**1. Introduction**

Alcohol-related liver disorder is one of the most common diseases in the world [1]. Chronic alcohol consumption leads to liver diseases including hepatic steatosis, hepatocellular injury, and inflammation, and can cause liver fibrosis, cirrhosis, and hepatocellular carcinoma [2,3]. In particular, as one of the most common forms of alcoholic liver disease, hepatic steatosis is generally considered to be a benign and reversible process that can progress to a more serious condition [4,5]. AMP-activated protein kinase (AMPK) is known to be a major factor of energy metabolism and is involved in lipid metabolism [6]. When AMPK activity is inhibited by several causes, lipogenic transcription factors increase, resulting in fat generation [7]. In addition, AMPK phosphorylates the serine residues of acetyl CoA carboxylase 1 (ACC1), activating catabolic pathways, including fatty acid oxidation [8]. Thus, the regulation of lipogenesis and fatty acid oxidation through AMPK signaling is a therapeutic mechanism for alcoholic steatosis.

After drinking, alcohol is metabolized by cytochrome P450 2E1 (CYP2E1), an enzyme mainly expressed in the liver [3]. Increased CYP2E1 by alcohol consumption causes oxidative stress and inflammation in the kupffer cells, resulting in liver damage [9,10]. In the liver, kupffer cells play a key role in an innate immune system and they accelerate the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1β, and IL-6 [11]. Recently, it has been found that these cytokines induce the imbalance of lipid metabolism in the liver and cause the more serious liver diseases [12]. Limuro et al. reported that inhibition of TNF-α inhibited fatty liver in alcohol-treated rats [13]. Moreover, hepatic injury and steatosis induced by alcohol were alleviated in TNF-α knockout mice [13]. Other cytokines, such as IL-1β and IL-6, have been also reported to interfere with lipid metabolism in the liver [14]. Therefore, suppression of the pro-inflammatory cytokines production might have a therapeutic effect against hepatic inflammation and steatosis induced by alcohol consumption.

Recently, there has been interest in edible insects, and they have been highlighted as a future food [15]. The silkworm, *Bombyx mori*, contains high amounts of protein, omega-3 fatty acids, vitamins, and minerals [16]. Silkworms are traditionally used for the treatment of spasms, phlegm, and flatulence [15]. In addition, the silkworm has recently received scientific attention and several studies have reported its health advantages against liver injury [16], Parkinson's disease [17], and diabetic hyperglycemia [18]. However, after the third day of the fifth instar, the protein composition of silkworm glands rapidly increases, hence, there is a problem insofar as the biological activity is lowered [19]. In addition, dried or refrigerated silkworms are difficult to consume as food because the silk protein is denatured to a high strength [19]. Therefore, Ji et al. have developed a new method of processing silkworms into an easy-to-eat form, in which the mature silkworms are steamed for 130 min at 100 ◦C before lyophilizing and grinding [19]. We previously reported that steamed and freeze-dried mature silkworm larval powder (SMSP, 0.1, 1, and 10 g/kg) alleviated fatty liver disease in rats treated with alcohol [20]. However, in terms of human dosage, the dose of SMSP reported in the previous studies is burdensome to consume large amounts of silkworm powder per day. In the present study, we aimed to determine whether low dosage of SMSP attenuates hepatic steatosis in rats with ethanol-induced fatty liver and to explore the potential molecular mechanisms of the health benefits of SMSP, focusing on the gene expression involved in lipid metabolism and inflammation in liver.

#### **2. Materials and Methods**

### *2.1. Preparation of Steamed Mature Silkworm Larval Powder*

SMSP was made as previously described [19]. Briefly, live mature larvae of *Bombyx mori*, 7th day of 5th instar silkworms, were immediately steamed for 130 min at 100 ◦C using an electric pressure-free cooking machine (KumSeong Ltd., Boocheon, Korea) and freeze-dried using freeze-drier (FDT-8612, Operon Ltd., Kimpo, Korea) for 24 h. Silkworms were then grinded using a disk mill (Disk Mill01, Korean Pulverizing Machinery Co. Ltd., Incheon, Korea) and a hammer mill (HM001, Korean Pulverizing Machinery Co. Ltd., Incheon, Korea). And the SMSP was stored at −50 ◦C and then used for oral gavage for rats.

#### *2.2. Animal and Experimental Design*

All animal experiment procedures were conducted in accordance with the guidelines and approval of the Institutional Animal Care and Use Committees (IACUC) of the CHA University (reference number: 180002). Three-week-old male Sprague-Dawley (SD) rats weighing 120–130 g were purchased from Orient bio (Seoul, Korea). All animals were housed in a standardized laboratory environment with a 12 h light/dark cycle at constant temperature of 24 ◦C. After a week of acclimatization, all animals randomly divided into 3 groups (*n* = 8). The normal group was fed with rodent chow diet (Haran 2018s) and orally received 0.2 mL of distilled water. The ethanol-treated group was fed with rodent chow diet and orally received 25% ethanol (3 g/kg body weight) once a day for four weeks. The SMSP-treated group was also fed rodent chow diet and orally received 25% ethanol and SMSP (50 mg/kg body weight) once a day for four weeks. Rats were monitored daily for body weight change for the experimental period. At the end of the experiment, all rats were sacrificed at 4 weeks by carbon dioxide anesthesia. Blood was drawn from the abdominal aorta into a heparin tube. Serum was subsequently obtained by centrifuging the blood at 3000 rpm for 15 min at 4 ◦C. The livers were excised, rinsed with PBS, and weighed, and the portion of liver was fixed in 10% formalin. Serum and liver samples were stored at −80 ◦C until analysis.

#### *2.3. Serum Analysis*

The serum levels of triglyceride, gamma glutamyl transpeptidase (GGT), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were analyzed using Hitachi automatic analyzer 7600-210 (Hitachi High-Technologies Corporation, Tokyo, Japan). The serum concentration of TNF-α and IL-1β were measured by using a commercial rat ELISA kit (R&D Systems, Minneapolis, MN, USA) following manufacturer's instruction.

#### *2.4. Hepatic Triglyceride Analysis*

For the measurement of triglyceride levels in liver tissues, we used the commercial triglyceride assay kit (AB 65336, Abcam, Cambridge, UK). The liver triglyceride was determined as following the manufacturer's instruction.

#### *2.5. Histological Analysis in the Liver*

Formalin-fixed liver samples were embedded in paraffin, sliced at 5 μm, followed by sectioning and hematoxylin and eosin (H&E) staining by standard procedures. Histopathological scoring was assessed by an experienced pathologist, who was blinded to the treatment groups. Levels of fatty infiltration and steatosis were graded as 0 point for no hepatocytes affected, 0.5 point for slightly affected (0–5%), 1 point for mildly (5–20%), 2 points for moderately (20–50%), and 3 points for severely (>50%) [21].
