| Objective: The effect of Xiaomudan Granule on histopathological changes of liver,serum liver enzymes,lipid metabolism indexes,and the proteins and genes of inositol requiring enzyme 1-alpha(IRE1α)-X-box binding protein 1(XBP1)signaling pathway in endoplasmic reticulum stress(ERS)will be observed in high-fat diet-induced non-alcoholic fatty liver disease(NAFLD)mouse model,and to explore the possible mechanism of action of Xiaomudan Granule in the treatment of NAFLD.Methods: Sixty male SPF-grade C57BL/6 mice were acclimatized for one week,and ten were randomly selected into the normal group and the remaining 50 into the high-fat chow group.After the mice in the normal group and the mice in the high-fat diet group were given the normal diet and high-fat diet,respectively,for 12 weeks,three mice were randomly selected in the normal group,and five mice were randomly selected in the high-fat diet group,killed and liver tissues were taken for pathological staining observation(HE staining and oil red O staining).After confirming the successful modeling,the mice in the normal and high-fat diet groups continued to be fed a normal and high-fat diet.Meanwhile,the mice fed a high-fat diet were randomly divided into five groups using the random number table method: model group,low,medium,and high dose groups of Xiaomudan Granule,and metformin group,with nine mice in each group and seven mice remaining in the normal group.Mice in each dosing group were treated by gavage of the corresponding drug intervention at 1 mL/100 g body weight per day starting from the 13 th week.Distilled water gavage was given to the model and normal groups similarly.The changes in the mice’s body weight,diet,stool,and mental status were observed and recorded daily.After eight weeks of continuous gavage,blood,and liver tissue specimens were collected.HE staining and oil red O staining were performed to observe the histopathological changes in the liver of each group of mice.Serum ALT and AST levels and liver TG,TC,VLDL,and FFA levels were detected by enzyme marker.The expression levels of p-IRE1α/IRE1α,XBP1,MTP,PDI,and ApoB100 in the liver tissues of each group of mice were detected by Western blot(WB).The expression levels of XBP1,MTP,PDI,and ApoB100 mRNA in the liver tissues of mice in each group were detected by real-time fluorescence quantitative PCR(qPCR).Results: 1.No significant abnormalities were observed in the diet,mental condition,fur color,movement,and urination and defecation of the mice in the normal group during the experiment.The body weight showed normal growth.In contrast,the mice in the high-fat diet group showed apparent changes in behavioral activities and living habits.Their body weight showed a trend of obese body growth,while the overall situation of the mice in each treatment group was better than that of the model group after the intervention of Xiaomudan Granule and metformin,and their body weight was significantly reduced.2.HE staining and oil red O staining showed that compared with the normal group,the liver of the model group exhibited significant pathological changes of non-alcoholic steatohepatitis: abnormal morphology of hepatocytes,the structural disorder of liver lobules,evident hepatic lipid accumulation,showing diffuse hepatocellular macrovesicular steatosis and ballooning enlargement of hepatocytes.After the drug administration intervention,different degrees of hepatocyte steatosis were still seen in each drug group.However,their inflammatory infiltration,diffuse area of steatosis cells,and degree of macrovesicular steatosis were significantly better than those in the model group and tended to be normal.3.The results of serum liver enzyme indexes showed that the ALT and AST contents of the model group were significantly higher than those of the normal group(P<0.01).Those of each dose of Xiaomudan Granule and metformin group were lower than those of the model group(P<0.05 or P<0.01).The overall difference between each dose group of Xiaomudan Granule and the metformin group for ALT was not significant(P>0.05),and the metformin group for AST was lower than the low and medium dose groups of Xiaomudan Granule(P<0.05).4.The results of Liver tissue lipid metabolism indicators showed that the contents of TC,TG,FFA,and VLDL in liver tissues of mice in the model group were significantly higher than those in the normal group(P<0.01).The contents of TC,TG,and VLDL in all dose groups of Xiaomudan Granule and the metformin group were lower than those in the model group(P<0.05 or P<0.01).Those in the metformin group were lower than those in the low-dose group of Xiaomudan Granule(P<0.05).The medium,high dose groups of Xiaomudan Granule and metformin groups with FFA were lower than the model group(P<0.05),and the metformin group with FFA was lower than the low and medium dose group of Xiaomudan Granule(P <0.01 or P <0.05).5.The results of WB showed that expression levels of p-IRE1α/IRE1α,XBP1,MTP,PDI,and ApoB100 in liver tissues of mice in the model group were significantly higher than those in the normal group(P < 0.01),the protein expressions of p-IRE1α/IRE1α,XBP1,and MTP in the high and medium dose groups of Xiaomudan Granule and metformin group were all down-regulated compared with those in the model group(P<0.01).The protein expression of ApoB100 was down-regulated in all dose groups of Xiaomudan Granule and metformin group compared with the model group(P<0.01),and the protein expression of PDI was down-regulated in the high dose group of Xiaomudan Granule and metformin group compared with the model group(P<0.05).The down-regulation of p-IRE1α/IRE1α and MTP protein in the metformin group was better than that in the low and medium dose groups of Xiaomudan Granule(P<0.01 or P<0.05);the down-regulation of XBP1 and ApoB100 protein expression in the metformin group was better than that in the low dose group of Xiaomudan Granule(P<0.01);while the down-regulation of XBP1 protein expression in the high dose group of Xiaomudan Granule was better than that in the metformin group,but the differences were not statistically significant(P>0.05).6.The qPCR results showed that XBP1,MTP,PDI,and ApoB100 mRNA expression in liver tissues of mice in the model group were significantly higher than those in the normal group(P<0.01).XBP1 and MTP mRNA expression were down-regulated in the high-dose Xiaomudan Granule group and the metformin group compared with the Model group(P<0.05 or P<0.01).The mRNA expression of PDI and ApoB100 was down-regulated in the high and medium dose groups of Xiaomudan Granule and the metformin group compared with the Model group(P<0.05 or P<0.01).Among them,the down-regulation of MTP and ApoB100 mRNA expression in the metformin group was better than that in the low-dose group of Xiaomudan Granule(P<0.05 or P<0.01).The down-regulation of PDI mRNA expression in the metformin group was better than in the low and medium-dose groups of Xiaomudan Granule(P<0.01).In comparison,the down-regulation of XBP1 mRNA expression in the high-dose group of Xiaomudan Granule was better than that in the metformin group.However,the difference was not statistically significant(P>0.05).Conclusion: 1.Xiaomudan Granule can improve the physical obesity status of NAFLD mice,reduce the excessive deposition of lipids,inflammatory infiltration,and necrosis degree in the liver,and have certain hepatoprotective effects.2.Xiaomudan Granule may alleviate the endoplasmic reticulum stress status of the liver by inhibiting the expression of IRE1α-XBP1 pathway and downstream proteins MTP,PDI,and ApoB100,improving Lipid metabolism disorder,thus reducing the degree of hepatocyte steatosis,which may be one of the mechanisms of Xiaomudan Granule to prevent and treat NAFLD. |
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