Gastrodin Improves Nonalcoholic Fatty Liver Disease Through Adenosine Monophate-activated Protein Kinase Signaling Pathway

Nonalcoholic fatty liver disease(NAFLD)is a general term for a series of liver diseases including simple steatosis,steatohepatitis,liver fibrosis,liver cirrhosis and liver cancer,which pose a huge threat to the health of humans and animals.Nonalcoholic fatty liver disease poses a huge threat to human and animal health.NAFLD is the most prevalent chronic liver disease in the world,with a prevalence of approximately 25% of the global population,and is one of the most common causes of liver transplantation and hepatocellular carcinoma.Fatty liver in dairy cows occurs frequently in the perinatal period,often accompanied by complications such as decreased milk production,endometritis and production paralysis,causing serious economic losses to intensive farms.There are currently no approved drugs for the treatment of nonalcoholic steatohepatitis(NASH)in humans and animals.Gastrodin is a low molecular weight phenolic glycoside extracted from the rhizome of Gastrodia elata.Studies have shown that gastrodin can treat a variety of liver diseases and has hepatoprotective effects.However,the therapeutic mechanism of gastrodin on liver diseases has not been fully elucidated.In this study,a mouse model of nonalcoholic fatty liver was used to investigate whether gastrodin could improve NAFLD,further explore the relevant molecular mechanism,and provide theoretical support for the development of natural medicines.(1)In this study,high-fat diet(HFD)or high-fat and high-cholesterol diet(HFHC)were used to induce NAFLD and NASH models in mice.The test results showed that compared with the model group,gastrodin treatment significantly reduced liver weight,liver-to-body ratio,liver and serum triglyceride(TG)and total cholesterol(TC)contents,and serum alanine aminotransferase and aspartate aminotransferase activity levels(P< 0.01).Oil red O,H&E,CD11 b fluorescence and PSR staining were respectively performed on liver tissue,and it was found that gastrodin could significantly reduce liver lipid accumulation,inflammation level and fibrosis.The results of q PCR and Western blot showed that gastrodin treatment significantly down-regulated the m RNA and protein levels of those genes which are related to lipid metabolism(Cd36,Fasn,Scd1,etc.),inflammation(Ccl2,Cxcl2,TNF,etc.)and fibrosis(Col1a1,Ctgf,SMAD,etc.)(P<0.01).(2)In this study,PO(palmitic acid:oleic acid=0.5:1m M)was used to stimulate mouse primary hepatocytes and hepatocyte cell lines to investigate the effect of gastrodin on lipid accumulation and inflammation.Oil red O staining indicated that gastrodin reduced lipid accumulation in hepatocytes;q PCR results showed that gastrodin treatment significantly down-regulated m RNA levels of those genes which are related to fatty acid synthesis(Fasn,Acaca,Cd36,etc.)and inflammation(Il1β,Cxcl10,Jun,etc.)(P<0.01);the contents of TG and TC in hepatocytes were significantly decreased after gastrodin treatment(P<0.01).(3)In this study,transcriptome sequencing was used to analyze the livers of mice in the HFHC model group and the gastrodin group,as well as the primary hepatocyte samples of mice in the PO model group and the gastrodin group.GSEA and GSVA analysis showed that gastrodin significantly inhibited those pathways that were related to lipid metabolism,inflammation and fibrosis,such as PPARγsignaling,NF-κB signaling pathway and TGF-β signaling pathway,etc.It was found that gastrodin treatment significantly inhibited those genes that were related to lipid metabolism(Scd1,Cd36,Fasn,etc.),inflammation(Ccl2,Cxcl10,Tnf,etc.)and fibrosis(Col1a1,Acta2,Ctgf,etc.).Using GSEA to screen the most significantly changed KEGG pathway in primary hepatocytes and mouse liver,it was found that AMPK pathway is the main pathway regulated by gastrodin.At the same time,Western blot results showed that gastrodin treatment significantly up-regulated the protein level of p-AMPK(P<0.01).(4)In this study,the effects of gastrodin on lipid metabolism and inflammation in hepatocytes were observed after inhibiting the AMPK pathway.Using recombinant adenovirus or AMPK inhibitor(Compound C,CC)to block the AMPK pathway,and then giving gastrodin treatment,Oil Red O staining showed that gastrodin did not reduce the accumulation of lipid droplets in hepatocytes induced by PO stimulation.q PCR showed that the m RNA levels of lipid metabolism-related genes(Scd1,Fasn,Acaca,etc.)and inflammation-related genes(Cxcl10,Il1β,Jun,etc.)were nonsignificant difference between gastrodin and model group.Transcriptomic sequencing of primary hepatocytes in PO group,PO+ gastrodin group and PO+gastrodin+CC group showed that CC blocked the effect of gastrodin which down-regulate lipid metabolism and inflammatory pathways and key genes.(5)In this study,the effect of gastrodin on the liver of NASH mice was detected after inhibiting the AMPK pathway.The results showed that CC blocked the effect of gastrodin in reducing liver weight,liver weight/body weight,liver and serum lipid content(TG and TC).Oil red O,H&E and PSR staining showed that CC blocked the effect of gastrodin in reducing liver lipid accumulation,inflammation and fibrosis.q PCR assay showed that CC blocked the effect of gastrodin in downregulating the m RNA levels of genes related to fatty acid synthesis(Cd36,Fasn,Scd1,Pparg),inflammation(Cxcl10,Il1β,Ccl2)and fibrosis(Col3a1,Acta2,Ctgf).In conclusion,gastrodin improves lipid accumulation,inflammation and fibrosis in nonalcoholic steatohepatitis by activating the AMPK signaling pathway,providing a new therapy for the treatment of NASH.