Therapeutic Intervention And Potential Mechanisms Of Calycosin In The Mouse Models With Ulcerative Colitis

Objective:Ulcerative colitis is a kind of inflammatory disease of the intestine caused by abnormal immunity,Calycosin has the biological activity of regulating immunity and anti inflammation.This study was to investigate the therapeutic effect of Calycosin in the mice models with ulcerative colitis and the potential mechanisms.Methods:For in vitro model,the hemolysis rate of Calycosin with various concentrations was tested with red blood cells,Raw264.7 cells proliferation was also evaluated after the stimulation in the culture by Calysosin.Accordingly,the dose of Calycosin for its anti-inflammatory effect in vitro was determined.The effect of Calycosin on lipopolysaccharides(LPS)-induced inflammatory genes mRNA expression in Raw264.7 cells was measured by real-time polymerase chain reaction(RT-PCR).Influence of Calycosin on inflammatory cytokine production in Raw264.7 cells was determined by enzyme linked immunosorbent assay(ELISA).The effect of Calycosin on LPS-induced reactive oxygen species(ROS)production in Raw264.7 cells was determined by flow cytometry.Western blot assay was adopted to determine the effect of Calycosin on NF-k B and MAPK pathway activation in LPS-inducedRaw264.7 cells.For in vivo models,Balb/c male mice were used to evaluate the effect of Calycosin on experimental ulcerative colitis.Thirty-five mice were divided into five groups(7 mice per group).To establish dextran sodium sulfate(DSS)models,mice were administered with 2.5%DSS dissolved in drinking water ad libitum for 7 days,followed by normal water for 3 days to induce experimental colitis.Normal mice treated with DSS-free drinking water were administered with 0.5%carboxymethylcellulose sodium(CMC-Na)solution by gavage as negative control.For DSS-induced colitis groups,mice were respectively administered with0.5%CMC-Na,25mg/kg Calycosin suspended in 0.5%CMC-Na,or 50mg/kg Calycosin suspended in 0.5%CMC-Na once daily from day 1st to day 10th;meanwhile administration of 50mg/kg 5-aminosalicylic acid(5-ASA)suspended in0.5% CMC-Na for 10 days was used as positive control.To establish trinitrobenzenesulfonic acid solution(TNBS)models,mice were administered with100 ?l 2.5%TNBS and 50% ethanol solution through the soft catheter(which was pre-inserted from the anus of mice)to induce experimental colitis.Normal mice were treated with only 100 ?l 50% ethanol solution as negative control.From day 1st on,the control mice were administered with 0.5%CMC-Na solution by gavage.For TNBS-induced colitis groups,mice were treated respectively with 0.5%CMC-Na,25mg/kg Calycosin suspended in 0.5%CMC-Na,or 50mg/kg Calycosin suspended in0.5%CMC-Na once daily from day 1st to day 3rd.At the same time,treatment with50mg/kg 5-ASA suspended in 0.5%CMC-Na was as positive control.Body weights of all mice were measured and disease activity index(DAI)were evaluated every day.After DSS induction for 10 days,or TNBS induction for 3 days,mice were sacrificed and the length of colon was measured.Hematoxylin-eosin(H&E)staining was used to evaluate the effect of Calycosin on DSS-induced or TNBS-induced colon damage.Total RNA from colon was isolated and the inflammatory genes m RNA expression were determined by RT-PCR.Protein was isolated form colon and the levels of inflammatory cytokines production were determined by ELISA.The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in colon were determined by the commercial kits.The effect of Calycosin on NF-kB pathway activation wasevaluated by measuring the levels of total and phosphorylated IKK?,IKKb,IkB? and p65 in colon tissue by western blot.The effect of Calycosin on MAPK pathway activation was tested by measuring the level of total and phosphorylated JNK,p38 and Erk1/2 in colon tissue by western blot as well.Patients with ulcerative colitis were given Astragalus injection and Mesalazin Enteric-coated Tablets,or given only Mesalazin Enteric-coated Tablets.The patients' condition changes were recorded,and the concentration of HB,ALB,CRP,ESR,SOD,MDA,TNF-? and IL-6 were detected,to evaluate the therapeutic effect.Results:In vitro model,the reaults showed that treatment with 160?M and 320?M Calycosin induced red blood cells hemolysis.Treatment with 160?M and 320?M Calycosin inhibited murine macrophage cell line Raw264.7 cell proliferation.20?M,40?M and 80?M Calycosin were selected for in vitro experiments.LPS-induced inflammatory cytokines IL-6 and TNF-? mRNA expression in Raw264.7 cells was significantly inhibited by the challenge with 40?M and 80?M Calycosin.40?M and80?M Calycosin significantly inhibits IL-6 and TNF-? protein production.Treated with 40?M and 80?M Calycosin,LPS-induced ROS production was significantly inhibited.Calycosin significantly inhibited LPS-induced IKK?,IKKb,IkB? and p65 phosphorylation levels in Raw264.7 cells.Calycosin significantly inhibited LPS-induced JNK phosphorylation levels,without effect on p38 and Erk1/2phosphorylation levels in Raw264.7 cells.In the mouse models of DSS-induced and TNBS-induced ulcerative colitis,50mg/kg Calycosin,same to 5-ASA(positive control),protected DSS-induced or TNBS-induced ulcerative colitis in mice from reducing weight loss,the length of colon shorting,disease activity index increasing.H&E staining showed Calycosin protected goblet cells from damage and crypt morphology complete,reduced mucosal injury and decreased inflammatory cell infiltration.RT-PCR and ELISA results showed that 50mg/kg Calycosin,similar to the effect of 5-ASA,significantly inhibited DSS-induced or TNBS-induced inflammatory cytokine mRNA expressionand protein production.50mg/kg Calycosin,similar to the effect of 5-ASA,significantly increased DSS-induced or TNBS-induced SOD activity and decreased MDA levels.Western blot analysis showed that 50mg/kg Calycosin,similar to the effect of 5-ASA,inhibited DSS-induced or TNBS-induced IKK?,IKKb,IkB? and p65 phosphorylation levels,and significantly inhibited JNK phosphorylation with no effect on p38 and Erk1/2 phosphorylation level in colon tissue.The clinical studies showed that Astragalus injection and Mesalazin Enteric-coated Tablets had significant effect in the treatment of ulcerative colitis.The effective rate of the treatment group was higher than that of the control group,and the recurrence rate was lower than that of the control group.The change range of all indexes(HB,ALB,CRP,ESR,IL-6,TNF-?,SOD and MDA)in treatment group is larger than that in control group.Conclusions:1.Calycosin significantly inhibited LPS-induced inflammtory cytokines in the mRNA and protein expression level,and reduced LPS-induced oxidative stress production in vitro.2.Calycosin siginificantly ameliorated the development of DSS-induced or TNBS-induced ulcerative colitis mice.The underlying mechanism may involve the inhibition of NF-kB and JNK pathways activation.3.Astragalus injection and Mesalazin Enteric-coated Tablets had significant effect in the treatment of ulcerative colitis.