The Study Of Chicken Bile-derived Exosomes Protect Against LPS-induced Liver Injury

Exosomes are extracellular vesicles with multiple biological functions secreted by living cells and play an important role in various physiopathological processes such as inflammation,apoptosis,and intercellular information exchange.As a special body fluid,bile contains exosomes secreted by hepatic interstitial cells,hepatocytes,and bile duct epithelial cells.our team has shown that chicken bile exosomes have a certain targeting effect on promoting the proliferation of immune cells in the liver and enhancing the immune function of the liver,suggesting that chicken bile exosomes have good potential and advantages for application in the process of liver injury.To clarify the role of chicken bile exosomes in inhibiting liver injury,this study assessed the protective effect of chicken bile exosomes on liver injury by establishing an LPS-induced ex vivo liver injury model and giving chicken bile exosomes.We collected 480 m L of fresh healthy chicken bile from a large-scale,modern slaughterhouse,and exosomes were extracted by graded centrifugation and ultracentrifugation.exosomes morphology and size were observed by electron microscopy,and the characteristic protein TSG101 was detected by Western Blot.in vitro and in vivo LPS liver injury models were established.in vitro: different concentrations of LPS were applied to hepatocytes.The hepatocyte activity and ALT and AST enzyme activities were detected by CCK-8 method and glutathione aminotransferase（ALT）and glutathione aminotransferase（AST）assay kits,respectively,and the hepatocyte morphology was observed by light microscopy,and the hepatocyte survival rate,ALT and AST activities and hepatocyte morphological damage indexes were detected.AST enzyme activity.To screen the optimal concentration of chicken bile exosomes,different concentrations of chicken bile exosomes were co-cultured with hepatocytes for 24 h.The cells were collected,and the effects of bile exosomes on hepatocyte activity and cell cycle were detected by CCK-8 method and flow cytometry.To clarify the protective effect of chicken bile exosomes on LPS-induced liver injury,an ex vivo liver injury model was constructed with LPS,treated with chicken bile exosomes,light microscopic observation,ghrelin,and ghrelin assay kits,q RT-PCR,gross anatomical and histopathological observation were applied to investigate the ALT and AST enzyme activities in cell supernatant and serum,hepatocyte morphology and liver histomorphology observation,as well as m RNA expression levels of inflammation and apoptosis-related factors in hepatocytes and liver,were examined.The results showed that exosomes extracted from chicken bile were teacup shaped vesicles with a diameter of 50-150 nm and carried characteristic protein Tsg101.LPS caused significant hepatocyte injury,as evidenced by decreased hepatocyte activity and increased ALT and AST enzyme activities in cell supernatants and serum,and successfully constructed hepatocyte injury models（600 μg/m L LPS）and In vivo liver injury model（6 mg/kg LPS）was successfully constructed.Chicken bile exosomes were not cytotoxic and could affect the cell cycle and promote hepatocyte proliferation and division at concentrations ≥20 μg/m L.In the case of LPS injury,the uptake of chicken bile exosomes by hepatocytes increased;chicken bile exosomes attenuated LPS-induced hepatocyte injury and significantly reduced the activities of two enzymes,ALT and AST,in cell supernatant and serum;chicken bile exosomes significantly inhibited LPS-induced liver injury in chickens,improved the damage of liver histological structures and exerted The q RT-PCR results showed that chicken bile exosomes could reduce the m RNA expression levels of LPS-induced inflammatory factors（IL-6,TNF-α,i NOS）and apoptotic factors（Caspase-3,Bax）and increase the m RNA expression of anti-apoptotic factors（Bcl-2）as well as hepatocyte growth factor（HGF）m RNA expression levels in liver tissue.In conclusion,this study successfully established an ex vivo model of LPS-induced liver injury and clarified that chicken bile exosomes can effectively reduce LPS-induced injury and enhance the expression of inflammatory factors（TNF-α,IL-6,i NOS）and apoptotic factors（Bax,caspase-3,Bcl-2）and HGF in hepatocytes and liver by regulating The anti-inflammatory and anti-apoptotic abilities of the liver,promote hepatocyte proliferation and division,enhance liver repair and regeneration,and exert its anti-LPS injury effects.Therefore,chicken bile exosomes have significant protective effects on LPS-induced liver injury.