Study On The Mechanism Of Jianpi Qingchang Decoction Regulating NF-κB/HIF-1α Signalling Pathway In The Treatment Of Ulcerative Colitis

Objective: To explore the potential pharmacodynamic mechanism of Jianpi Qingchang decoction in the treatment of ulcerative colitis,which enriching the scientific connotation of traditional Chinese medicine on ulcerative colitis,and providing a new theoretical basis for its clinical application.Methods: 1.The murine model of colitis was induced by DSS and Jianpi Qingchang decoction was given intragastrically.The disease activity index scores used to calculate the severity of colitis daily,then the body weight and colon length were observed.Histopathological changes of colon were assessed by hematoxylin-eosin staining and immunohistochemistry.The levels of TNF-a and IL-1β in serum were evaluated through ELISA.The m RNA expression of IL-1β,TNF-a and IL-10 in colon were detected by RTPCR.2.Systematic pharmacology approach was used to systematically explore the potential pharmacodynamic mechanism of Jianpi Qingchang decoction on ulcerative colitis using a holistic process of active compound screening of Chinese herbal medicine by relevant databases,target matching,network construction and analysis as well as enrichment analysis of potential pharmacodynamic targets.3.Bone marrow-derived macrophages was isolated from mice.In addition,to better simulate the physiological environment in vivo,Caco2 cells were co-cultured with bone marrow-derived macrophages.Cells were incubated with TNF-α alone or together with Jianpi Qingchang decoction or positive control drugs pyrrolidinedithiocarbamate and rapamycin.The m RNA expression of IL-1β,TNF-a and IL-10 were detected by RT-PCR.The protein and m RNA expression of HIF-1a,NF-κB and m TOR as well as upstream events TRAF2,IκB,PI3 K and Akt were detected by Western blot,RT-PCR and immunofluorescence staining.The expression of tight junction proteins Claudin-1 and ZO-1 were detected by Western blot and immunofluorescence staining in Caco2 cells.4.The protein and m RNA expression of HIF-1a,NF-κB and m TOR as well as upstream events TRAF2,IκB,PI3 K and Akt in colon were detected by Western blot,RTPCR and immunofluorescence staining.The expression of tight junction proteins Claudin-1 and ZO-1 in colon were detected by Western blot and immunofluorescence staining.Results: 1.The DSS-induced colitis model were successfully established.Jianpi Qingchang decoction could improve the general obseravation and reduce the pathological changes of inflammatory cell infiltration,crypt abscess and ulcer in the model mice.The expression of TNF-a and IL-1β in serum as well as TNF-a,IL-1β and IL-10 in colon were reduced by Jianpi Qingchang decoction.2.Systematic pharmacology predicted 107 candidate active ingredients in Jianpi Qingchang decoction and 170 potential active ingredients targets.Enrichment analyses showed that the pharmacodynamic mechanisms of Jianpi Qingchang decoction against ulcerative colitis contained 652 putative targets,mainly involving the metabolic processes related to the NF-κB and m TOR signalling pathways.3.The stimulation of TNF-a induced the increase of IL-1β,TNF-a,IL-10 m RNA expression level as well as HIF-1a,NF-κB,m TOR,TRAF2,IκB,PI3 K and Akt m RNA and protein expression level,while Jianpi Qingchang decoction reversed the situation,which suggested that Jianpi Qingchang decoction could inhibited the inflammatory response stimulated by TNF-a via NF-κB/HIF-1α signalling pathway in bone marrowderived macrophages.The stimulation of TNF-a induced the increase of IL-1β,TNF-a,IL-10 m RNA expression level as well as HIF-1a,NF-κB m RNA and protein expression level,whereas down-regulated the expression of Claudin-1 and ZO-1,while Jianpi Qingchang decoction reversed the situation,which suggested that Jianpi Qingchang decoction could improved the inflammation activation and intestinal epithelial barrier defect stimulated by TNF-a via NF-κB/HIF-1α signalling pathway in Caco2 cells.4.DSS induced the increased expression of HIF-1α,NF-κB,m TOR,TRAF2,IκB and Akt,as well as down-regulated the expression of Claudin-1 and ZO-1,while Jianpi Qingchang decoction reversed the situation,which suggested that Jianpi Qingchang decoction could improved the inflammation activation and intestinal epithelial barrier defect via NF-κB/HIF-1α signalling pathway in DSS induced colitis.Conclusions: 1.The potential pharmacodynamic mechanisms of Jianpi Qingchang decoction against ulcerative colitis mainly involving the NF-κB and m TOR signalling pathways.2.Jianpi Qingchang decoction could improve the inflammatory response and permeability of inflammatory model cells induced by TNF-α via NF-κB/HIF-1α signalling pathway.3.Jianpi Qingchang decoction could improve the inflammatory response and intestinal epithelial barrier dysfunction of colitis model mice induced by DSS via NF-κB/HIF-1α signalling pathway.