Investigation Of Anti-Inflammatory Mechanisms Of Taurolithocholic Acid Through Network Pharmacology-based Analysis And Experimental Validation

Aims: The present study was to explore the anti-inflammatory effect and possible mechanisms of taurolithocholic acid(TLCA)through network pharmacology-based analysis,and to verify with in vitro macrophage study.Method: The molecular structure and potential targets of TLCA were acquired from public database,and then the protein-protein interaction(PPI)networks against inflammation were constructed from STRING database and visualized by using Cytoscape.Gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment were performed.The binding activity of TLCA and its target(TGR5)was evaluated through molecular docking analysis.Lastly,RAW264.7 cells were divided into control group,taurolithocholic acid(TLCA,30 (?)M)group,lipopolysaccharide(LPS,100 ng/ m L)and interferon-γ(IFN-γ,20 ng/m L)group,and taurolithocholic acid complex lipopolysaccharide and interferon-γ group,and cultured for 24 h to verify the network pharmacological analysis results.The m RNA expression levels of M1 and M2 polarization related markers IL-1β,IL-6,TNF-α,i NOS,Arg1 and IL-10 in RAW264.7 cells were detected by RT-q PCR.The expression levels of CD86 on cell membrane were detected by immunofluorescence.The protein expressions of P65,phosphorylated P65(P-P65),IκBα,phosphorylated IκBα(P-IκBα)and TGR5 in RAW264.7 cells were detected by Western blot.Results: There were 87 anti-inflammatory potential targets were screened by matching 232 targets of TLCA with 1565 inflammation targets.The core targets were SRC,EGFR,MAPK3,PIK3 CA,JAK2,IL10,MMP2,KDR and ESR1.GO analysis revealed gene functions were mainly involved in biological process(regulation of inflammatory response),cellular components(membrane raft)and molecular functions(protein tyrosine kinase).The results of KEGG pathway analysis suggest that PI3K-Akt signaling pathway,human cytomegalovirus infection,proteoglycans in cancer might be the critical pathway of TLCA against inflammation.The m RNA expressions of IL-1β,IL-6,TNF-α,i NOS,Arg1 and IL-10(P<0.01)were significantly increased by lipopolysaccharide and interferon-γ in RAW264.7 cells,the protein expression of CD86,P-P65 and P-IκBα(P<0.05)were increased,while the m RNA expression levels of IL-6,TNF-α and i NOS were decreased after administration of taurocholic acid(P<0.05),the expression of IL-1β was unchanged,the expression of IL-10 and Arg1 were decreased,but there was no statistical difference,the protein expression of CD86,P-P65 and P-IκBαwere decreased(P<0.05),furthermore,the expression of TGR5 protein was increased after TLCA treatment.Conclusion: This study revealed the pharmacological mechanism of taurolithocholic acid,obtained some potential therapeutic targets related to inflammation,and selected the TGR5 target for in vitro experimental verification.It shows that TLCA may inhibit the inflammatory response of RAW 264.7 macrophages induced by lipopolysaccharide and interferon-γ through the TGR5 receptor,which provides a basis for the development of new drugs for the treatment of inflammation-related diseases.