| Objective:Non alcoholic steatohepatitis(NASH)has become an important cause of chronic liver disease worldwide,as obesity and related metabolic syndromes are becoming an epidemic trend,which has brought a serious impact and economic burden to the society.Preliminary studies have found that Pueraria lobata possesses hepatoprotective and anti-inflammatory bioactivities,but the pharmacodynamic.Previous studies have found that Pueraria lobata has hepatoprotective and anti-inflammatory biological activities,but its pharmacodynamic basis and mechanism of action are not yet completely clear,which has hindered its application.Therefore,this experiment will explore the pharmacodynamic substances and molecular mechanisms of Pueraria lobata in interfering with NASH,and lay the foundation for the development of drugs to treat NASH.Methods:(1)Extraction,isolation and purification of the chemical components of Pueraria lobata were used silica gel column chromatography and preparative thin layer chromatography.(2)Screening of the main active components of Pueraria lobata against NASH by determining the changes in TG and TC content of each chemical component in Pueraria lobata on FFA-induced Hepg2 cell model using a kit and confirming the results using web-based pharmacological analyses.(3)Evaluation of the pharmacodynamic effect of puerarin on the improvement of NASH in a zebrafish model using the changes in the content of TG,TC,AST,and ALT,the oil-red O staining,and HE staining assays.(4)Evaluate the effect of puerarin on macrophage numbers using macrophage transgenic fluorescent zebrafish.(5)Use Clod-lipo clearance reagent to clear macrophages in mice,and evaluate the clearance efficiency using flow cytometry experiments.(6)Detect the effects of puerarin on M1 and M2 by using Western Blot,immunofluorescence,flow cytometry and q RT-PCR experiments.Detect the effect of puerarin on the level of polarization protein in M1 and M2 type macrophages.(7)Use transcriptomics to explore the target and signaling pathway of puerarin to intervene in macrophage polarization.(8)Use si RNA knockdown and plasmid overexpression experiments to verify the target and signaling pathway.(9)Use Western Blot to detect the effect of puerarin on the level of autophagy protein in macrophages.(10)Western Blot assay was used to detect the changes in polarized protein levels after activation or inhibition of macrophage autophagic flow.(11)si RNA knockdown and immunoprecipitation assays were used to detect the relationship between ULK1 and PAI-1.(12)In vivo experiments were used to validate the molecular mechanism of puerarin in interfering with NASH.Results:(1)A total of 20 compounds were isolated from Pueraria lobata,among which 8 compounds were isolated for the first time from Pueraria lobata:Linolei cacid,Sandwicensin,Isovanillin,Ethyl ferulate,Isopterofuran,Haginin A,3’,7-Dihydroxyisoflavan,3-2′,4′-Dihydroxyphenyl-4,7-dihydroxy-2H-1-benzopyran-2-one;the other compounds are:Lupenone,Lupeol,β-sitosterol,Medicarpin,Coniferyl aldehyde,Genistein,Ayapin,Syringaldehyde,4’,7-Dihydroxy-3’-methoxyisoflavone,Formononetin,Daidzein,Puerarin.(2)The detection of the kit revealed that Puerarin was able to significantly reduce the TG and TC contents in the FFA-induced Hepg2 cell model,indicating that Puerarin is the main active ingredient of Puerarin to improve NASH.(3)The results of in vitro experiments and zebrafish experiments showed that Puerarin was able to reduce the contents of TG,TC,AST,and ALT in the FFA-induced Hepg2 cell model,and was also able to reduce the lipid droplet Accumulation.(4)The results of in vitro experiments and zebrafish experiments demonstrated that puerarin was able to reduce FFA-induced TG,TC,AST,and ALT content and.(5)After using Puerarin to treat macrophage transgenic fluorescent zebrafish,it was found that Puerarin was able to significantly reduce the number of macrophages in the inflammation area.(6)Clod-lipo removal reagent was able to effectively remove the macrophages in the mice,but when the macrophages were removed from the mice,there was no significant reduction in the number of red lipid droplets in the mice’s liver area,and the degree of structural abnormality in the liver tissue did not change,the area of blue collagen did not change,and the area of blue collagen did not change.changes,the blue collagen area was not significantly weakened,and the symptoms of NASH were not improved,suggesting that Puerarin can treat NASH by regulating macrophages.(7)Puerarin can significantly reduce the release of NO,IL-6,and IL-1β,and elevate the release of IL-10in LPS+IFN-γ-induced RAW264.7 cells,suggesting that Puerarin can reduce the level of inflammatory factors and it has anti-inflammatory effects.(8)The proportion of CD86~+and i NOS~+M1-type macrophages decreased while the proportion of CD206~+and Arg1~+M2-type macrophages increased after treatment with puerarin,indicating that puerarin was able to transform M1-phenotype macrophages into M2-phenotype macrophages.(9)Transcriptomics and in vitro and in vivo experiments showed that puerarin could inhibit the Stat3/Hif-1αsignaling pathway to promote to the decrease of M1-type macrophages and activate the PI3K/AKT signaling pathway to promote to the increase of M2-type macrophages by PAI-1.(10)Puerarin was able to elevate the protein expression level of autophagy cell-associated markers LCII/LCI and decrease the protein expression level of P62,suggesting that puerarin was able to promote macrophage autophagy,thereby attenuating the inflammation induced by LPS+IFN-γ.In addition,the expression of p-AMPK/AMPK and p-ULK1(Ser757)/ULK1 was up-regulated and that of p-m TOR/m TOR was down-regulated after Puerarin treatment,suggesting that Puerarin may inhibit the activity of m TOR by promoting the activation of AMPK,reduce the binding of m TOR to the Ser757 site of ULK1,and thus promote the phosphorylation of Ser757-ULK1 and promote autophagy.(11)After macrophage autophagic flow was inhibited,the number of M1-type macrophages increased and the number of M2-type macrophages decreased,and after macrophage autophagic flow was activated,the number of M1-type macrophages decreased and the number of M2-type macrophages increased,suggesting that Puerarin can exert anti-inflammatory effects by regulating macrophage autophagy-mediated polarization.(12)After p-ULK1 was overexpressed,the PAI-1 expression level decreased,while CD86protein level decreased and CD206 protein level increased,suggesting that p-ULK1 can regulate the expression of PAI-1,which in turn regulates the ratio of M1-type and M2-type macrophages.However,immunoprecipitation experiments showed that there was no interaction between p-ULK1 and PAI-1,which may function through indirect regulation.Conclusion:Puerarin is the main active ingredient of Pueraria lobata to intervene in NASH,and its molecular mechanism is that puerarin can activate macrophage autophagy by regulating the AMPK-m TOR-ULK1 signaling pathway,and the phosphorylation of ULK1can inhibit the activity of PAI-1,and then inhibit the Stat3/Hif-1αsignaling pathway to promote the reduction of M1-type macrophages and activate the PI3K/AKT pathway to promote the increase of M2-type macrophages,leading to the reduction of pro-inflammatory factors and the increase of anti-inflammatory factors,thus exerting pharmacological effects to improve NASH. |
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