Pharmacodynamic Evaluation And Mechanism Of Hederasaponin C In Anti-inflammatory Bowel Disease

Inflammatory bowel disease(IBD),a chronic disease characterized by intestinal inflammation and tissue damage,including two types: ulcerative colitis(UC)and crohn’s disease(CD).The clinical symptoms of IBD are as follows: diarrhoea,abdominal pain and hemafecia,causeinggreat pain to patients.But its pathogenesis isunclear.Pulsatilla chinensis,a traditional Chinese medicine with a long history,exhibits “blood-cooling” and detoxification activities.It has been extensively used for adjunctive treatment of intestinal amebiasis,malaria,vaginal trichomoniasis,bacterial infections and malignant tumors.Hederasaponin C(HSC)is one of the main components of Pulsatilla chinensis,accounting for 1 %.However,there are few studies on pharmacological activit of HSC and no reports on its therapeutic effect on IBD.In this study,UC and CD models were established to evaluate the anti-inflammatory bowel disease effect of HSC in vivo and elucidate its mechanism,providing lead compounds or candidate drug molecules for the development of new IBD therapeutic drugs.1.Inhibitory effect of HSC on LPS-induced inflammatory response.HSC inhibits LPS-induced expression of inflammatory cytokines in RAW264.7 cell: ELISA and RT-PCR were used to detect the difference of gene expression of inflammatory cytokines.After LPS stimulation,the expression of IL-6 and TNF-α in cells increased significantly,and HSC could significantly reduce the expression of inflammatory factors,which showed the dose-dependent manner.HSC inhibits LPS-induced activation of NF-κB in RAW264.7 cell: Western blot and immunofluorescence analysis were used to study the effect of HSC on activation of NF-κB phathway.The results showed that HSC could decrease the expression of p-IKKa/β in RAW264.7 cell induced by LPS in a dose-dependent manner,increase the expression of IκBα,and significantly inhibit the nuclear tanslacationof NF-κB.HSC inhibits LPS-induced TLR4 dimerization: HA-TLR4 and FLAG-TLR4 were transfected into HEK293 T cells simultaneously.The effect of HSC on TLR4 dimerization was studied by immunoprecipitation(IP).The results showed that HSC could inhibit LPS-induced receptor dimerization.2.Inhibitory effect of HSC on inflammatory bowel disease in ratsIn this study,we firstly established a rat model of UC induced by acetic acid and TNBS-induced CD,evaluating the therapeutic activity of HSC for IBD.The results showed that HSC(2.5 mg/kg,5 mg/kg,intraperitoneal injection)or mesalazine(200 mg/kg,oral administration)could significantly inhibit the weight loss rate,improve the DAI score and reduce the intestinal mass coefficient of the model animals,which was significantly different from that of the model group.Colon histopathological sections showed that ulcer formation and inflammatory cell infiltration were observed in the model group.There were more neovascularization,blood vessel congestion and expansion,and erythrocyte exudation in model group.Inflammatory cell infiltration was reduced in HSC group and mesalazine group,goblet cell number was relatively increased,no tissue edema and congestion was found in mucosal muscle layer,and the structure of colon was obvious.The colon recovery effect of HSC group was similar to that of mesalazine group.3.Preliminary safety evaluation of HSCHSC incubated with rabbit red cell suspension for 3 hours without obvious hemolysis.It showed that HSC had no hemolytic activity.MTT assay was used to detect the effect of HSC on cell survival rate.It was found that HSC had no effect on the proliferation of RAW264.7,HT-29 and HEK293 T cells.ICR mice were injected with HSC(5 mg/kg)intraperitoneally.The occurrence of behavioral abnormalities and toxicity in HSC mice was observed.There was no difference in body weight between HSC and normal group.In conclusion,HSC can inhibit the dimerization of TLR4,block the signal transduction of TLR4/NF-κB,down-regulate the expression of inflammatory factors IL-6 and TNF-a,and play an anti-inflammatory role.HSC has an inhibitory effect on colitis in vivo,showing significant anti-inflammatory bowel disease activity.This study provides a candidate drug molecule for treatment of IBD.