| Purpose:Dracocephalum heterophyllum(DH)is a chinese herbal medicine used to treat diseases such as hepatitis,myocardial hypertrophy,and hypertension.It can inhibit the production of inflammatory cytokines.Uveitis is a type of autoimmune disease mainly mediated by CD4~+T cells.Our previous research found that DH can significantly inhibit the proliferation of T cells in vitro,therefore,we carried out this experiment,the purpose is to study the effect of DH on the function of CD4+T cell and whether DH has a therapeutic effect on autoimmune uveitis and its mechanism,also provide new methods for the prevention and treatment of autoimmune diseases especially autoimmune uveitis.Methods:(1)Prepare the DH extract:Use ethanol and ethyl acetate to prepare the DH extract,and make it into dry powder by vacuum freeze dryer for use.(2)CD4~+T cells were isolated from mesenteric lymph nodes of normal mice,and treated with different concentrations of DH extract(10,20,40mg/L),the control group did not add the extract.Collect the supernatant after 48 hours of culture,and then using ELISA method to determine the DH effect extract on cytokines produced by CD4~+T cells.(3)CD4~+T cells were isolated from mesenteric lymph nodes of normal mice,and treated with different concentrations of DH extract(10,20,40mg/L),the control group did not add the extract.Staining the cell after 48 hours of culture,and then using flow cytometry to detect the effect of the DH extract on the ability of CD4+T cells to produce cytokines.(4)CD4~+T cells were isolated from mesenteric lymph nodes of normal mice,some of the CD4~+T cells were treated with different concentrations of DH extract(10,20,40mg/L),the control group did not add the extract.After 48h,the cells were counted and stained Annexin-?/PI to study the effect of DH extract on the apoptosis of CD4~+T cells in vitro.The other part of CD4~+T cells were labeled with CFSE dyes,after 72 hours of culture,they were detected the effect of DH extract on CD4~+T cell proliferation in vitro by flow cytometry.(5)The 6-8 weeks old normal mice were divided into two groups.The DH group was injected with 20 mg/kg of DH extract in the tail vein,the DMSO group was injected with the same dose of DMSO,and all the mice were injected with BrdU intraperitoneally 12hours later.After continuous administration for 3 days,staining the PP(Peyer's patch),iEC(intestinal epithelial cells)and LP(intrinsic layer cells)of DMSO group and DH group mice,and measuring the number of CD4~+BrdU~+T cells and CD4~+T cells by flow cytometry,to study the effect of DH extract on the proliferation of CD4~+T cells in vivo.(6)Induce the BMDC and BMDM cells,and isolate Treg cells from Foxp3-YFP transgenic mice,then treat them with different concentrations of DH extract(10,20,40mg/L),the control group did not add the extract.Count the total number of cells and collect the supernatant after 48 hours,measure the concentration of cytokines in the supernatant by ELISA,and study the effect of DH extract on the proliferation of other types of immune cells and the production of cytokines.(7)Construct the EAU mouse model.The DH group was injected with 20 mg/L of DH extract in the tail vein,the DMSO group was injected with the same dose of DMSO,and the untreated group was the control group.The pathological sections of mouse eyeballs were scored by HE staining,the total cells of mouse eyeballs were isolated and cultured.Collect the supernatant and the cytokine concentration in the supernatant was detected by ELISA to investigate whether DH extract can prevent or reduce autoimmune uveitis.(8)Total mouse eyeball cells were isolated from the DH,DMSO and control group induced EAU,and detecte the number and percentage of CD4+T cells and apoptotic CD4~+T cells in the mouse eyeballs by flow cytometry to investigate the effect of DH extract on proliferation and apoptosis of CD4~+T cells in mouse eyes.(9)Total cervical lymph nodes(CLN)cells were isolated from the DH,DMSO and control group induced EAU,and detect the number and percentage of CD4~+T cells in the mouse eyeball by flow cytometryto investigate the effect of DH extract on the proliferation of CD4+T cells in mouse CLN.(10)Total mouse eyeball cells were isolated from the DH,DMSO and control group induced EAU,and detect the percentage of MDSC cells in the eyeballs of mice by flow cytometry to study the impact of DH extract on the number of MDSC cells in the eyes of mice.Results:(1)The inflammatory cytokines produced by CD4~+T cell decrease with the increase of the concentration of DH extract(P<0.01),the difference is statistically significant.(2)CD4~+T cells'ability of produce inflammatory cytokines does not change with the increase concentration of DH extract,and the difference is not statistically significant.(3)The number of CD4~+T cells in vitro decreases as the concentration of DH extract increases(P<0.01),the difference is statistically significant,the apoptosis of CD4+T cells has shown no significant difference between groups.(4)In vivo,the proliferation of CD4~+T cells in PP,iEC and LP of DH group is less than that in DMSO group(P<0.05),the difference is statistically significant.(5)In the DH group and the DMSO group,the number of BMDC,BMDM and Treg cells and the cytokine expression was not statistically significance.(6)The incidence of EAU in DH group was lower than that in DMSO group(P<0.01),the difference was statistically significant.(7)The number of CD4~+T cells in the eyes of mice treated with the DH extract is less than that in DMSO group(P<0.01),the difference was statistically significant,however,the number of apoptotic CD4+T cells in the eyes of mice was not statistically significant.(8)Compared with normal mice,the number of CD4~+T cells in CLN of DH and DMSO group was increased,but there is no difference between these two groups.(9)The percentage of MDCS cells in the eyes of DH group did not increase compared with the DMSO group,and the difference was not statistically significant.Conclusions:(1)The DH extract inhibit the proliferation of CD4~+T cell in vitro which reduces the level of inflammatory factors but does not affect the apoptosis of CD4+T cells.(2)DH extract also inhibits the proliferation of CD4~+T cells in vivo.(3)DH extract does not affect the proliferation of other types of immune cells and its cytokine expression.(4)DH extract can significantly improve the incidence of EAU in mice,the mechanism is to reduce the level of inflammatory factors by inhibiting the proliferation of CD4~+T cells without affecting the apoptosis and the number of MDSC cells in the eye. |
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