Effect Of Ginsenoside Derivative AD-1 On CD4~+ T Cells In Mice With Acute Colitis Induced By TNBS

Background:Ginseng is listed as the top grade of Shennong’s herbal medicine and is often referred to as a tonic,but it is also valuable for medicinal purposes,such as anti-inflammatory and anti-cancer.The ginsenoside derivative AD-1 is extracted from ginseng pulp,which also has various pharmacological effects such as anti-inflammatory and anti-tumor.Inflammatory bowel disease(IBD)is a chronic,nonspecific intestinal inflammation in which various components of the mucosal immune system are involved in the pathogenesis of IBD,including intestinal epithelial cells and the adaptive cellular immune system and their secreted mediators.CD4+T cell subsets in IBD may play an important role in the disease,among which Th1/Th2 and Th17/Treg imbalances are considered to be the main causes of IBD.Objective:The efficacy of AD-1,a novel ginsenoside derivative based on the immunomodulatory effect of CD4~+T cells,on TNBS-induced acute colitis model was clarified.Methods:C57BL/6 female mice were divided into five groups:normal control group(Control),ethanol control group(Ethanol),model group(TNBS),AD-1treatment group(TNBS+AD-1),5-amino group Salicylic acid positive control group(TNBS+5-asa),control group and Ethanol group with 5 rats each,TNBS group,TNBS+AD-1 group and TNBS+5-asa group with 10 rats each.On the zeroth day,the TNBS group,TNBS+AD-1 group and TNBS+5asa group were given 2.5%TNBS ethanol solution enema,the Control group was given normal saline enema,and the Ethanol group was given 25%ethanol solution enema.On the first day,TNBS+AD-1group and TNBS+5asa group were given intragastric administration,AD-1administration dose was 10mg/kg,5-asa administration dose was 30mg/kg,and the intragastric solvent used 0.5%carboxylate Methylcellulose sodium(CMC)solution,Control group,Ethanol group and TNBS group were given 0.5%CMC solution by gavage for four days,and the body weight and clinical score of mice in each group were recorded.Before all the mice were sacrificed on the fifth day,the blood of the eyeball was collected,and the serum was separated to detect the cytokines interferon(IFN)-γ,interleukin(IL)-4,IL-17A,and IL-10 by enzyme-linked immunosorbent assay(ELISA).After being sacrificed,the spleen and colon were separated,the length of the colon was measured,and a part of the colon was fixed in formaldehyde solution,made into paraffin section for hematoxylin-eosin(HE)staining,and the colon tissue was observed under microscope after section staining.The expression levels of cytokines IFN-γ,IL-4,IL-17A and IL-10 in the colon were detected by RT-PCR;Western blotting(WB)was used to detect the transcription factor T box transcription protein in the colon(Expression levels of T-bet),Gata-binding protein 3(Gata3),retinoic acid-related orphan receptorγt(ROR-γt)and forkhead box protein P3(Foxp3);The percentage of CD4~+T cell subsets differentiated in the spleen was measured by flow cytometry.Results:1.Compared with the TNBS group,TNBS+AD-1 significantly improved the weight loss,shortened colon length,and decreased the DAI score of the IBD mice.2.The results of HE staining of colon histopathological sections showed that the colonic mucosa of the mice in the TNBS group was severely damaged,the crypt structure was destroyed,the goblet cells disappeared,and a large number of inflammatory cells were infiltrated.However,in the TNBS+AD-1 group,the colonic mucosal structure was relatively complete,the infiltration of inflammatory cells was reduced,the disappearance of goblet cells,and the destruction of the crypt structure were greatly improved.3.RT-PCR method was used to detect the expression of IFN-γ,IL-17A,IL-4 and IL-10 cytokine genes in mouse colon tissue.Compared with TNBS group,IFN-γand IL-The expression of 17A gene was significantly decreased(P<0.05,P<0.01),and the expression of IL-4 and IL-10 genes was significantly increased(P<0.01,P<0.001).4.The protein expression levels of T-bet,ROR-γt,Gata3 and Foxp3 in the colon tissue of mice were detected by WB method.Compared with the TNBS group,the protein expressions of T-bet and ROR-γt in the TNBS+AD-1 group were significantly decreased(P<0.01),and the protein expressions of Gata3 and Foxp3 were significantly increased(P<0.05,P<0.01).5.The cytokine secretion of IFN-γ,IL-17A,IL-4 and IL-10 in the serum of mice was detected by ELISA.Compared with the TNBS group,the IFN-γand The content of IL-17A was significantly decreased(P<0.05,P<0.01,P<0.001),and the content of IL-4 and IL-10 was increased(P<0.05,P<0.01,P<0.001).6.The differentiation rates of Th1,Th2,Th17 and Treg cells were detected by flow cytometry,and the results showed that compared with the TNBS group,CD4~+IFN-γ~+and CD4~+IL-17A~+T in the spleen of mice in the TNBS+AD-1 group The cell differentiation rate was significantly down-regulated(P<0.01,P<0.001),and the differentiation rate of CD4~+IL-4~+and CD4~+Foxp3~+T cells was significantly increased(P<0.05,P<0.001).Conclusion:1.Ginsenoside derivative AD-1 can alleviate TNBS-induced acute IBD in mice.2.The therapeutic effect of ginsenoside derivative AD-1 on IBD may be related to regulating the balance of Th1/Th2 and Th17/Treg cells.