The Role Of Tfh Cells In The Pathogenesis Of Type 1 Diabetes And The Intervention Effect Of Oxymatrine

Objective To investigate the role of follicular helper T cells(Tfh)in type 1 diabetes mellitus(T1DM)and the protective effect of oxymatrine(OMT)on isletβcells and its molecular mechanism.In this study,we collected the clinical laboratory examination indexes of patients with T1DM and established non-obese diabetic(NOD/Lt J)mice model.We detected the percentage of Tfh in peripheral blood and the expression of anti-isletβcell antibody(ICA),zinc transporter 8 antibody(Zn T8Ab),glutamic acid decarboxylase antibody(GADA65-Ab),protein tyrosine phosphatase antibody(PTP-Ab),insulin antibody(IA),IL-21,IL-6 and IL-1βin serum of patients with T1DM,and analyzed the correlationship between the percentage of Tfh cells and serum factors.NOD mice was treated with Oxymatrine(OMT)and measured the changes of blood glucose,Oral glucose tolerance,insulin,spleen germinal center,pancreas and kidney.And we detected the level of IL-21,IL-6 and IgG1 in serum,and the expression level of Bcl-6 and IL-21 mRNA in spleen lymphocytes of NOD mice.To provide experimental basis for finding new treatment methods.Methods(1)The clinical data of 158 T1DM patients in the Department of Endocrinology,General Hospital of Ningxia Medical University from January 2016 to December 2019 were assembled to observe whether the T1DM patients had immune disorder.(2)30 cases of T1DM from October 2019 to 2020 were selected as subjects(T1DM group),and 20 healthy volunteers were recruited(HC group).(3)The percentage of Tfh cells in peripheral blood of T1DM patients was detected by flow cytometry.(4)The expression levels of cytokines IL-1β,IL-6,IL-21 and autoantibodies IA,GAD65Ab,PTP-Ab,Zn T8Ab,ICA in peripheral blood serum of patients with T1DM were detected by ELISA.(5)The correlation between Tfh cell percentage and IL-1β,IL-6,IL-21 the expression of IA,GAD65Ab,PTP-Ab,Zn T8Ab,ICA was analyzed by Spearman.(6)12 NOD/Lt J mice were stochastically divided into model group and oxymatrine group(OMT group),6 ICR mice were set as normal group.(7)We measured the weight of mice,the random blood glucose of mice,oral glucose tolerance test and urine glucose test.(8)The pathological changes of islet,spleen and kidney were observed by HE staining.(9)The positive area of insulin in mouse islet cells was concluded by immunohistochemistry.(10)In spleen of NOD mice,the percentage of Tfh cells was analysed by flow cytometry.(11)In serum of mice,the contents of cytokines insulin,IL-6,IL-21 and autoantibody IgG1 were tested by ELISA.(12)In mouse splenocytes,the levels of Bcl-6 and IL-21 were detected by RT-qPCR.Results(1)In T1DM patients,the content of C-peptide decreased significantly,while the contents of glycosylated hemoglobin,postprandial blood glucose,blood ketone body,urine glucose and urinary Microalbumin increased,and the positive rate of GADA was the highest.(2)The percentage of CD4~+CXCR5~+T cells(20.01±5.14),CD4~+ICOS~+T cells(4.39±0.81),CD4~+PD1~+T cells(24.92±5.24)and CD4~+CXCR5~+ICOS~+PD1~+T cells(3.57±0.68)in peripheral blood lymphocytes of T1DM patients were obviously raised than those of healthy controls.(3)The contents of ICA,Zn T8Ab,GADA65-Ab,PTP-Ab,IA,IL-21,IL-6and IL-1βin T1DM patients were increased distinctly than those in healthy controls.(4)In T1DM patients,the proportion of Tfh cells had a positive relation with serum auto-antibodies and cytokines.(5)In comparison with the model group,OMT group gained weight(p<0.05),lowered blood glucose(p<0.01),and increased insulin secretion(p<0.01).(6)In the model group,the percentage of Tfh cells(5.38±0.27)was obviously higher than that in the normal group(2.95±0.23)(p<0.001).The proportion of CD4~+CXCR5~+ICOS~+Tfh cells in the OMT group was apparently lowered against model group(3.26±0.33)(p<0.001).(7)As a result of HE staining,isletβcells were agenesis in the pancreas of the model group,and atrophied isletβcells were occasionally observed.The formation and morphology of isletβcells in OMT group were intact and inerratic.The number of germinal centers much more in the model group.However,there was no difference between the OMT group and the normal group.With the increase of blood glucose,the glomeruli in the kidney of the model group were enlarged and the basement membrane thickened.The kidney tissue of the OMT group was arranged clearly,and there was no difference between the OMT group and the normal group.(8)The result of immunohistochemical showed that the number of isletβcells in model mice was minified,the morphology was irregular,the cells was atrophied,and the positive rate of Insulin protein diminished significantly.But in OMT group,the stained area of insulin protein was positive in isletβcells.(9)To contrast the model group,IL-21,IL-6 and IgG1 were lessened in the serum of OMT group,but the content of insulin increased significantly.(10)The contents of Bcl-6 and IL-21 mRNA in the OMT group was reduced evidently against model group.Conclusion(1)Tfh cells may promote humoral immune response and drive the occurrence of type 1 diabetes.(2)OMT may alleviate the pathogenesis of type 1 diabetes by regulating Tfh cells to protect isletβcells.