Study On The Protective Effect And Mechanism Of Angelica Sinensis Polysaccharide On Liver And Intestine Based On "Gut-Liver Axis" In Alcoholic Fatty Liver Mice

Objective: Angelica sinensis polysaccharide(ASP),one of the noteworthy active components from Angelica sinensis(Oliv.)Diels,showed antioxidant,hepatoprotective and hypolipidemia activities.However,the research on the improvement effect and mechanism of ASP on fatty liver was not enough in-depth.Therefore,the mice model of alcoholic fatty liver damage(AFLD)was established to investigate the hypolipidemia and hepatoprotective activities of ASP and explore the mechanism from the perspectives of liver and “gut-liver axis” in current research.Methods: Firstly,the high-purity ASP was prepared by extraction and alcohol precipitation,dialysis,freeze-thaw and chromatographic column separation.The structure of ASP was identified by FT-IR.Secondly,the AFLD mice model was established by gavage of excessive alcohol and the intervention of ASP was carried out in BALB/c mice.The hepatoprotective activity of ASP,including the levels of liver injury,lipid metabolism and oxidative stress,were evaluated by using the histological analysis,the kit detection,flow cytometry and so on.The effects of ASP on the hepatic related proteins and genes in AFLD mice were studied by using Western blot,q RT-PCR,and immunohistochemistry.Simultaneously,the AFLD cell models and inhibitor control groups were constructed in vitro to verify the lipid-lowering mechanism of ASP.Thirdly,the effects of ASP on the liver in AFLD mice based on the “gut-liver axis” were studied by Western blot,16 S r RNA gene sequencing of gut microbiota and ultra performance liquid chromatography coupled with mass spectrometry from three perspectives: the uptake of intestinal free fatty acid,gut microbiota and the metabolism of bile acid.To further investigate the “gut-liver axis”,the effects of ASP on ileum and colon were studied by the immunofluorescence and gas chromatography.Finally,a two-step synthesis method was used to prepare the nuclide technetium-labeled ASP and the absorption and distribution of technetium-labeled ASP was detected by a γ counter after the oral administration in normal mice and AFLD mice.Results: The levels of alanine aminotransferase and aspartate aminotransferase in serum and the content of the lipid parameters in liver and serum,including free fatty acid,triglyceride and total cholesterol,were significantly reduced after ASP administration,and the levels of reduced glutathione and glutathione peroxidase were also restored,suggesting the obvious improvement of ASP on the liver injury,lipid metabolism disorder and reactive oxygen species levels.The in vitro and in vivo results showed that ASP could activate the AMPK/SIRT1 pathway of hepatocytes to inhibit the protein levels of ACC,FAS and LIPIN1 and reduce the synthesis of free fatty acid by the hepatic de novo lipogenesis pathway.On the other hand,ASP could inhibit the free fatty acid uptake of CD36-mediated in liver,thus exerting the hypolipidemia and hepatoprotective activities.For “gut-liver axis”,ASP could(1)activate the AMPK/SIRT1 pathway to reduce CD36 levels and inhibit the free fatty acid absorption in ileum,leading to the reducement of the hepatic free fatty acid levels;(2)improve the homeostasis of intestinal microflora to reduce the levels of gram-negative bacteria,resulting in the improvement of the liver damage and increased LPS caused by alcohol;(3)increase the activity of FXR in ileum to inhibit the levels of BA transporters such as ASBT,and promote the FGF15/FGFR4 pathway,thereby leading to the inhibition of the hepatic BA synthesis and the improvement of cholestasis.For gut,ASP could reduce alcohol-induced apoptosis and intestinal leakage,which might result from the regulation of the alcohol metabolism pathway in ileum and short-chain fatty acid contents in colon,respectively.The absorption and distribution results showed that both the normal mice and AFLD mice showed the significant absorption after oral administration of ASP,and the liver was the main distributing organ.However,the peak time of serum technetium-labeled ASP was delayed in AFLD mice with the significantly increased relative amount.Conclusion: The paper delved into the hepatoprotective effect and its underlying mechanism of ASP in AFLD mice from two aspects of the liver and the “gut-liver axis”,and the absorption and distribution behavior of ASP under the AFLD model was studied.Overall,this paper provides a reference for the potential pharmacological applications and pharmacokinetic research under pathological models of ASP.