Calcitriol Ameliorating Th17/Treg Imbalance Elicited By AGEs In Diabetic Nephropathy Through STAT3/HIF-1 Signaling Pathway

Objective:The immune inflammation mechanism may play a significant role in diabetic nephropathy.The Treg/Th17 balance would be broken in patients with diabetic nephropathy,but the underlying mechanism has not been elucidated yet.The advanced glycosylation products(AGEs)may accumulate in patients with diabetic nephropathy and cause a series of damage through the RAGE signaling pathway.Calcitriol has been used as a drug for the treatment of renal osteodystrophy to regulate calcium homeostasis,and Calcitriol also has the effect on the regulation of immune system.To find out whether AGEs could cause imbalance of Th17/Treg through RAGE signaling pathway and whether Calcitriol could regulate the balance of Th17/Treg caused by AGEs through VDR signaling pathway,we designed this experiment.Methods:Both healthy volunteers and patients with diabetic nephropathy were recruited in the study,the concentration of the AGEs and vitamin D3 in the serum of them were measured by ELISA and the ratio of Th17/Treg cells in peripheral blood was detected by flow cytometry to define the correlation between Th17/Treg ratio and the concentration of the AGEs and vitamin D3.AGE-HSA was prepared in vitro and the CD4+T cells were isolated from peripheral blood of healthy volunteers.CD4+T cells were treated with AGE-HSA and/or Calcitriol,the ratio of Th17 cells was measured by flow cytometry,the expression of RORyt,Foxp3 and STAT3/HIF-a was measured by qPCR and/or Western-blot;CD4+T cells were treated with AGE-HSA and Calcitriol with or without anti-RAGE,the expression of RORyt,Foxp3 and STAT3/HIF-a was measured by qPCR and/or Western-blot;after the effect of si-VDR had been defined by Western-blot,CD4+T cells were treated with AGE-HSA and Calcitriol with or without si-VDR,the ratio of Th17 cells was measured by flow cytometry.Results:1)Compared with healthy volunteers,the concentration of AGEs and the ratio of Th17/Treg was higher(P<0.05)and the concentration of VitD3 was lower(P<0.05)in patients with diabetic nephropathy.In patients with diabetic nephropathy,the ratio of Th17/Treg was positively correlated with the concentration of AGEs(r= 0.607,P<0.05)and negatively correlated with the concentration of VitD3(r=0.594,P<0.05).2)AGE-HSA promoted the differentiation of Th17 cells(P<0.05);AGE-HSA up-regulated the expression of RORyt(P<0.05),but had no effect on the expression of Foxp3(P>0.05).3)Anti-RAGE inhibited the differentiation of Th17 cells(P<0.05)and the expression of RORyt(P<0.05)induced by AGE-HSA.4)AGEs up-regulated the expression of STAT3 and HIF-la(P<0.05);anti-RAGE inhibited the expression of STAT3 and HIF-1?(P<0.05)induced by AGE-HSA.5)Calcitriol inhibited the differentiation of Th17 cells(P<0.05)and the expression of RORyt(P<0.05)induced by AGE-HSA.6)Si-VDR inhibited the expression of VDR(P<0.05);si-VDR attenuated the inhibition of Calcitriol on the differentiation of Th17(P<0.05).7)Calcitriol inhibited the expression of STAT3 and HIF-a(P<0.05)induced by AGE-HSA.Conclusion:In patients with diabetic nephropathy,AGEs could induce the differentiation of Th17 cells and Th17/Treg imbalance through RAGE-STAT3/HIF-1 pathway;Calcitriol could inhibit the differentiation of Th17 cells and regulate Th17/Treg balance caused by AGEs through VDR-STAT3/HIF-1 pathway,.