Estrogen Regulates Th17/Treg Cell Immune Balance To Protect Renal Ischemia Reperfusion Injury

Objective:Through observing the damage degree of estrogen（E₂）on the renal function and kidney,the expression level of T lymphocyte subsets and related cytokines and the influence of Cx43 expression of rats in each group,the research was aimed at exploring whether the estrogen regulate the balance of T lymphocyte and influence the expression of Cx43 and the secretion of cytokines to improve the renal injury induced by ischemia/reperfusion（I/R）.Methods:（1）Female Sprague–Dawley（SD）rats were randomly divided into 6 groups:normal rats（Control group）,normal rats with I/R injury（I/R group）,ovariectomized rats（OVX group）,ovariectomized rats with I/R injury（OVX+I/R group）and treatment with estrogen（OE+I/R group）and Gap27（OG+I/R group）before ovariectomized rats with I/R injury respectively.Blood and kidney samples were collected at 6 hours after reperfusion in rats of each group,and blood urea nitrogen（BUN）and serum creatinine levels were detected by automatic biochemical analyzer;Hematoxylin-eosin（HE）Staining and Paller score were used to quantify the degree of renal damage.（2）Flow cytometry was used to detect the positive rate of CD3⁺CD4⁺and CD3⁺CD8⁺T cells in peripheral blood and renal tissues.In addition,the expression and distribution of CD4⁺T cells in renal tissues were observed by immunofluorescence after I/R injury.（3）Serum and homogenate concentrations of IL-17 and IL-10 were measured by ELISA.（4）Western-blot and flow cytometry were applied to detect the expression of Cx43 and CD4⁺Cx43⁺in peripheral blood mononuclear cells（PBMCs）respectively.Results:（1）In the early stage of renal IRI,the levels of BUN,Scr and the damage degree of renal tissue in rats were significantly higher than that in control group（p<0.01）.Compared with I/R group,the levels of BUN,Scr and pathological damage in OVX+I/R rats were significantly increased（p<0.01）,but these indexes were significantly decreased after administration with estrogen and Gap27（p<0.05）.（2）（1）After I/R injury,the positive expression rate of CD3⁺CD4⁺T cells in peripheral blood of rats increased significantly（p<0.05）,the rate of CD3⁺CD8⁺T cells decreased significantly（p<0.05）,and the ratio of CD4⁺/CD8⁺increased significantly（p<0.05）.The positive rate of CD3⁺CD4⁺T cells in OVX+I/R group were significantly higher than I/R group（p<0.05）,the proportion of CD3⁺CD8⁺T cells were significantly lower（p<0.05）,and the ratio of CD4⁺/CD8⁺was significantly increased（p<0.05）,after giving estrogen and Gap27 respectively,this phenomenon was reversed（p<0.05）.（2）After I/R injury,the positive expression rates of CD3⁺CD4⁺and CD3⁺CD8⁺T cells in rat renal tissue were significantly increased（p<0.05）.The positive rate of CD3⁺CD4⁺and CD3⁺CD8⁺T cells in OVX+I/R rats was significantly higher than that in I/R group（p<0.05）,given estrogen and Gap27,the positive expression rate was significantly reduced（p<0.05）.（3）The results of immunofluorescence revealed that CD4⁺T cells infiltrated in the kidney tissue in the early stage of ischemia-reperfusion injury.（3）（1）After I/R injury,the concentration of IL-17 in rats increased significantly（p<0.01）,while IL-10 decreased significantly（p<0.05）;the concentration of IL-17 in rats in OVX+I/R group was significantly higher than that in I/R group（p<0.05）,however,IL-10 was significantly lower than that of the I/R group,when intervention with estrogen and Gap27,IL-17 levels were significantly reduced and IL-10 levels were significantly increased.（2）Correlation analysis showed that IL-17 was positively correlated with BUN,Scr and Paller scores,while IL-10 was negatively correlated with the above indicators.（4）Western-blot and flow cytometry showed that the expression of Cx43 and CD4⁺Cx43⁺in PBMCs were significantly up-regulated after ischemia-reperfusion injury,and the expression of OVX+I/R group was significantly higher than that of I/R group.After intervention with Gap27 and estrogen,its expression was significantly downregulated.Conclusions:（1）Estrogen inhibits the inflammatory response induced by renal I/R injury by modulating the balance of CD4⁺/CD8⁺.（2）Estrogen inhibits the inflammatory response induced by renal I/R injury by modulating the balance of Th17/Treg in CD4⁺T cell.（3）Cx43is involved in the regulation of Th17/Treg balance of CD4⁺T cell subsets by estrogen.