| Kinsenoside(KD)is an important active ingredient extracted from the traditional Chinese herbal Anoectochilus roxburghii(Wall.)Lindl.Studies have shown that KD has anti-inflammatory,antioxidant and liver protection effects on autoimmune hepatitis.Acetaldehyde,reactive oxygen species,etc.produced during the metabolism of alcohol in liver can cause oxidative stress,leading to liver inflammation and damage.However it is still unknown whether KD plays a protective role in alcohol-induced liver injury,this study intends to explore the protective effect and mechanism of KD on alcoholic fatty liver and liver fibrosis.The results of the study are as follows:(1)After KD treatment of alpha mouse liver 12(AML12),the Cell Counting Kit-8method was used to detect that KD promoted the proliferation of AML12 cells.Oil red O and Bodipy staining results showed that intracellular lipids were significantly increased after alcohol treatment in AML12 cells,however,KD treatment significantly attenuate the lipid increase.The peak areas of alcohol and tert-butanol were obtained by headspace gas chromatography,and the blood alcohol concentration of rats at different time points after gavage was calculated according to the alcohol standard curve.Compared with model group,KD promoted the metabolism of alcohol in rats,but it was slightly slower than the positive drug metadoxine.These results suggest that KD has a protective effect on alcohol-induced hepatocyte damage and can promote alcohol metabolism in vivo.(2)Using the mouse model of alcoholic fatty liver,the effect of KD on alcohol-induced liver injury was analyzed.Through hematoxylin-eosin staining and oil red O staining of liver tissue sections,it was found that there was a large area of lipid deposition in the liver of alcoholic fatty liver mice.However,after administration of KD or silymarin,the deposition area was significantly smaller than that of the model group,which proved that KD had a protective effect on alcohol-induced hepatocyte damage.In addition,after KD administration,it was found that the serum alanine aminotransferase,aspartate aminotransferase,tumor necrosis factor-α and interleukin-6 levels of the mice were significantly reduced,suggesting that KD attenuates alcohol-induced liver damage and liver inflammation.Compared with model group,it was found that superoxide dismutase in liver tissue homogenate was significantly increased and malondialdehyde was significantly decreased after KD administration,indicating that KD can inhibit alcohol-induced oxidative stress and enhance the body’s antioxidant capacity.The above results indicated that KD alleviates liver injury in mice with alcoholic fatty liver.(3)In the model of alcoholic liver fibrosis mice,after KD administration,liver tissue sections were prepared,and it was found by hematoxylin-eosin and Masson staining that it was related to the obvious liver inflammation and fibrosis in mice of model group.Compared with that,the degree of liver inflammation and fibrosis in mice after KD administration was significantly reduced,and the ratio of liver weight to body weight and triglyceride in liver tissue homogenate of mice in KD administration group were significantly reduced,indicating that KD significantly inhibited liver weight and fat accumulation in fibrotic mice.Serum alanine aminotransferase,aspartate aminotransferase,tumor necrosis factor-α and interleukin-6 were significantly decreased in KD administration group,showing that KD improved liver function and reduced inflammation in fibrotic mice.After KD administration,the liver homogenate superoxide dismutase,reduced glutathione/glutathione(oxidized),and nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide were increased significantly,indicating that KD can alleviate oxidative stress in the fibrosis mice.In brief,KD can alleviate alcoholic liver fibrosis in mice.(4)Proteomics was used to detect the expression of all proteins in fibrotic mice liver tissue,and the potential mechanism of KD protecting liver was explored by bioinformatics methods such as protein cluster analysis,gene ontology function analysis,and Kyoto Encyclopedia of Genes and Genomes pathway analysis.The results of proteomics showed that compared with model group,there were a large number of significantly different proteins in control group,and there were also many significantly different proteins in KD group.Gene ontology analysis showed that the differential proteins between KD and model group were mainly concentrated in biological processes such as acute inflammation and extracellular structure organization.The results of enzyme activity and Western blotting showed that compared with the model group,the activity of alcohol dehydrogenase was decreased significantly but the expression level was increased significantly,the activities of catalase and aldehyde dehydrogenase were increased significantly,and the expression level of cytochrome P450 2E1 was decreased significantly after KD administration.All of these indicating that KD promotes metabolism of alcohol.Compared with model group,the expression levels of key endoplasmic reticulum stress proteins such as Bip and eIF2α were decreased significantly;the expression levels of key AMP-activated protein kinase(AMPK)signaling pathway proteins such as LKB1,STRAD,and p-AMPK were increased significantly;and the autophagy marker proteins p-mTOR and SQSTM1 were decreased,and LC3A/B was increased significantly,suggesting that KD reverses the sustained endoplasmic reticulum stress and inhibition of AMPK and autophagy pathways induced by alcohol,plays a protective role against alcohol-induced liver damage.In summary,this study found for the first time that KD has a protective effect on alcoholic fatty liver and liver fibrosis.KD alleviates inflammation and fibrosis in hepatic fibrotic mice by promoting alcohol metabolism,reducing oxidative stress and endoplasmic reticulum stress,and activating AMPK-dependent autophagy,which can be used as a potential drug for the treatment of alcoholic liver disease. |
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