Supplementary Materialsfoods-09-00285-s001. 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- and IL-1 were measured by using a commercial rat ELISA kit (R&D Systems, Minneapolis, MN, USA) following manufacturers 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 manufacturers instruction. 2.5. Histological Analysis in the Liver Formalin-fixed liver samples were embedded in paraffin, sliced at 5 m, 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 (0C5%), 1 point for mildly (5C20%), 2 points for moderately (20C50%), and 3 points for severely ( 50%) [21]. 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 manufacturers instructions. The mRNA levels were analyzed by quantitative real-time 520-36-5 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. 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 BACH1 4 weeks. SMSP administration 520-36-5 for 4 weeks significantly reduced the total liver 520-36-5 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 520-36-5 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 2BCE). In the meantime, SMSP significantly reversed the ethanol-treated hepatic accumulation of triglyceride by as much as 35% (Shape 2A), aswell as decreasing the serum triglyceride, GGT, ALT, and AST actions by 15%, 41%, 8.3%, and 9.4%, respectively (Shape 2BCE). Furthermore, steatosis ratings had been evaluated in H&E staining pictures of liver organ cells from all combined organizations. As a total result, hepatic lipid build up was remarkably improved in ethanol-treated rats (Shape 3A). As demonstrated in Shape 3B, the ethanol-induced elevation from the steatosis score was normalized in the SMSP-treated rats significantly. Open in another window Shape 1 Aftereffect of steamed and freeze-dried mature silkworm larval natural powder (SMSP) on bodyweight and liver organ pounds in rats treated with ethanol (EtOH) for four weeks. (A) Bodyweight changes, (B) liver organ weight by the end of test, and (C) liver organ/body weight percentage had been measured. The info represent mean SD (= 8); ** 520-36-5 0.01 and *** 0.001 vs. regular group; ## 0.01 and ### 0.001 vs. EtOH group. Open up in another window Shape 2 SMSP administration alleviates hepatic steatosis in EtOH-treated rats. (A) Hepatic triglyceride and serum degrees of (B) triglyceride, (C) gamma-glutamyl transpeptidase (GGT), (D) alanine aminotransferase (ALT), (E) aspartate aminotransferase (AST) had been measured. The info represent mean SD (= 8); * 0.05 and *** 0.001 vs. regular group;.