The Effects And Mechanism Of Sinomenine On Experimental Colitis-induced By 2,4,6-trinitrobenzene Sulfonic Acid (TNBS) In Rat

Research background Inflammatory bowel disease(IBD),a significant health problem both in the developed and developing countries,especially in the developed countries,which comprised two chronic relapsing and remitting inflammatory disorders of the gastrointestinal tract,which was a non-specificity inflammatory disease in gastrointestinal tract,mainly included ulcerative colitis(UC) and Crohn's disease(CD). Up to now,little was known about the underlying pathogenesis of IBD,however,it was considered a disease of multifactorial origin reflecting an interplay of genetic, immunological,infection,psychology and environmental factors,which might underlie the initiation and perpetuation of gastrointestinal inflammation.With the heightening of incidence and prevalence in IBD,the difficulty of prevention and therapeutics evoked extensive attention.At present,pharmacologic management of IBD had relied mainly on 5-aminosalicylates,corticosteroids,and immunosuppressants,including purine antimetabolites and cyclosporine.However,corticosteroid dependence was a clinically important problem,and other immunosuppressive agents such as azathioprine and cyclosporine A also exhibited serious side effects.Recently,biological products such as anti-TNF-αand anti-IL-12 antibody had been used with some success in IBD.This approach,however,was also not without side effects,and treatment protocols were complicated and expensive.Therefore,additional studies were warranted to identify new anti-inflammatory agents to treat IBD.Sinomenine was a selective alkaloidal monomer which extracted from sinomenium acutum.Sinomenine possessed anti-inflammatory effects,depressed humoral immunity and cellular immunity,removed nitric oxide free radicals,produced anti-inflammatory effection,and almost without toxic and side-effect,was easily absorbed in intestinal tract.Extensively clinically applying sinomenine in therapeutics of arthritis,and acquired good therapeutic effects. But in the study of IBD,there was almost no report which be concerned with sinomenine.Objective To observe the anti-inflammatory and protective effects of sinomenine on experimental colitis-induced by 2,4,6-trinitrobenzene sulfonic acid(TNBS)/ alcohol in rats,and investigate it's mechanism of action on colitis.In order to supply determinate experimental theory for clinical application of sinomenine in therapeutics of IBDMethods Sixty Sprague-Dawley(SD) rats were randomly divided into six groups (n=10):normal control group(N group),model colitis group(M group),5-aminosalicylic acid treatment group(5-ASA treatment group,C group),High dose sinomenine treatment group(HD group),Middle dose sinomenine treatment group(MD group) and Low dose treatment sinomenine group(LD group).Excepted normal control group,other group were colitis models of rats which were established by intracolon enema with trinitrobenzene sulfonic acid(TNBS)/ ethanol.In the normal control group,no colitis induction was performed,but colitis was induced in all other groups.Followed the induction of colitis,5-ASA and sinomenine were applied to homologous treatment groups of rats.Began to proceeding intragastric administration, after twenty-four hours by establishing colitis models.The each of rats in normal control group and model colitis group with physiological saline(2 mL);the each of rats in 5-ASA treatment group with 5-ASA(100 mg/kg·d);the each of rats in High,Middle and Low sinomenine treatment group with sinomenine in dose of 120,80 and 40 mg/kg·d respectively.Disease activity index(DAI) and colon macroscopic damage index(CMDI) as well as tissues damage index(TDI) of rats in each group were evaluated respectively.The serum levels of IL-1β,TNF-α,MMP-2 as well as MMP-9 were detected by enzyme-linked immunosorbent assay(ELISA).The expressions of MMP-2,MMP-9 as well as pp38MAPK in colonic tissues were detected using immunohistochemical method.Results In normal control group,model colitis group,5-ASA treatment group, High,Middle and Low dose of sinomenine treatment group,the DAI were 0.00±0.00, 3.47±0.48,1.00±0.55,0.93±0.54,1.60±0.81 and 2.43±0.75,respectively;the CMDI were 0.30±0.48,6.50±1.27,1.30±1.16,1.00±0.82,2.30±1.70 and 4.40±2.01 respectively;the TDI were 0.20±0.42,5.10±0.74,1.10±1.20,0.90±0.99,2.80±1.40 and 4.10±1.10 respectively;the serum levels of IL-1βwere 24.39±5.08 pg/mL, 88.48±5.12 pg/mL,35.44±4.31 pg/mL,35.80±3.05 pg/mL,51.77±2.73 pg/mL and 62.20±5.07 pg/mL respectively;the serum levels of TNF-αwere 23.54±4.20 pg/mL, 74.72±5.30 pg/mL,35.37±4.44 pg/mL,32.50±4.91 pg/mL,49.76±4.38 pg/mL and 61.63±7.16pg/ mL respectively;the serum levels of MMP-2 were 0.76±0.44 ng/mL, 10.40±2.10 ng/mL,2.44±0.83 ng/mL,2.50±0.82 ng/mL,3.76±1.44 ng/mL and 5.34±1.07 ng/mL respectively;the serum levels of MMP-9 were 2.83±1.15 ng/mL, 15.85±2.39 ng/mL,5.88±1.84 ng/mL,5.32±1.81 ng/mL,7.69±2.16 ng/mL and 10.14±2.61ng/mL respectively;the expressions of MMP-2 in colonic tissues of rats were 0.60±0.52,5.00±1.41,1.30±1.25,1.00±0.94,2.20±1.03 and 3.50±1.58 respectively;the expressions of MMP-9 in colonic tissues of rats were 0.50±0.53,6.30±0.67,1.50±1.35,1.40±0.97,2.90±1.20 and 4.50±1.43 respectively;the expressions of PP38MAPK in colonic tissues of rats were 1.30±0.48,5.80±1.03,2.10±0.74,2.00±0.82,3.10±1.37 and 4.30±0.95 respectively.Compared with normal control group,all index above-mentioned significantly increased in model colitis group (all P＜0.01).Compared with model colitis group,the DAI,CMDI as well as TDI in 5-ASA treatment group,High and Middle dose of sinomenine treatment group were significantly ameliorated(all P＜0.01);The serum levels of IL-1β,TNF-α,MMP-2 as well as MMP-9 were significantly reduced(all P＜0.01).The expressions of MMP-2, MMP-9 as well as pp38MAPK in colonic tissues of rats in 5-ASA treatment group, High and Middle dose of sinomenine treatment group significantly decreased(all P＜0.01),however,equalitily effects between 5-ASA treatment group and High dose sinomenine treatment group(P＞0.05).Compared with High dose sinomenine treatment group,all index above-mentioned in Low dose sinomenine treatment group significantly increased(all P＜0.01),suggested that the therapeutics of sinomenine on experimental colitis-induced by 2,4,6-trinitrobenzene sulfonic acid(TNBS)/ alcohol in rats presented dose-response relationship.All above results were determined by using one-way analysis of variance(ANOVA) and Student's t test.Conclusions The present results of study indicates that sinomenine can ameliorate the colonic inflammation,reduce the serum levels of IL-1β,TNF-α,MMP-2 and MMP-9, and decrease the expression of MMP-2,MMP-9,pp38MAPK.It's mechanism of anti-inflammatory effects is possiblely related with reducing the expression of cytokine, MMP-2,MMP-9,as well as pp38MAPK,alleviating epithelial cells cytoclasis,and protecting intestines mucous membrane barrier.