To Investigate The Effect And Mechanism Of TIM Signal Path On The Proliferation Of Human Mesangial Cells Stimulated By IgA1 In Patients With IgA Nephropathy

Objective:The T-cell immunoglobulin and mucin domain(TIM)family are involved in the regulation of innate and adaptive immune responses,studies have identified its role in diverse disease processes and increasingly in alloimmunity.However,no studies thus far have investigated the roles of TIMs on IgAN.However,the role of TIMs in IgAN has rarely been investigated.In this study,we observed the effect of tim-1 on abnormal glycosylated IgA1 molecules of spleen B lymphocytes and on IgA1 induced proliferation and activation of Human mesangial cell(HMC),and further revealed the role of tim-1 in local immunity of IgAN system and kidney.Method:1.serum IgA1 of polymerized IgAN patients(aIgA1)and normal subjects was purified by Jacalin affinity chromatography combined with sephacry1s-200 gel filtration.2.spleen lymphocytes and HMC of mice were studied.After the intervention of tim-1 antibody and tim-4 antibody on mouse lymphocytes,they were co-cultured with B cells under LPS stimulation.Then the HMC was stimulated with aIgA1 of IgAN patients and IgA1 of healthy people respectively.3.determine the action time and concentration of aIgA1,take the HMC cells in the logarithmic growth period and in good growth condition,and inoculated 2×105 HMC cells per well in the 6-well plate of cell culture,and cultured overnight in the incubator of 37℃ and 5% CO2.Six experimental groups were set up:1)normal serum IgA1 culture2)normal serum IgA1+TIM1 antibody culture3)normal serum IgA1+TIM4 antibody culture4)serum IgA1 culture of IgAN patients5)serum IgA1+TIM1 antibody culture of IgAN patients6)serum IgA1+TIM4 antibody culture of IgAN patients4.MTT method was used to detect the proliferation of mesangial cells,the increased expression of tim-1,TLR4 and related cytokines was detected by flow cytometry,real-time PCR and immunohistochemistry,and the contents of TGF-TGF 1,IFN-TGF,IL4,IL10 and IL12 in the supernatant of cell culture were detected by ELISA.5.All experimental data were expressed as mean ± standard deviation(x(-)±s),and t was detected by the statistical software IBM SPSS Statistics 22,and p<0.05 was considered statistically significant.Results:1)to join TIM-1 antibodies,TIM-4 deal with lymphocytes in mice after 24 hours,compared with blank control group,determined by MTT method to detect the antibody treatment or TIM,TIM-1-4 of spleen lymphocyte proliferation in the model group was significantly lower(p < 0.05),statistically significant differences,TIM-1 antibodies in mice lymphocyte proliferation response has certain inhibition;After stimulation of tim-1 antibody and tim-4 antibody,the contents of IL4 and IL10 decreased compared with the control group(p<0.05),the difference was statistically significant.Tim-1 antibody could inhibit the abnormal production of IgA and the glycosylation of IgA molecules in B cells by inhibiting the up-regulation of Th2 cell-related cytokines after IgA1 stimulation.2)After 24 hours of treatment with tim-1 antibody and tim-4 antibody,aIgA1 and normal serum IgA1 with IgAN in HMC were interfered.Compared with the blank group,the cell proliferation degree was significantly reduced by MTT assay(p<0.05).Tim-1 antibody had a certain inhibitory effect on the proliferation response of IgA1 to stimulate mesangial cells.Total proteins and total RNA were extracted from each group to detect the changes of related proteins and mRNA.The expression of TLR4,TGF-TGF 1 protein and mRNA in tim-1 antibody and tim-4 antibody treatment group was significantly lower than that in the blank control group(p<0.05).Conclusions:1)tim-1 antibody and tim-4 antibody can inhibit the proliferation of mouse lymphocytes.2)the contents of IL4 and IL10 decreased in mouse lymphocytes after tim-1 treatment,and the expression of th2-related cytokines was up-regulated after tim-1 antibody inhibited IgA1 stimulation.3)aIgA1 in IgAN patients can stimulate the proliferation of HMC,while tim-1 antibody has a certain inhibitory effect on the proliferation response of IgA1 stimulating mesangial cells.4)tim-1 antibody can inhibit aIgA1 stimulation-induced HMC proliferation,and down-regulate the expression of TLR4 TGF-1 and tim-1,thus playing a protective role on the kidney.