The Effect Of Cdc42 On CD4+T Helper Cells Of Inflammatory Bowel Diseases

Background and aimInflammatory bowel disease(IBD)is a chronic nonspecific inflammation of the intestinal,including Crohn’s disease(CD)together with ulcerative colitis(UC),the causes are not entirely clear.Recent studies have found that immune dysfunction especially CD4+T helper cell is one of the important factors.Cdc42(cell division cycle 42)is a member of Rho GTP enzyme protein family.Activated Cdc42 binding with GTP can regulate cell polarity,an active form by overexpression or mutation-negative regulator of actin cytoskeleton rearrangement,cell migration,differentiation and survival.In T cells,over-expression mutation of Cdc42 influences the polarization,migration and differentiation of cytoskeleton of actin and tubulin.Cdc42 is also involved in positive selection and maturation of lymphocytes.In our previous work,we made use of two-dimensional gel electrophoresis and immunoblotting to analyze colonic mucosa of patients with UC.The results showed that level of Cdc42 was significantly higher than that of health co at the same time,both p38 and Cdc42 were upregulated.In detecting the relationship between them,lipopolysaccharide(LPS)activateed p38MAPK pathway of murine macrophages RAW264.7,as while as the Cdc42 expression;however,SB203580,a special inhibitor of p38,downregulated both p38 and Cdc42.The activity of Cdc42 had relationship with p38 degree.Based on the preliminary findings,we assume that Cdc42 participated in the regulation of T cells and intestinal mucosal immune cells.Further more,it will be altered as the improvement of the inflammation.Methods1.Experiments were conducted in healthy C57 mice weighted 22-25g,6-8w.12 mice were divided into control group and DSS group.For mouse’s spleen and MLNs,the ration of each subgroup of CD4+T helper cells were detected by flow cytometry,the RNA levels of Cdc42 were detected by qPCR and the protein levels of Cdc42 were measured by western blot.HE staining is for colon inflammation analysis.Cdc42 protein expression in colon was assessed by immunohistochemistry.2.In vitro culture,the effect of inhibiting the function of Cdc42 on CD4+T helper cells differentiation were detected by flow cytometry.The master transcription factors of each subpopulation were measured by western blot3.In DSS model,after applying ML141 to mice by intraperitoneal injection,we detected the ration of CD4+T helper cells of spleens and MLNs by flow cytometry.HE staining is for colon inflammation analysis.4.Statistics:data were statistically analyzed using SPSS 17.0 software and homogeneity of variance was conducted first.Measurement data were expressed as meantstandard deviation.Gray value of bar charts was analyzed by Gelpro analysis software.Measurement data were gathered and also expressed as mean±standard deviation.One-way ANOVA was used for parametric test whilst Welch or Kruskal-Wallis tests were used for non-parametric tests.Multiple comparisons between groups used LSD-t test.The body weight and DAI score needed sphericity test.If P>0.05,the data should be analyzed by One-way ANOVA;if P<0.05,a repeated measurement was required and we choose Greenhouse-Geisser to analyze.The indexes of induced-cells were analyzed by Two-way ANOVA.P<0.05 was considered as significant difference and there was statistically significant.Results:1.Comparing DSS group with blank group,the ration of Th17 and Thl cells in mouse’s spleen and MLNs were elevated while no difference between Th2 and Treg cells.2.Comparing DSS group with blank group,the protein levels of Cdc42 in spleen and MLNs were up-regulated in DSS group while the RNA levels were reduced.3.For in vitro culture of polarization condition,inhibiting the function of Cdc42 has no effect on the proliferation and apoptosis of CD4+T cells4.For in vitro culture of polarization condition,inhibiting the function of Cdc42 can promote the differentiation of CD4+T helper and pathogenetic Th17 cells5.Inhibiting the function of Cdc42 in DSS group can aggravate the inflammation in gut6.In DSS model,inhibiting the function of Cdc42 can promote the differentiation of Th17 cells and Thl cells in mouse’s MLNs7.In DSS model,inhibiting the function of Cdc42 has no effect on CD4+T cells in mouse’s spleenConclusionThe protein levels of Cdc42 in spleen and MLN is elevated in DSS group,While the RNA levels were reduced;Inhibiting the function of Cdc42 can promote the differentiation of CD4+T helper cells,thus,we hypothesized that the Cdc42 may play an important role in protecting the gut from inflammation in DSS model.