Study On The Therapeutic Effect Of Folk Unilateral X On Dextran Sodium Sulfate Induced Ulcerative Colitis In Mice

Objective: To verify the therapeutic effect of folk unilateral X on inflammatory bowel disease;to explore the effect and possible mechanism of active fraction of X medicinal materialon dextran sodium sulfate(DSS)-induced ulcerative colitis(UC)in mice.Methods: The UC model was established by the chemical induction of DSS.The UC mouse model was employed to discover the active fraction of the X medicinal material.After one week of adaptive feeding,54 C57BL/6 mice were randomly divided into the normal control group(Control-S),model group(DSS-S)(mass fraction 2.5% DSS),positive control group of 5-aminosalicylic acid(5-ASA-S),raw material drug group,decoction group,methanol extract group,chloroform extract group,ethyl acetate extract group and water extract group with 6 mice in each group;Based on the change of body weight of mice,disease activity index(DAI)score,and pathological and inflammatory factors,the therapeutic evaluationeffect of different experimental groups on UC mice was completely assessed,Subsequently,the dose-effect relationship and possible mechanism of active fraction chloroform extract(CE)was further investigated.After one week of adaptive feeding,Forty-eight C57BL/6 mice were randomly divided into normal control group(Control),model group(DSS)(mass fraction 2.5% DSS),positive drug group(5-ASA,75 mg/kg +DSS),CE low-dose group(30 mg/kg + DSS),CE medium-dose group(60 mg/kg +DSS),and CE high-dose group(90 mg/kg + DSS)with 8 mice in each group,and they were administered by gavage for 8 consecutive days.The general observations included monitoring mouse activity,defecation,and weighing regularly during testing,and these indices was used to calculate the DAI score.After stopping the administration,the colon length and spleen weight was measured;HE staining was used to observe the pathological changes in liver and kidney tissues of mice;The degree of liver fibrosis in mice was detected using Masson staining;ELISA was used to detect the level of TNF-?,IL-6 and IL-1? in the plasma;Colorimetric method was used to detect the content of myeloperoxidase(MPO)in the colon.The transcription levels of TNF-?,IL-1?,IL-6,mucins(mucin-1 and mucin-2),and apoptosis-related genes(Bax and Bcl-2)in mouse colon tissue were detected by RT-q PCR,Western blotting was used to detect the expression of tight junction-associated proteins(ZO-1,ZO-2,Claudin-1 and Occludin)and apoptosis-related proteins(Bax and Bcl-2).TUNEL method was used to detect the apoptosis of intestinal epithelial cells in mouse colon tissue;The GC-MS combined technology was used to analyze the chloroform extract.Results: The therapeutic effect of different parts from X medicinal material showed that the CE was the best active fraction against UC mouse model.Furthermore,CE at different concentrations(30 mg/kg,60 mg/kg,and 90 mg/kg)showed similar to the therapeutic effect of the positive drug 5-ASA(75 mg/kg).CE significantly alleviated symptoms of UC,including inhibiting weight loss,extending the length of the colon,lowering the DAI score,inhibiting splenomegaly,and reducing colon tissue damage.Meanwhile,similar to 5-ASA,CE could significantly decrease the levels of pro-inflammatory factors such as TNF-?(p <0.05),IL-1?(p <0.01)and IL-6(p <0.01)in plasma and colon tissue cells,reduce inflammatory cell infiltration,and effectively inhibit MPO content.In addition,90 mg/kg CE showed manifold inhibitory capacity on the expression of IL-1? and IL-6 than 5-ASA at the level of transcription.On the basis of the the pathological changes of mouse liver and kidney,it was found that CE did not damage to the liver and kidney of mice by HE and Masson stainings.Further research showed that the CE-administered group(60,90 mg/kg)significantlyreduceed the expression of tight junction proteins ZO-1(p < 0.05),ZO-2(p < 0.01),Claudin-1(p < 0.01),and Occludin(p < 0.05),and the transcription levels of mucin-1(p < 0.01)and mucin-2(p < 0.01)in comprision to the DSS group,and the inhibitory ability was even better than 5-ASA.In addition,TUNEL analysis results indicated that CE could reduce DSS-induced cellular apoptosis in colon tissue.At the level of transcription and translation,CE significantly down-regulated the pro-apoptotic factor Bax and up-regulated anti-apoptotic factor Bcl-2,and showed more potent regulatory capacity than 5-ASA,especially in the medium and high-dose groups.CE has been preliminarily isolated and identified 19 known compounds,of which Pulegone,Lupeol and Heptadecane have been reported to have anti-inflammatory effects.Conclusion: Folk unilateral X shows obvious therapeutic effect against DSS-induced mouse UC.The CE was the best active fraction in the X medicinal material.Pulegone,Lupeol,Heptadecane,5-hydroxymethylfurfural,4-di-tert-butylphenol,and 4-vinyl-2-methoxy-phenol are important components of anti-inflammatory activity.The underlying mechanism of CE against UC is closely related to reducing intestinal inflammation,inhibiting intestinal epithelial cell apoptosis,and maintaining the integrity of the intestinal mucosal barrier.