Effect And Mechanism Of Calculus Bovis Sativus On Acute And Chronic Ulcerative Colitis

Backgrounds:Ulcerative colitis（UC）belongs to inflammatory bowel disease,and is manifested by relapsing intestinal reactions such as abdominal pain,diarrhea and rectal bleeding.Acute explosive UC and chronic recurrent UC are two common types of human UC.The former presents as acute onset with severe illness accompanied by inflammatory reaction,while the latter presents as alternation of attack stage and remission stage.Dextra sulfate sodium（DSS）solution is a classic drug that induces UC model.Acute and chronic animal models,can be established by changing the concentration of DSS and the delivery cycle to mimic the pathogenesis of human UC.Pathogenesis of UC may be related to oxidative stress,inflammatory response and apoptosis,but the molecular mechanism has not been elucidated,and effective therapeutic drugs remains studying.Calculus Bovis（CB）has been proved to have anti-inflammatory,anti-apoptosis,anti-oxidative stress and other pharmacological activities.Calculus Bovis Sativus（CBS）has been approved by CFDA to be an ideal substitute for CB,and may have potential curative effect on UC.Aims:To establish acute and chronic UC model for mimicing acute explosive UC and chronic recurrent UC in human,and explore the effect and possible mechanism of CBS on acute and chronic UC,providing a reference for the pathogenesis and treatment of acute and chronic UC.Methods:（1）In the first part,5%DSS solution was used to establish the acute UC model.Twenty-eight male C57BL/6 mice were randomly divided into four groups（7 mice in each group）:control group,DSS group,CBS 50mg/kg group and CBS 150mg/kg group.Mice in DSS group and CBS group were exposed to 5%DSS in drinking water for 7 days,and mice in control group received only drinking water.At the same time,CBS was given orally at 50 and 150 mg/kg once per day.During the model,body weight,diarrhea and rectal bleeding were recorded every day.After sacrifice in 8^thday,disease activity index（DAI）was evaluated.The colon tissues were removed,and colon length was measured.Subsequently,colonic myeloperoxidase（MPO）activity,superoxide dismutase（SOD）activity,malondialdehyde（MDA）and nitric oxide（NO）levels were assayed.Moreover,mRNA levels of inflammatory cytokines TNF-α,IL-1βand IL-6 were determined by RT-PCR;（2）In the first part,2%DSS solution was used to establish the chronic UC model.Mice in control group received drinking water,and mice in DSS group and CBS group were given three cycles of 5-day 2%DSS solution with two intervals of 10-day drinking water for recovery,totaling 35 days.For the last 7 days of modeling,mice were given CBS(50 or 150 mg·kg^-1per day)intragastrically.Body weight,water intake,diarrhea,and blood in stool of the mice were measured regularly.Mice were sacrificed after fasting,Hematoxylin and eosin（HE）and Periodic Acid-Schiff（PAS）staining were applied to evaluate the damage of colons.TdT-mediated dUTP nick end labeling（TUNEL）assays were performed to evaluate the apoptosis of colonic cells.Then levels of apoptosis-associated proteins,Caspase 3,Bcl-2 and Bax,were determined by western blot.Results:（1）In the acute UC model,CBS evidently reversed the damage caused by DSS,including the weight loss,the increase of DAI,and the shortening of colon.Meanwhile,CBS significantly reduced MPO activity,MDA and NO content in colon tissues,and increased SOD activity.The mRNA levels of inflammatory cytokines such as TNF-α,IL-1βand IL-6 in colitis mice were significantly decreased.These results imply that CBS may play a protective role in DSS-induced acute UC through its antioxidant and anti-inflammatory activities;（2）In the chronic UC model,we found that CBS significantly reduced the pathological characteristics of colitis mice,such as weight loss,increased DAI,and colon injury,and significantly improved the apoptosis rate of colon cells.Furthermore,CBS significantly inhibited the increase of Caspase 3 and Bax,the decrease of Bcl-2and the increase of Bax/Bcl-2 in chronic UC mice.It suggests that CBS may play an anti-apoptotic role in the treatment of chronic UC by regulating apoptosis-related proteins.Conclusions:CBS showed an effective effect on acute UC,and its potential mechanisms may be related to its anti-inflammatory and antioxidant activities.But it has complex components,and the exact targets need to be further studied;CBS may play a protective role in chronic UC through its anti-apoptosis effect,but its material basis is not yet clear,and the specific apoptosis pathway is worth further exploration.