Study On The Mechanism Of Astragalus Polysaccharide Against Alcoholic Liver Injury Based On Network Pharmacology

Objective: Alcoholic liver injury is a liver injury disease caused by long-term or shortterm excessive intake of alcohol in the body,under the influence of ethanol and ethanol metabolites.The initial manifestation is fatty liver,and then it is transformed into liver fibrosis.Liver cirrhosis or liver cancer may occur after the stage of liver fibrosis.Early intervention is very important for blocking the development of the disease and delaying the course of the disease.Astragalus has many pharmacological properties,such as inhibiting tumor growth,protecting cardio-cerebrovascular,improving immune function and protecting lung,kidney and liver tissue.In particular,the main active ingredient Astragalus polysaccharide has shown a protective effect on liver injury,but the underlying molecular mechanism is still unclear.This study aims to predict the mechanism of Astragalus polysaccharide on alcoholic liver injury by using network pharmacological methods and molecular docking technology,and establish an animal model to evaluate the effect of Astragalus polysaccharide on alcohol-induced liver injury.Methods: First,the active ingredients and potential targets of astragalus in the treatment of alcoholic liver injury were predicted by network pharmacology.Then,the SD rat model of alcoholic liver injury was established by intragastric administration of 50% alcohol on rats,which were randomly divided into control group,1-week group,2-week group and4-week group.We detected the effect of alcohol on the expression of PTRF and TLR4 in rat liver tissue by immunofluorescence staining.The expression levels of PTRF,TLR4,p-JNK1/2,NF-κB p65 and My D88 were detected by western blotting.The levels of IL-1β and TNF-α were determined by enzyme-linked immunosorbent assay,and the effect of alcohol treatment on hepatic fibrosis was observed by Masson staining.In addition,after the modeling method was determined,the rats were randomly divided into control group,alcoholic liver injury group,APS low-dose group(200 mg/kg),APS high-dose group(400 mg/kg),AAV9-NC group,AAV9-PTRF overexpression group,and the expression of PTRF and TLR4 were detected by immunofluorescence staining.The changes of PTRF,TLR4,p-JNK1/2,NF-κB p65 and My D88 in rat liver were detected by western blotting.Masson staining was used to observe the effect of APS on hepatic fibrosis.Immunohistochemical staining was used to detect the expression of PTRF in AAV9-NC group and AAV9-PTRF overexpression rats,and HE staining was used to evaluate the histopathologic changes of liver in AAV9-NC group and AAV9-PTRF overexpression rats.Results: Compared with the control group,the levels of serum IL-1β and TNF-α were significantly up-regulated after 50% alcohol administration,and the expressions of TLR4,PTRF,P-JNK1/2,NF-κB p65 and My D88 were significantly up-regulated in 1 week group and 2-week group(P<0.05).The expressions of TLR4,PTRF,P-JNK1/2,NF-κB p65 and My D88 in the liver of rats in the 2-week group were higher than those in the 1-week group(P<0.05),and the immunofluorescence results showed that the expressions of PTRF and TLR4 in the 2-week group were higher than those in the 1-week group.After alcohol treatment,liver fibrosis injury of different degrees was observed in the 1-week group,the 2-week group and the 4-week group.After Astragalus polysaccharide administration(400 mg/kg),alcohol-induced liver fibrosis was reversed,especially in the2-week group.After administration of Astragalus polysaccharide(200 mg/kg,400 mg/kg),the protein expression levels of TLR4,PTRF,p-JNK1/2,NF-κB p65 and My D88 were down-regulated,and the immunofluorescence expressions of PTRF and TLR4 were reduced.Adenovirus-packed plasmids were injected into caudal vein for PTRF overexpression in rats.After 4 weeks,PTRF was stably overexpressed in both AAV9-NC group and AAV9-PTRF overexpression group.Astragalus polysaccharide treatment fully inhibited the expression of PTRF in AAV9-PTRF overexpression group and AAV9-NC group.HE staining showed that astragalus polysaccharide treatment group significantly reduced the degree of liver injury.In addition,liver damage was more severe in the alcohol /AAV9-PTRF overexpression group than in the alcohol /AAV9-NC group,which was improved by high dose of astragalus polysaccharide(400 mg/kg).Conclusion: APS may reduce alcohol-induced liver injury by inhibiting the expression of PTRF and TLR4-JNK/NF-κB/My D88 pathway genes,providing a scientific basis for elucidating the mechanism of APS anti-injury activity and a promising new therapeutic method for the treatment of liver injury.