*2.6. RNA Isolation and Gene Expression Analysis*

Total mRNA was isolated from the rat livers using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using Labopass cDNA synthesis kit (Cosmogenetech Co., Ltd., Seoul, Korea) according to the manufacturer's instructions. The mRNA levels were analyzed by quantitative real-time PCR (qRT-PCR). The qRT-PCR was assessed as previously reported [22] and was performed on a ViiATM 7 real-time PCR system (Life Technologies Corporation, Carlsbad, CA, USA) using Luna universal qPCR master mix (New England Biolabs, Beverly, MA, USA). 18S ribosomal RNA (18s rRNA) were used as an internal control. The primer sets for qRT-PCR and RT-PCR are listed in Table 1.


#### **Table 1.** List of primers.

#### *2.7. Western Blotting*

Liver tissues were grinded with cell lysis buffer containing protease inhibitor (Roche Applied Science, Mannheim, Germany) and samples were incubated on ice with frequent vortexing for 10 min and centrifuged for 15 min at 13,000 rpm. The protein concentration of each supernatant was quantified using PierceTM BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer's instructions. The proteins were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). After transfer, membranes were blocked with bovine serum albumin (BSA) solution and probed with the specified primary antibodies (diluted 1:1000) overnight at 4 ◦C. The membranes were washed and incubated with the appropriate secondary antibodies for 40 min. The blots were then developed using an enhanced chemiluminescence system (Thermo Fisher Scientific, Waltham, MA, USA). Antibodies for p-AMPK, AMPK, peroxisome proliferator-activated receptor gamma (PPARγ) and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies for ACC1 and p-ACC1 were purchased from Cell Signaling Technology (Danvers, MA, USA).

#### *2.8. Statistical Analysis*

All data are expressed as means ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA). Statistical significance was accepted at *p* < 0.05.

#### **3. Results**

#### *3.1. SMSP Supplementation Alleviates Hepatic Steatosis in Ethanol-Treated Rats*

To examine the hepatoprotective role of SMSP, we used an ethanol-induced hepatic steatosis rat model. The ethanol (3 g/kg) and SMSP (50 mg/kg) were orally injected into SD rats for 4 weeks. SMSP administration for 4 weeks significantly reduced the total liver weight compared with ethanol-treated rats without affecting body weight change (Figure 1A,B). The SMSP group, as compared with the ethanol group, had a reduction in liver weight to body weight ratio (Figure 1C). Ethanol treatment for 4 weeks successfully induced fatty liver and liver injury in rats, which were manifested by significant increases in serum triglyceride, GGT, ALT, and AST activities compared with those of normal diet-fed rats (Figure 2B–E). In the meantime, SMSP significantly reversed the ethanol-treated hepatic accumulation of triglyceride by as much as 35% (Figure 2A), as well as lowering the serum triglyceride, GGT, ALT, and AST activities by 15%, 41%, 8.3%, and 9.4%, respectively (Figure 2B–E). In addition, steatosis scores were evaluated in H&E staining images of liver tissues from all groups. As a result, hepatic lipid accumulation was remarkably increased in ethanol-treated rats (Figure 3A). As shown in Figure 3B, the ethanol-induced elevation of the steatosis score was significantly normalized in the SMSP-treated rats.

**Figure 1.** Effect of steamed and freeze-dried mature silkworm larval powder (SMSP) on body weight and liver weight in rats treated with ethanol (EtOH) for 4 weeks. (**A**) Body weight changes, (**B**) liver weight at the end of experiment, and (**C**) liver/body weight ratio were measured. The data represent mean ± SD (*n* = 8); \*\* *p* < 0.01 and \*\*\* *p* < 0.001 vs. normal group; ## *p* < 0.01 and ### *p* < 0.001 vs. EtOH group.

**Figure 2.** SMSP administration alleviates hepatic steatosis in EtOH-treated rats. (**A**) Hepatic triglyceride and serum levels of (**B**) triglyceride, (**C**) gamma-glutamyl transpeptidase (GGT), (**D**) alanine aminotransferase (ALT), (**E**) aspartate aminotransferase (AST) were measured. The data represent mean ± SD (*n* = 8); \* *p* < 0.05 and \*\*\* *p* < 0.001 vs. normal group; # *p* < 0.05 and ## *p* < 0.01 vs. EtOH group.

**Figure 3.** SMSP administration attenuates hepatic lipid accumulation in EtOH-treated rats. (**A**) Histopathological sections of liver were stained with H&E (magnification, ×40 and 100). (**B**) Hepatic steatosis scores were quantified from H&E-stained sections. The data represent mean ± SD (*n* = 4); \*\*\* *p* < 0.001 vs. normal group; # *p* < 0.05 vs. EtOH group.

#### *3.2. SMSP Supplementation Attenuates Inflammation in Ethanol-Treated Rats*

Chronic alcohol consumption leads to liver injury, fat metabolism, inflammation, and hepatocellular carcinogenesis [3]. In addition, chronic alcohol consumption induces the production of reactive oxygen species (ROS), regulated by cytochrome P450 2E1 (CYP2E1), that lead to the production of reactive aldehydes with potent pro-inflammatory properties [11]. Based on this rationale, we analyzed the serum levels of TNF-α and IL-1β. As shown in Figure 4A,B, the TNF-α and IL-1β were significantly increased in ethanol-treated rats, while SMSP treatment attenuated the serum levels of TNF-α and IL-1β. Moreover, the hepatic mRNA expression of the CYP2E1 and IL-1β were significantly decreased by SMSP administration (Figure 4C,D). These results indicate that SMSP supplementation ameliorates liver inflammation induced by ethanol.

**Figure 4.** SMSP administration attenuates inflammation in EtOH-treated rats. Serum levels of (**A**) tumor necrosis factor-alpha (TNF-α) and (**B**) interleukin 1 beta (IL-1β) were measured by ELISA. The mRNA expressions of (**C**) cytochrome P450 2E1 (CYP2E1) and (**D**) IL-1β were detected by qRT-PCR in liver tissues from each group. The data represent mean ± SD (*n* = 4); \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 vs. normal group; # *p* < 0.05 and ### *p* < 0.001 vs. EtOH group.

#### *3.3. SMSP Supplementation Improves Lipid Metabolism in Ethanol-Treated Rats*

To examine the underlying molecular mechanism, we examined the effect of SMSP supplementation on hepatic lipid metabolism such as lipogenesis and free fatty acid (FFA) oxidation. AMPK is one of the key activators of the lipogenesis and FFA oxidation catabolism [23]; we tested whether prolonged SMSP supplementation could modulate the AMPK pathways in the liver of alcohol-treated rats. Western blot results showed that SMSP administration significantly increased the phosphorylation of

AMPK reduced by ethanol treatment. However, PPARγ, the well-known lipogenic factor, significantly decreased in the livers of SMSP-fed rats (Figure 5A). The expression of lipogenic nuclear transcription factors, such as sterol regulatory element-binding protein 1c (SREBP1c), PPARγ, and fatty acid synthase (FAS), were down-regulated in the liver of SMSP-treated rats compared with the ethanol-treated rats (Figure 5B). However, SMSP supplementation restored the mRNA levels of adiponectin receptor 1 (AdipoR1) and sirtuin 1 (Sirt1) in the livers of ethanol-treated rats (Figure 5B).

**Figure 5.** SMSP administration improves lipid metabolism in ethanol-treated rats. (**A**) The expression of phosphorylation of AMP-activated protein kinase (p-AMPK), AMPK and peroxisome proliferator-activated receptor gamma (PPARγ) in the liver tissues were determined by Western blotting and normalized to that of β-actin. (**B**) The mRNA expression of adiponectin receptor 1 (AdipoR1), sirtuin 1 (Sirt1), sterol regulatory element-binding proteins (SREBP1c), PPARγ and fatty acid synthase (FAS) in liver tissues were determined by qRT-PCR and normalized to that of 18s rRNA. The data represent mean ± SD (*n* = 4); \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001 vs. normal group; # *p* < 0.05 and ## *p* < 0.01 vs. EtOH group.

The inhibition of ACC1 has been reported to reduce hepatic triglyceride accumulation by decreasing lipogenesis and increasing FFA oxidation [24]. Therefore, we investigated the expression of ACC1 and the phosphorylation. As shown in Figure 6A,B, the phosphorylation of ACC1 was significantly inhibited in ethanol-treated rats while SMSP supplementation recovered the phosphorylation of ACC1. Moreover, the decreased hepatic expressions of ACC1 and peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) mRNA by ethanol treatment were recovered in SMSP-administered rats, although not statistically significant (Figure 6B,C). These results suggest that SMSP ameliorates EtOH-induced fatty liver through the regulation of lipogenesis and FFA oxidation mediated by AMPK and ACC1.

**Figure 6.** SMSP administration affects the expressions of lipogenesis-related molecules in ethanol-treated rats. (**A**) The phosphorylation of acetyl-CoA carboxylase 1 (ACC1) in the liver tissues was determined by Western blotting and normalized to that of β-actin. The mRNA expression of (**B**) ACC1 and (**C**) peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) in liver tissues was determined by qRT-PCR and normalized to that of 18s rRNA. The data represent mean ± SD (*n* = 4); \* *p* < 0.05 and \*\* *p* < 0.01 vs. normal group; # *p* < 0.05 vs. EtOH group.

#### **4. Discussion**

Insects are used for food and animal feed because they are rich in beneficial ingredients, such as proteins, fats, vitamins, minerals, and fiber [25]. In particular, the silkworm *Bombyx mori* has traditionally been used as food in Asia. Our colleagues have compared the nutrient composition of SMSP with those of freeze-dried mature silkworm powder (FMSP) and freeze-dried 3rd day of 5th instar silkworm powder (FDSP) [16]. A proximal analysis revealed that SMSP shows the highest protein contents compared to FMSP and FDSP. In addition, as shown in Table S1, amino acids such as glycine, alanine, and serine, which are the major components of silk protein are very abundant in SMSP. Moreover, the amount of unsaturated fatty acids is more than twice as high than that of saturated fatty acids in SMSP [16]. In addition, the amount of polyphenol, flavonoids, and minerals were examined. Since the amino acids and n-3 fatty acids have been shown to be effective in preventing metabolic diseases and promoting human health [26,27], in line with this notion, we suggest that the administration of SMSP may have beneficial effects on health based on its nutritional composition.

In our previous study, we confirmed the hepatoprotective effects of SMSP 0.1, 1, and 10 g/kg in diethylnitrosamine (DEN)-induced acute liver injury rat model and ethanol-induced liver damage rat model [20,28]. We did not show any side effects such as weight change or decreased dietary intake in rats treated with SMSP. In addition, the previous study with DEN-induced hepatocellular carcinoma rat model have shown that the administration of 1 g/kg of SMSP for 16 weeks does not induce any toxicity compared to the normal group [29]. These results demonstrate that the supplementation of 1–10 g/kg rat body weight of SMSP for 4–16 weeks would be safe. However, 1 g/kg of SMSP in rats corresponds to an intake of approximately 9.73 g/60 kg adult/day, when calculated on the basis of normalization to body surface area [30]. Therefore, this study was conducted to investigate the hepatoprotective effect of the lower dose of SMSP, 50 mg/kg rat body weight, corresponding to an intake of 0.487 kg/60kg in human dose.

Chronic alcohol intake can cause the progress of hepatic steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) [17]. Alcohol is a small molecule that can spread easily through the cell membrane [17]. The major enzymes of alcohol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) [31]. These enzymes are known as phase 1 xenobiotic metabolize enzymes [31]. ADH rapidly oxidizes alcohol to acetaldehyde, and then ALDH converts acetaldehyde to acetate [32]. This metabolism uses NAD<sup>+</sup> and is based on CYP2E1. The ADH, CYP2E1, and ALDH are mainly produced in hepatocytes [2]. We previously reported that SMSP supplementation alleviated the serum levels of ADH and ALDH in acute alcohol-induced liver injury rat models [33]. In this study, to explore the protective effect of SMSP in rats, we evaluated the anti-hepatosteatotic effect of SMSP using the fatty liver model induced by ethanol (3 g/kg, daily, 4 weeks). Based on our previous study, this dosage was enough to induce hepatic steatosis in SD rats [20]. We found that rats fed the rodent chow diet and oral gavage of SMSP induced no changes, such as in body weight (Figure 1A), but the mass of liver tissue and liver weight/body weight ratio from rats treated SMSP were 8% and 10% respectively lower than those received ethanol only (Figure 1B,C). In addition, SMSP significantly alleviated the levels of liver triglycerides, serum triglycerides, and liver injury markers, such as GGT, ALT, and AST (Figure 2C–E).

Alcoholic liver disease is characterized by the accumulation of large amounts of lipids in the liver with inflammation [11]. In alcoholic liver diseases, chronic ethanol consumption stimulates Kupffer cells to activation by lipopolysaccharides through diverse signal such as Toll-like receptors [11]. This regulation induces the production of various pro-inflammatory cytokines, such as TNF-α, interferon gamma (IFN-γ), and IL-1β, which are critically regulated hepatic inflammation, steatosis, fibrosis, and HCC [34]. It has been reported that both patient and animal model with alcoholic liver disease, the levels of IL-1β are significantly increased in the liver and the serum [35,36]. The present study showed that SMSP treatment can attenuate the serum levels of TNF-α and IL-1β as well as mRNA levels of IL-1β (Figure 4). These results indicate that SMSP can reduce the production of pro-inflammatory cytokines by the chronic alcohol consumption.

In addition, chronic alcohol intake also induces the oxidative stress that induces lipid peroxidation, intracellular membrane damage, and lead to the production of pro-inflammatory cytokines and pro-fibrotic effects. Oxidative stress and ROS are caused through the CYP2E1 signaling [11]. It has been reported that CYP2E1 knock-out mice exhibit ethanol-induced liver disease [37], and we previously demonstrated that SMSP supplementation restored total antioxidant concentration levels and significantly reduced hepatic malondialdehyde levels [20]. In this study, we also found that rats fed SMSP reduced CYP2E1 mRNA levels even the low dosage of SMSP (Figure 4C). These results suggest that low dosage of SMSP can diminish oxidative stress through inhibition of CYP2E1 induced by ethanol.

The accumulation of triglycerides in hepatocytes causes hepatic steatosis, because of the imbalance between lipogenesis and FFA oxidation. Considerable evidence has reported that adiponectin plays a key role in alcoholic fatty liver in several animal models [38–41]. Circulating adiponectin binds to adiponectin receptors in the liver and thereby regulates lipid metabolism through AMPK [42]. In hepatocytes, AMPK is a crucial regulator of intracellular energy sensors that has been reported in the regulation of lipid homeostasis [43]. The inactivation of AMPK negatively regulated FFA oxidation through the inhibition of the transcription factors ACC1 and PGC-1α, and increased activation of SREBP1c, a lipogenic transcription factor [44]. Several studies indicate a relationship between SIRT1 and the AMPK signaling pathway [45]. The upregulation of SIRT1 acts on AMPK upstream, suggesting that the regulation of the SIRT1/AMPK signal may act as a key mechanism for lipid homeostasis in liver. In this study, the protein and mRNA levels of genes involved in SIRT1/AMPK-mediated lipogenesis and FFA oxidation were regulated in rats administered SMSP. These results indicate that SMSP improves lipid metabolism in the livers of rats treated with ethanol.

In summary, the present study suggests that the low dose of SMSP (50 mg/kg) effectively reduced the hepatic steatosis through the activation of SIRT1/AMPK-mediated signaling cascades in ethanol-treated rats. Increased hepatic SIRT1 and AMPK activity appears to be associated with these

beneficial effects of SMSP. Further studies on the effect of SMSP in vitro and in humans should confirm that SMSP could serve as an effective therapeutic agent in treating human alcoholic fatty liver disease.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2304-8158/9/3/285/s1, Table S1: The nutrient composition of SMSP.

**Author Contributions:** Conceptualization, D.-Y.L., K.-S.H., and E.-H.K.; methodology, D.-Y.L., K.-S.H., and E.-H.K.; formal analysis, D.-Y.L., K.-S.H., M.-Y.S., and S.-M.Y.; investigation, D.-Y.L., K.-S.H., M.-Y.S., and S.-M.Y.; resources, S.-D.J. and J.-G.S.; writing—original draft preparation, D.-Y.L.; writing—review and editing, D.-Y.L., and E.-H.K.; project administration, E.-H.K.; funding acquisition, E.-H.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Agri-Bio industry Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (317004-4).

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