The Effect And Mechanism Of Zhikang Capsule On Dextran Sodium Sulfate-induced Colitis In Mice

Aims:To identify the primary chemical compounds in Zhikang Capsule.Methods:Zhikang Capsule weighing 0.4 g was dissolved in 50 ml methanol.All solvents were treated by ultrasonic wave for 40 min and filtered twice through a 0.45 mm filter before analysis.To confirm the authenticities of all components in Zhikang Capsule,a Dionex UltiMate 3000 high performance liquid chromatography system equipped with a photodiode array detector was employed.The chromatographic separation was carried out at a flow rate of 1.0 ml/min on an Agilent TC-C18 column(4.6*150 mm,5 mm)at 30℃.The mobile phase consisted of Methanol(A)and water(B),using a gradient elution of 10-90%A at 0-60 min,then 90%A at 60-75 min.The sample injection volume was 10ul and the detection wave length was 270 nm.Results:By comparing the retention time and UV spectra between the standards and the samples,ten compounds including dencichine,glycyrrhizic acid,imperatorin,berberine,isoimperatorin,rhein,aloe emodin,emodin,chrysophanol,and physcion were identified in Zhikang Capsule.The contents of the above compounds were:0.42 mg/g for dencichine,0.37 mg/g for glycyrrhizic acid,0.041 mg/g for imperatorin,4.58 mg/g for berberine,0.026 mg/g for isoimperatorin,0.40 mg/g for rhein,0.162 mg/g for aloe emodin,0.347 mg/g for emodin,0.617 mg/g for chrysophanol,0.282 mg/g for physcion.Dencichine is extracted from Panax notoginseng(Burk.)F.H.Chen.Glycyrrhizic acid is derived from Glycyrrhiza uralensis Fisch.Imperatorin and isoimperatorin are mainly contributed by Angelica dahurica(Fisch.ex Hoffm.)Benth.et Hook.f.Berberine is derived from Coptis chinensis Franch.Rhein,aloe emodin,emodin,chrysophanol,and physcion are contributed by Rheum palmatum L.Conclusion:Zhikang Capsule mainly contains ten chemical compounds including dencichine,glycyrrhizic acid,imperatorin,berberine,isoimperatorin,rhein,aloe emodin,emodin,chrysophanol,and physcion.Aims:To study the effect of Zhikang Capsule(ZKC)on dextran sodium sulfate(DSS)-induced colitis.Methods:C57BL/6 mice were fed with 3%DSS in drinking water for one week to induce experimental colitis.Mice were randomly divided into 6 groups:(1)control group receiving only tap water(CON group),(2)DSS-treated mice(DSS group),(3)DSS-treated mice receiving sulfasalazine per day(SASP group),(4)DSS-treated mice receiving low dosage of ZKC per day(ZKC-L group),(5)DSS-treated mice receiving middle dosage of ZKC per day(ZKC-M group),(6)DSS-treated mice receiving high dosage of ZKC per day(ZKC-H group).Animals were observed daily for weight changes,diarrhea,and rectal bleeding.The disease activity index(DAI)was determined.The histological changes of colon tissues were observed by hematoxylin and eosin(H&E)staining.The histological activity index(HAI)was determined.Colonic tissues were homogenized in phosphate buffered saline(PBS)and detected by Myeloperoxidase(MPO)assay kits.Results:DSS-induced animals exhibited evident weight loss.The control animals showed no weight loss.Administration of high-dose ZKC significantly protected from obvious body weight loss.We evaluated the severity of weight change,stool condition,and fecal blood based on the DAI score.The DAI score revealed a significant decrease in ZKC-treated mice(ZKC-L:7.40±0.96;ZKC-M:4.70±0.94;ZKC-H:1.50±0.52)compared with DSS model mice(DSS:8.60±0.96).Besides,the colon from DSS mice appeared shorter compared with the colon from the control and ZKC-treated mice.Three dosages of ZKC significantly reduced the degree of colonic shortening in a dose-dependent manner.The colon from SASP group appeared longer in comparison with the DSS group.The potential protective effect of ZKC was further confirmed by examining H&E-stained colon slices.Tissue sections of control group indicated intact surface epithelium,crypt,muscularis mucosa,and submucosa.The DSS-induced group revealed serious inflammatory cell infiltration,large loss of goblet cells as well as crypts,and extensive submucosal edema,indicating a high HAI score(DSS:4.80±1.03)in histological analysis.However,the colon from mice in ZKC-H group showing relatively well histological structure,obtained drastically low HAI scores(ZKC-H:2.30±1.16).Administration of middle dose of ZKC also moderately diminished the HAI score(ZKC-M:3.30±1.49).The MPO value increased significantly during DSS administration.However,ZKC-treated and SASP-treated mice had statistically lower MPO activity compared with the DSS-induced mice.Conclusion:This research demonstrated the protective effect of ZKC on DSS-induced colitis.ZKC can ameliorate inflammation,edema and ulceration in colon and treat DSS-induced colitis in mice.Aims:To explore the underlying mechanisms of Zhikang Capsule(ZKC)on dextran sodium sulfate(DSS)-induced colitis.Methods:C57BL/6 mice were fed with 3%DSS in drinking water for one week to induce experimental colitis.They were randomly assigned to six groups according to the treatment conditions.The histological changes of colon tissues were observed by hematoxylin and eosin(H&E)staining.The serum concentration of pro-inflammatory cytokines(TNF-a,IFN-γ,IL-1β,and IL-12)and anti-inflammatory mediators(IL-4 and IL-10)was detected by enzyme-linked immune sorbent assays(ELISAs).The production of MPO,SOD,MDA,NO,and caspase-3 was assessed by biochemical assay kits.The expression of iNOS,ICAM-1,and NF-KB was evaluated by immunohistochemistry staining.The levels of TLR4,MyD88,and TRAF6 were determined by western blot.Results:ZKC significantly suppressed the pro-inflammatory cytokines(TNF-α,IFN-γ,IL-1β,and IL-12)and promoted the anti-inflammatory mediators(IL-4 and IL-10).The antioxidation of ZKC was indicated by increased activity of SOD and reduced production of MDA,NO,and iNOS in ZKC-treated mice.ZKC can decrease the expression of ICAM-1,and NF-KB.Furthermore,ZKC repressed the colonic expression of caspase-3 and the activity of the MyD 8 8-dependent TLR4 signaling pathway.Conclusion:This research demonstrated the protective effect of ZKC on DSS-induced colitis.For the first time,we identified four therapeutic mechanisms of ZKC,including effective inhibition of the inflammatory responses,significant alleviation of intestinal epithelium apoptosis,considerable prevention of oxidative stress,and selective down-regulation of the MyD88-dependent TLR4 signaling pathway.With high therapeutic effects and low toxic effects,ZKC exhibits great superiority over western medicines in IBD treatment.