Study On The Mechanism Of Kangxian Pill In The Treatment Of Chronic Liver Injury Based On Network Pharmacology

ObjectThis study screened the active ingredients and its key targets and predicted pathways of Kangxian Pill for the treatment of chronic liver injury based on network pharmacology.In addition,we established in vivo experiment to further validate the effects and mechanisms of Kangxian Pill for the treatment of chronic liver injury,providing a molecular biological basis for the clinical application of Kangxian Pill.MethodsEfficacy evaluation:BALB/c mice were injected intraperitoneally with 20%CCL₄ olive oil solution（1:4 v/v）to establish mice model of chronic liver injury,while normal mice were injected intraperitoneally with an equal volume of olive oil.After mice models with chronic liver injury were successfully established,all mice models were divided into Model group,diammonium glycyrrhizate group（positive group）（60mg/kg）and Kangxian Pill group（3g/kg）.After 4 weeks of administration,the therapeutic effect of KXP in the treatment of CCL₄-induced chronic hepatic injury is investigated by calculating liver index,measuring transaminase levels as well as detecting the content of SOD,GSH-Px and MDA.In addition,we performed HE staining and Masson staining to observe the histopathological changes and the aggregation of collagen fibers as well as conducted immunohistochemistry to observe the expression ofα-SMA in liver tissues.Target prediction:To predict the potential targets of Kangxian Pill for the treatment of chronic hepatic injury using network pharmacology,the following steps were performed:（1）To find the active ingredients and related targets of Kangxian Pill using the TCMSP and CRDCAS databases.（2）In order to collect the disease targets,we searched in Dis Ge NET database using the keyword‘chemically-induced liver toxicity’as well as searched in Online Mendelian Inheritance in Man（OMIM）database and Gene Cards database with the keyword‘chronic hepatic injury’.（3）In the Venny database,potential targets of Kangxian Pill in the treatment of CHI were obtained intersection of disease targets and medicines targets.（4）The GO function enrichment analysis,and representative pathways related to Kangxian Pill in the treatment of CHI were analyzed using the Metascape database.（5）Networks of Component-Target,Protein-protein interaction and Target-Pathway were performed based on the core targets for further analysis.In vivo experiment validation:q PCR was performed to validated Bax,Bcl2 and Caspase-3gene expression level in liver tissues;Protein expression levels of NRF2,Nuclear NRF2,HO-1,GCLC,NQO1,Bax,Bcl2,Caspase-3,PI3K,Akt and p-Akt in liver tissues was performed by Western-blot according to the potential molecular mechanisms analyzed based on network pharmacology analysis.ResultsThe results showed that the mice with chronic liver injury showed significantly higher levels of aminotransferase,decreased the content of MDA and increased the activity of SOD and GSH-Px,disturbed liver tissue structure,extensive edema and partial necrosis of hepatocytes,aggregation of collagen fibers and up-regulation ofα-SMA expression in liver tissue compared with the Normal group.After treatment with KXP,the results showed that the levels of aminotransferase decreased,content of MDA increased and the activity of SOD and GSH-Px decreased,the liver tissues structure normalized,collagen fibers dereased and the expression ofα-SMA down-regulated.These results showed that Kangxian Pill has a significant therapeutic effect on the mice with CCL₄-induced chronic liver injury.Based on network pharmacology to predict the targets of Kangxian Pill for chronic liver injury,a total of 218 active ingredients and 385 targets of Kangxian Pill were obtained;1088disease targets of chronic liver injury were obtained,and 165 potential targets of Kangxian Pill for chronic liver injury were obtained after taking the intersection,and the drug-compound-potential target network was constructed.Based on the‘Degree’value greater than 33,we screened 81 potential targets of Kangxian Pill for the treatment of chronic liver injury and conducted PPI network.GO functional enrichment analysis of 81 potential targets revealed that Kangxian Pill may treat chronic liver injury through oxidative stress and apoptosis.KEGG pathway enrichment analysis and’target-pathway’network analysis revealed that the mechanism of Kangxian Pill for chronic liver injury is closely related to PI3K/Akt.The results of in vivo experiments showed that the gene expression of Bax and Caspase-3were significantly upregulated in the liver tissues of mice in the model group,while the gene expression of Bcl2 was significantly downregulated;meanwhile,the protein expression of PI3K,p-Akt,NRF2,Nuclear NRF2,GCLC and NQO1 were down-regulated,while the protein expression of Bax and Caspase-3 were more significantly up-regulated,and the difference was significant compared with the normal group.In contrast,the administration of Kangxian Pill reversed the abnormal expression of genes and proteins in mice with chronic liver injury to varying degrees.ConclusionOur results suggest that Kangxian Pill could regulat PI3K/Akt signaling pathway and inhibit oxidative stress and apoptosis to alleviate chronic liver injury based on network pharmacology predictions.