The Study On The Mechanism Of Huaihuasan In The Treatment Of Ulcerative Colitis Based On The Network Pharmacology

OBJECTIVE: Network pharmacology combined with in vitro and in vivo experiments were conducted to explore the therapeutic effects and to clarify the mechanism of Sophora San on ulcerative colitis.METHODS: 1.Network pharmacology analysis.the active compounds,targets and related signaling pathways of HHS were enriched and analyzed.TCMSP database was used to search for the compounds in four herbs(Sophora japonica,Platycladus orientalis,Schizonepeta tenuifolia and Fructus aurantii)contained in HHS.The compounds were evaluated and screened according to drug likeness(DL)and oral bioavailability(OB);Pharm Mapper database was used to collect the potential targets of compounds,and Gene Cards database was used to collect and screen the targets affected by UC;Go analysis and KEGG pathway analysis were performed with KOBAS3.0database;The complex "compounds-targets-pathways"(C-T-P)topological network and protein-protein interaction network(PPI)were constructed by using Cytoscape 3.6.1 software and STRING 10.5 database.The binding ability of active compounds with key targets was predicted by molecular docking.2.Experimental studies in vitro.The inflammatory cell model of RAW264.7 was induced by lipopolysaccharide(LPS).The levels of IL-6 and TNF-α in the model cells were detected by ELISA kit.The effects of rutin,quercetin,quercitrin and kaempferol on IL-6 and TNF-α were detected.To verify its anti-inflammatory effect,Preliminary study on the mechanism of action in vitro.Western blot was used to investigate the effect of HHS on the expression of key proteins of EGFR / PI3 K / Akt / HIF-1 pathway in colonic inflammation.3.In vivo validation test.5% DSS solution was used to induce UC mice model.DAI score,H&E staining,myeloperoxidase(MPO)activity,inflammatory factors IL-6,IL-1β and TNF-α were detected.To evaluate the efficacy of HHS,the effect of HHS on EGFR / PI3 K / Akt / HIF-1 / VEGF pathway axis was further studied by Western blotting.RESULTS: 1.Network pharmacology analysis.23 compounds were selected from HHS,239 targets and 450 UC targets were obtained,and there are 97 common targets.The "C-T-P" topology network was constructed,and the top ten potential targets were screened according to degree value: PLA2G2 A,PIK3R1,EGFR,KDR,GSK3 B,ODC1,BACE1,SRC,CA2 and CA1.in PPI,AKT1,KDR,ALB,EGFR,PIK3R1 AND MAPK1 have higher degree value.we considered EGFR,KDR and PIK3R1 as key targets.A total of 185 pathways were obtained by KEGG pathway enrichment,and 58 pathways were screened according to the set value(P < 0.05,count > 8).The signal pathways in the top three were HIF-1,PI3K/AKT and VEGF signaling pathway.The results of the analysis of GO enrichment showed 843 items,including protein transport,fatty acid metabolism,active oxygen metabolism,peptide tyrosine self phosphorylation and other biological processes.The results showed that the docking score of active compounds and key targets EGFR,KDR and PIK3R1 were higher than the threshold,and the docking score of kaempferol,quercetin,quercitrin and rutin was higher.2.Study on anti inflammatory effect in vitro.At a rate of 10μg / m L concentration of LPS induced RAW264.7 cells can form a stable cellular inflammation model.The inflammatory factors were detected by ELISA Kit after the treatment of the cells with HHS for 24 hours in 0.5,1,2,4 mg/m L.The results showed that HHS of different concentrations significantly reduced the inflammatory cytokines IL-6 and TNF-α.The content was determined.Meanwhile,the active components kaempferol,quercetin,quercitrin and rutin were used to treat RAW264.7 inflammatory cells,The contents of IL-6 and TNF-α were significantly reduced.The results of Western blotting showed that HHS inhibited EGFR,P-PI3 K,P-AKT and HIF-1α.The expression of the drug was dose-dependent.3.In vivo validation experimental results indicated that HHS with low(0.52g/kg),middle(1.04g/kg)and high(2.08g/kg)dose could reduce DAI score,increase colon length of UC mice,and significantly reduce MPO activity of colon tissue and the content of inflammatory factors IL-6,TNF-αand IL-1β in UC mice.Western blot showed that HHS could inhibit the expression of EGFR,P-PI3 K,P-AKT,HIF-1α and VEGFR-2.CONCLUSION: HHS and its active compounds can reduce the content of inflammatory cytokines IL-6 and TNF-α in RAW264.7 cells under inflammatory conditions,indicating that HHS has the effect of alleviating colonic inflammation.At the same time,HHS has therapeutic effects on mice with UC.It suggested that HHS may play a therapeutic role in UC by inhibiting the EGFR/PI3K/Akt/HIF-1/VEGF signaling pathway thus playing a therapeutic role for UC and related inflammatory bowel diseases.HHS treatment of UC may be closely related to key targets such as EGFR,KDR,PIK3R1 and their co-expressed proteins.