, which belongs to a group called phenolic compounds, which are a naturally occurring chemical structure found abundantly in fruits and vegetables [21,22].

#### **2. Caffeic Acid as a Phenolic Compound**

Phenolic compounds provide protection against noncommunicable diseases not only by their means of antioxidant activity but also by regulating a variety of cellular processes at different levels, including enzyme inhibition, modification of gene expression, and protein phosphorylation [23]. An increase in phenolic compounds can alter their health benefits [24]. There are over 8000 phenolic compounds that can be classified into two main groups: flavonoids and nonflavonoids. Flavonoids contain a phenyl benzopyran skeleton: two phenyl rings joined through a heterocyclic pyran ring. Nonflavonoids, on the other hand, are mostly smaller and simpler in comparison to flavonoids [17].

Phenolic acids (PAs) are a group of nonflavonoid phenolic compounds that contain a single phenyl group substituted by a carboxylic group and one or more hydroxyl (OH) groups [25]. PAs are further divided according to the length of the chain that contains the carboxylic group into: hydroxybenzoic acids, hydroxycinnamic acids, and hydroxyphenyl acids. The group hydroxycinnamic acid has a C6-C3 (phenylpropanoid) basic skeleton.

Hydroxy derivatives of cinnamic acid are more effective as an antioxidant than the hydroxyl derivatives of benzoic acid as the presence of a CH<sup>2</sup> = CH-COOH group in the cinnamic acids ensures a greater antioxidant capacity than the COOH group in benzoic acid (Figure 1). One of the major hydroxycinnamic acids is CA [26–28]. droxyl derivatives of benzoic acid as the presence of a CH2 = CH-COOH group in the cinnamic acids ensures a greater antioxidant capacity than the COOH group in benzoic acid (Figure 1). One of the major hydroxycinnamic acids is CA [26–28].

Hydroxy derivatives of cinnamic acid are more effective as an antioxidant than the hy-

**Figure 1.** Chemical structure of PA, CA, and CAPE.

*Molecules* **2021**, *26*, x FOR PEER REVIEW 3 of 14

**Figure 1.** Chemical structure of PA, CA, and CAPE. CA is found in coffee, honey, potatoes, berries, herbs, and vegetables such as olives, Swiss chard, and carrot [29]. In vitro and in vivo studies have shown that CA not only possesses antioxidant capacity but also has immunomodulatory [30], antimicrobial [31], neuroprotective, antianxiolytic [32], antiproliferative, and anti-inflammatory activities [33], and has shown to improve inflammation and oxidative stress in chronic metabolic diseases. Besides the therapeutic potentials of CA, studies have also shown that the pure CA is found in coffee, honey, potatoes, berries, herbs, and vegetables such as olives, Swiss chard, and carrot [29]. In vitro and in vivo studies have shown that CA not only possesses antioxidant capacity but also has immunomodulatory [30], antimicrobial [31], neuroprotective, antianxiolytic [32], antiproliferative, and anti-inflammatory activities [33], and has shown to improve inflammation and oxidative stress in chronic metabolic diseases. Besides the therapeutic potentials of CA, studies have also shown that the pure form of CA has the availability to be absorbed in the intestines and form subsequent interactions with the target tissue [34]. This solidifies the potential of using CA as an oral route of administration as an appealing choice for a phytonutrient.

form of CA has the availability to be absorbed in the intestines and form subsequent interactions with the target tissue [34]. This solidifies the potential of using CA as an oral route of administration as an appealing choice for a phytonutrient. CA has also been found in Gelam honey and stingless bee honey through HPLC analysis [35,36]. The antioxidant capability of CA is due to its ability to scavenge ROS, including O2−, OH−, and H2O2 [37]. CA has shown to be an effective ABTS, DPPH, and superoxide anion radical scavenger, with a total reducing power and metal chelates on ferrous ion activities, in comparison to other standard antioxidant compounds such as BHA, BHT, alpha-tocopherol, and trolox in different in vitro antioxidant assays [38]. Multiple factors CA has also been found in Gelam honey and stingless bee honey through HPLC analysis [35,36]. The antioxidant capability of CA is due to its ability to scavenge ROS, including O2−, OH−, and H2O<sup>2</sup> [37]. CA has shown to be an effective ABTS, DPPH, and superoxide anion radical scavenger, with a total reducing power and metal chelates on ferrous ion activities, in comparison to other standard antioxidant compounds such as BHA, BHT, alpha-tocopherol, and trolox in different in vitro antioxidant assays [38]. Multiple factors influence PA efficacy in vivo, including the amount of consumed chemical, whether it is absorbed or metabolized, its plasma or tissue concentrations, PA type and dosage, and synergistic effects [39].

influence PA efficacy in vivo, including the amount of consumed chemical, whether it is absorbed or metabolized, its plasma or tissue concentrations, PA type and dosage, and synergistic effects [39]. Besides pure CA, its derivatives in the form of caffeic acid phenyl ester (CAPE) and caffeic acid phenylethyl amide (CAPA) have also been found to have a therapeutic effect against MetS. However, CAPA and CAPE are less stable in its form compared to CA [40]. Besides pure CA, its derivatives in the form of caffeic acid phenyl ester (CAPE) and caffeic acid phenylethyl amide (CAPA) have also been found to have a therapeutic effect against MetS. However, CAPA and CAPE are less stable in its form compared to CA [40]. CAPE is an active component of the propolis substance and has been known for its antiinflammatory, antioxidant, and anti-cancer effects [41]. The following section discusses the effects of CA and its derivatives on different components of MetS.

#### CAPE is an active component of the propolis substance and has been known for its anti-**3. CA vs. Obesity**

inflammatory, antioxidant, and anti-cancer effects [41]. The following section discusses the effects of CA and its derivatives on different components of MetS. **3. CA vs. Obesity** Obesity is a condition where excess body fat accumulates either due to the enlargement of lipids in existing adipocytes (hypertrophy), or through an increase in the number of adipocytes (hyperplasia) [42]. Adipose tissue in the human body functions as an energy Obesity is a condition where excess body fat accumulates either due to the enlargement of lipids in existing adipocytes (hypertrophy), or through an increase in the number of adipocytes (hyperplasia) [42]. Adipose tissue in the human body functions as an energy storage system, an endocrine gland, and a heat productor (nonshivering thermogenesis) [43]. In healthy slender individuals, adipocytes are smaller, more insulin-sensitive, and secretes anti-inflammatory mediators such as adiponectin, IL-10, IL-4, IL-13, IL-1 receptor agonist (IL-1Ra), apelin, and transforming growth factor beta (TGFβ). In contrast, the adipocytes of an obese individual are enlarged and infiltrated by a large number of

pro-inflammatory M1 macrophages that secrete pro-inflammatory cytokines such as TNFα, IL-6, visfatin, leptin, MCP-1, Ang-II, and plasminogen activator inhibitor-1 [44]. With the surplus of these pro-inflammatory compounds within the obese adipocyte, they are often referred to be in a state of inflammation. This state of chronic low-grade activation of the innate immune system is critical in the pathophysiology of obesity and MetS [45].

Visceral fat is localized within the abdomen and is metabolically active with the constant release of free fatty acids into the portal circulation [46]. In a state of caloric excess, the hypertrophied adipocytes will secrete adipokines that result in the increment of additional pre-adipocytes that will later mature. However, this compensatory act reaches its threshold and causes fat accumulation in the visceral depots. The accumulation and distribution of the fat depots play a key role in forming metabolic complications. A metabolically healthy obese individual that remains insulin-sensitive and displays a normal metabolic and hormonal profile and is physically different compares to a metabolically unhealthy obese person through their higher abdominal circumference measurement [47].

Metabolic changes in obesity are associated with a persistent low-grade inflammatory state that impairs energy homeostasis and glucose metabolism [44,48]. The c-Jun N-terminal kinase (JNK) and the nuclear factor-kappa B (NF-κB) signaling pathways contribute to inflammation and play a key role in obesity, insulin-resistance, and in regulating the expression of proinflammatory molecules [49]. Zhang and colleagues found that CA was able to exert anti-inflammatory effects in dextran sulfate sodium-induced colitis mice, showing a significantly suppressed secretion of IL-6 and TNFα and colonic infiltration of CD3+ T cells, CD177+ neutrophils, and F4/80+ macrophages through the activation of the NF-κB signaling pathway. Their study concluded that CA was able to amend the colonic pathology and inflammation, indirectly contributing toward reducing obesity [50].

Obesity may also be associated with adipocyte necrosis, which could be the start of a pro-inflammatory response. Adipocytes grow hypertrophic when their caloric intake and energy expenditure increase, which has been linked to cell hypoxia and death. These hypertrophic adipocytes will subsequently start secreting TNFα in small amounts, resulting in a chemotactic response that draws macrophages [48]. An in vitro study using adipose stem cells (ASCs) showed that CAPE had the ability to inverse the effects of high glucose and lipopolysaccharide exposure. Through this study, they found that CAPE treatment was able to restore the functions of adipocytes by increasing the adiponectin and peroxisome proliferator-activated receptor gamma (PPARγ), resulting in the reduction in pro-inflammatory factors [51]. CA also acts on adipogenesis by reducing intracellular lipid accumulation in an in vitro model [52].

Increasing evidence has shown that gut microbiota plays a role in the development of obesity and MetS through the modulation of energy absorption, and subsequently influences glucose and lipid metabolism [53,54]. It was recently postulated that gut microbiota producing t10,c12-conjugated linoleic acid induced lipogenesis [8]. Dietary polyphenols have been found to promote the growth of beneficial bacteria while inhibiting pathogenic bacteria [55]. In an in vivo study to determine the anti-obesity effect of CA, high-fat-diet (HFD)-induced mice were seen to have a positive effect after being given a daily dose of 50 mg/kg CA for a span of 12 weeks. The researchers noted a significant reduction in body weight and fat accumulation, increases in energy expenditure and beneficial gut bacteria (i.e., Muribaculaceae), and a decrease in pathogenic bacteriae (i.e., Lachnospiraceae) [56].

In another study, HFDs in nonalcoholic fatty liver disease (NAFLD)-induced mice were used to demonstrate the effectiveness of CA treatment and its effects toward the gut microbiota. CA was able to significantly reduce the body weight of the HFD-fed mice and attenuated the expression of lipogenesis-related protein expression (Srebp1, Fas, Acc, and Scd1) in the liver. It was concluded that CA exerted protective effects on the NAFLD mice by inhibiting gut dysbiosis, pro-inflammatory LPS release, and subsequent lipid synthesis [57].

#### **4. CA vs. Hyperglycemia and Insulin Resistance**

One of the primary causes of metabolic and endocrine abnormalities, as well as cellular damage in afflicted tissue, is hyperglycemia-related oxidative stress [15]. Nutrientinduced toxicity due to overnutrition may lead to insulin-resistance in tissues such as the heart and the skeletal muscle, which normally responds to insulin for glucose uptake [58]. Insulin resistance is a condition where the tissues use their adaptive mechanism to avoid toxic nutrient overload [59]. Over time, insulin resistance will cause an increase in fasting glucose and reduced insulin-mediated glucose clearance. Eventually, hyperinsulinemia will occur as a negative feedback from the target cells, signaling inadequate insulin response, and, in turn, the pancreatic β-cells will produce more insulin. The prolonged inability to correct the state of insulin resistance will eventually give rise to hyperglycemia and type 2 diabetes [60].

CA is found to increase insulin sensitivity through the reduction in proinflammatory cytokines and increase in adiponectins under the hyperglycemic state [51]. In a study that used MetS diet-induced rats, where it caused increases in BMI and abdominal circumference, blood glucose, triglycerides, and LDLc, and lowered the HDLc, the group that received a dose of 40 mg/kg oral gavage of CA daily for 6 weeks showcased a significant reduction in serum leptin, adiponectin, insulin, TNF-a, IL-6, and IL-8. The study showed that CA had the highest superoxide dismutase (SOD), catalase, and glutathione peroxidase antioxidant enzymes in the liver after 4 weeks of CA administration in comparison to ferulic acid, gallic acid, and protocatechuic acid under the same doses [61]. This suggests that the scavenging activity as a result of CA administration shows the most promising effectivity amongst the listed phenolic acids that protect against hyperglycemic damages.

Nasry et al. investigated the role of pioglitazone (a synthetic PPARγ agonist that causes a decrease in insulin resistance) on HFD-induced-MetS rats, and CA was able to show promising results. There was a significant reduction in insulin resistance, fasting blood glucose, and fasting serum insulin with an increase of insulin sensitivity and β cell function. CA also reduces the nitric oxide (NO) liver contents to almost half of those of the HFD-induced MetS rats [62]. This shows the efficacy of CA as scavenging activity toward correcting the insulin resistance through the reduction in oxidative stress caused by the HFD.

CA also suppresses the hepatic glucose output by enhancing its utilization and inhibiting overproduction [63]. This can be seen by the increase in glucokinase activity through an increase in its mRNA expression and glycogen content. It was also found to simultaneously lower the G6Pase and phosphoenolpyruvate carboxykinase activities together with their respective mRNA expressions, along with a decline in the GLUT2 expression in the liver [63].

CA methyl and ethyl esters exert antidiabetic activities in insulin-responsive cells through insulin-independent mechanisms involving AMPK and adipogenic factors [64]. A 2-week treatment of CAPA toward streptozotocin and diet-induced diabetic mice were able to protect them against hepatic inflammation and glucose intolerance associated with the NF-κB-mediated induction of inflammatory cytokines and the increase in the expression of antioxidant protein. HepG2 cell models were then used to further investigate CAPA's ability. They were able to show that CAPA was able to ameliorate TNFα-induced pIKKα/β expression and prevent TG accumulation in H2O2-treated HepG2 cells [40]. These findings strengthen the belief that chronic oral administration of CAPA is able to protect against MetS.

Stress-induced inflammation may cause the development of insulin resistance [65–67]. Stress activates the hypothalamic–pituitary–adrenal axis, renin–angiotensin system pathway, and sympathoadrenal system, all of which are involved in the production of proinflammatory cytokines, resulting in the negative downregulation of insulin signaling by either phosphorylating insulin resistance serine residues or inhibiting Akt, resulting in insulin resistance. CA given to chronic restraint stress-induced insulin-resistance mice

showed to reduce fasting blood sugar, systemic inflammation, and oxidative stress, and improve insulin sensitivity [68].

#### **5. CA vs. Dyslipidemia**

Dyslipidemia is described as an abnormal level of circulating lipids. It has been acknowledged that dyslipidaemia increases the risk of cardiovascular disease development [69]. This condition may be of primary cause (genetic) or secondary (diet, drugs, chronic diseases, and metabolic disorders, including MetS). Dyslipidemia is detected through a biochemical analysis of fasting lipid profile, which consists of TG, total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), and non-HDL-c. Dyslipidemia is diagnosed when there is an increased concentration of TG, TC, LDL-c, and non-HDL-c, along with a decreased level of HDL-c [70].

Free fatty acids (FFA) are abundantly released in an obese body due to the increase in the adipose tissue mass. FFA causes an increase in the synthesis of glucose and TG in the liver, as well as an increase in VLDL secretion. This occurs together with the reduction in HDL-C and increased density of LDL [71]. CA has shown improvement in the serum lipid profile, serum liver biomarker enzymes, and hepatic tissue architecture to normal in HFDinduced hyperlipidemic rat models by showing antihyperlipidemic and hepatoprotective activities. CA was found able to reduce the levels of endoplasmic reticulum stress markers in the liver after a HFD obese induction [72]. Besides CA's ability to revert dyslipidemia by reducing TG and TC, studies have shown that CA was able to revert hepatic steatosis in the long run [49,73–75]. In a recent in vivo study, a 12 week CA supplementation on HFD obese mice revealed that CA was able to reduce body weight and fat accumulation together with readings of improved lipid profile with an increased HDL [56]. This suggests that CA's ability to impair the formation of bad white fat tissue could subsequently reduce FFA production, thereby showing its hepatoprotective ability.

CA is capable of providing a TG-lowering, anticoagulatory, antioxidative, and antiinflammatory protection for the cardiac tissue and also downregulating the TNF-α and monocyte chemoattractant protein-1 mRNA expression in the kidney of diet-induced diabetic rats [76]. Studies on diet-induced hypercholesterolemic rats by Agunloye and Oboh compared the modulatory properties of CA and chlorogenic acid, proving that CA was a better candidate in ameliorating the pathological condition. They also tested two different dosages of the drug (10 mg/kg and 15 mg/kg of CA) and concluded the lipid-lowering effects were more effective at larger doses [77].

It is possible that an excessive amount of oxidative stress and/or inflammation can convert circulating LDL and HDL particles into oxidized LDL (oxLDL) and oxidized HDL particles (oxHDL). OxLDL and oxHDL both stay longer in the bloodstream due to their impaired interaction with their specific receptors. Their diminished clearance and imbalance of lipid profile ultimately contributes to the onset of atherosclerosis [19]. CA is thought to prevent atherosclerosis by lowering the functional and structural changes in the arteries [78]. This has been demonstrated by its ability to inhibit thrombogenic thromboxane A2 (TXA2) production together with other platelet-aggregating molecules [79,80]. CA also downregulated platelet-activating molecules such as COX-1, calcium ions, and P-selectin and upregulated platelet-inhibiting molecules such as cAMP and cGMP, resulting in an inhibition toward thrombogenic processes [81].

#### **6. CA vs. Hypertension**

Almost 80% of the individuals with MetS suffer from hypertension. Evidence concurred that 65–75% of the risk factor for primary hypertension is contributed by obesity and excess weight gain [82]. Besides, insulin resistance has also been linked to hypertension as insulin is able to cross the blood–brain barrier and subsequently activate the systemic nervous system, in addition to its ability to upregulate the angiotensin II (AT-II) receptor and reduce NO [60]. NO is one of the most important ROS in the cardiovascular

system. ROS are produced by NO synthase enzymatically, and they act as a prototype endothelial-derived vasodilator [83].

Nω-Nitro-L-arginine-methyl ester (L-NAME) is a well-known active inhibitor of NO production in the nerves and the endothelial cell. A study using L-NAME-induced hypertensive rats showed that a combination of caffeine and CA was able to reduce the systolic BP. A decrease in ACE and arginase activity coupled with high NO and low MDA levels might be associated with their antihypertensive effects [84,85]. In another study using CAPE against the high-fructose corn syrup diet-induced vascular damage in rats, blood pressure values were significantly reduced after a two-week intraperitoneal injection with CA derivative. This study also noted that CAPE has the ability to correct the reduced levels of endothelial NO synthase levels caused by the high-fructose corn syrup diet [73].

According to a more recent study, CA has a favorable effect on the vascular function and blood pressure stabilization. In this study, male SERCA2a knockout mice and its wild-type were surgically implanted with mini osmotic pumps filled with AT-II solutions and fed with a normal diet of 0.05% CA in drinking water. CA significantly attenuated the AT-II-induced increase in blood pressure reading in the wildtype mice but showed no hypotensive effect to the SERCA2a knockout mice. This suggests that the CA might act by activating the SERCA2a on the primary vascular smooth muscle cells [86].

CA has also been reported to be a potent antihypertensive agent and has been confirmed to have a nontoxic manifestation [87,88]. Agunloye and Oboh's in vitro study revealed that CA was capable of inhibiting key enzymes associated with hypertension that includes E-NTPDase, 50 -ectonucleotidase, ADA, ACE, arginase, and AChE. This study suggested that CA targets specific enzymes associated with hypertension [89]. Decreased ACE and arginase activity, as well as high NO and low MDA levels, might be associated with their antihypertensive effects [77]. The summary for MetS studies related to CA can be found in Table 1, whereas the proposed pathway for CA against MetS can be found in Figure 2.


**Table 1.** MetS studies related to CA.



L-NAME-induced Sprague Dawley rats

**Figure 2.** Proposed CA pathways against MetS. **Figure 2.** Proposed CA pathways against MetS.

#### **7. Conclusions 7. Conclusions**

50 µmol/kg/day intra-

peritoneally 14 days

There has been enormous progress in understanding the effect of CA through retrospective research. Strong evidence of the ability of CA to reverse the MetS effects through the reduction in inflammatory markers such as TNFα coupled with reduced oxidative stress parameters have guided researchers to a more proteomic and metabolomic approach. Besides the singular usage of CA, studies of using CA as an enhancer together with more commonly used drugs have surfaced. Through this review, we can conclude that CA holds strong potential to be used as MetS management by its anti-obesity, antidiabetic, hypolipidemic, and hypotensive activities. During the course of drafting this manuscript, we identified a substantial gap in which the wealth of knowledge about CA is limited to findings in animal models or cell lines. Further studies in the form of a clinical trial or a population cohort study would further strengthen the beneficial effect of CA on There has been enormous progress in understanding the effect of CA through retrospective research. Strong evidence of the ability of CA to reverse the MetS effects through the reduction in inflammatory markers such as TNFα coupled with reduced oxidative stress parameters have guided researchers to a more proteomic and metabolomic approach. Besides the singular usage of CA, studies of using CA as an enhancer together with more commonly used drugs have surfaced. Through this review, we can conclude that CA holds strong potential to be used as MetS management by its anti-obesity, antidiabetic, hypolipidemic, and hypotensive activities. During the course of drafting this manuscript, we identified a substantial gap in which the wealth of knowledge about CA is limited to findings in animal models or cell lines. Further studies in the form of a clinical trial or a population cohort study would further strengthen the beneficial effect of CA on MetS.

CA results were seen better in reversing the diabetic nephropathy in comparison to prevention.

Kidney tissue analysis shows that

—Unable to preserve PON1

[93]

[94]

—Unable to reduce NF-κB

CA was:

activity

—TG —HDL-c

significantly

Significantly reduced: —Total cholesterol

MetS. **Author Contributions:** Conceptualization, N.N.M.A.K. and M.F.Y.; writing—original draft preparation, N.N.M.A.K.; writing—review and editing, M.F.Y., F.A. and S.L.T.; supervision, M.F.Y., F.A. and S.L.T.; project administration, M.F.Y.; funding acquisition, M.F.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by PPUKM Fundamental Grant, grant number FF-2019-014.

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

#### **References**


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