Study On The Mechanism Of Triptolide In Improving Diabetic Nephropathy Via PDK1/Akt And MiR-137/Notch1 Pathway

Objective:Diabetic nephropathy(DN)is one of the most serious microvascular complications of diabetes and also the leading cause of end-stage renal disease worldwide,which is clinically characterized by the development of proteinuria followed by decreased glomerular filtration in association with glomerulosclerosis.Pathological features of DN include mesangial expansion,caused by the proliferation of mesangial cells and the excessive accumulation of extracellular matrix,which will lead to glomerulosclerosis.Therefore,inhibiting proliferation and ECM accumulation of mesangial cells is regarded as an effective strategy to ameliorate DN.Tripterygium wilfordii Hook F(TWHF),a traditional Chinese medicine,has been used for the treatment of chronic kidney disease for a long time.However,side effects caused by the complicate components limit its application.Thus,finding the active components,which mainly contribute to nephropathy improvement,will help reducing its side effects.Triptolide(TP)is a major active component of TWHF,which exerts immunosuppressive and anti-inflammatory therapeutic effects.And its relative potency is 100 to 200 times higher than TWHF.In this study,STZ/HFD induced diabetic rats and HRMCs treated with high glucose(HG)were used to discuss:1.Effects and safety of triptolide on glomerulosclerosis in the STZ/HFD-induced diabetic rats;2.Effects of triptolide on the proliferation of diabetic glomerular mesangial cells and its mechanism;3.Effects of triptolide on ECM accumulation in diabetic mesangial cells and its mechanism.Methods:1.SD rats were induced diabetic through a STZ/HFD method,and were randomly assigned to normal control group(NC),diabetic group(DM)and TP group(DM + TP).Twelve weeks after TP treatment,we evaluated the renal function,liver function and 24 h urine micro albumin in rats by biochemical method to evaluate the effect and safety of triptolide on diabetic nephropathy of the rats.HE and PAS staining were used to observe glomerulosclerosis in the rat kidney to evaluate the effect of TP on glomerulosclerosis.Immunochemistry and western blot were used to examine the proliferation and ECM accumulation of HRMCs and its related regulators.RT-PCR was used to examine the expression of microRNA in the rat kidney.2.Western blot and immunofluorescence(IF)were used to prove the role of TP in Ki-67 and PCNA expression in HRMCs.MTT assay was used to detect cell numbers,and cytometry was used to detect cell cycle analysis.Western blot was used to examine the effect of TP on the expression of PDK1/ Akt /mTOR pathway.To determine the specific role of PDK1 in TP-potentiated anti-proliferation,we applied 5 ?mol/L PS48 following the treatment of TP.3.Western blot and IF were used to prove the role of TP in ECM accumulation in HRMCs.The miRNAome was assessed by microRNA array analysis and further proved by RT-PCR.A luciferase reporter assay was conducted to confirm the target gene of miRNA.miRNA mimics and inhibitor were transfected to the cells to detect the possible role of TP in DN.DAPT and siRNA were used to evaluate the role of Notch1 in diabetic glomerulosclerosis.To verify the role of miR-137 in the ECM inhibition effect of triptolide,miR-137 inhibitor was transfected into the HRMCs pretreated with TP.Results:Effects and safety of triptolide on glomerulosclerosis in the STZ/HFD-induced diabetic rats:1.Animal modeling: After modeling,blood glucose in diabetic rats was significantly higher than that of normal rats,the body weight decreased significantly.Compared with NC group,24 h urinary albumin level in DM group was significantly increased(P < 0.05),glomerular basement membrane thickening,mesangial matrix increased,glomerular sclerosis index increased(P < 0.05).2.Effect of triptolide on glomerular sclerosis: Triptolide had no significant effect on blood glucose and body weight in DM rats(P > 0.05).It reduced the ratio of kidney weight to body weight in DM rats(P < 0.05)and decreased the 24 h urinary albumin(P < 0.05).It also improved kidney morphology,decreased mesangial matrix expression and glomerulosclerosis index in DM rats(P < 0.05).3.Triptolide had no effect on liver and kidney function in DM group(all P > 0.05).Effects of triptolide on the proliferation of diabetic glomerular mesangial cells and its mechanism:1.Triptolide significantly inhibited the elevated protein expression of Ki-67 and PCNA in STZ/HFD-induced diabetic rats and HRMCs treated with HG(P < 0.05).2.Triptolide significantly inhibited the number of HRMCs stimulated by HG.G0 / G1 phase ratio of TP group(55.4 ± 3.1)was significantly higher than that in the HG group(40.4 ± 2.1)(P < 0.05).S phase ratio of TP group(37.9±2.6)was lower than that in the HG group(40.4±2.1)(P < 0.05).The percentage of cells in G2/M phase of TP group(5.7 ± 1.2)was lower than that in the HG group(18.0 ± 1.9)(P < 0.05).3.Triptolide significantly inhibited the activation of PDK1 protein and the phosphorylation of Akt and mTOR protein(P <0.05)in STZ/HFD-induced diabetic rats and HRMCs treated with HG4.The inhibition effect of TP on the proliferation of HRMCs was suppressed by PS48(P < 0.05).The regulation of TP in cell cycle analysis was reversed by PS48.The inhibitory effect of Triptolide on Ki-67,PCNA and PDK1/Akt/mTOR pathway was reversed by PS48(P <0.05).Effects of triptolide on ECM accumulation in diabetic mesangial cells and its mechanism:1.Triptolide significantly inhibited the elevated protein expression of Col IV and FN in STZ/HFD-induced diabetic rats and HRMCs treated with HG(P < 0.05).2.Triptolide significantly inhibited the activated Notch1 pathway in STZ/HFD-induced diabetic rats and HRMCs treated with HG(P < 0.05).3.Notch1 pathway inhibitor DAPT and Notch1-siRNA both inhibited the protein expression of Col IV and FN in HRMCs(P < 0.05).4.Triptolide increased miR-137 level in STZ/HFD-induced diabetic rats and HRMCs treated with HG(P < 0.05).5.miR-137 mimics decreased the accumulation of ECM and the activation of Notch1 pathway in HRMCs induced by HG(P < 0.05).MiR-137 inhibitor increased the expression of ECM and Notch1 in HRMCs(P < 0.05).Dual-fluorescence assay revealed that miR-137 binds to the 3'-UTR region of Notch1.6.miR-137 inhibitor decreased the inhibitory effect of triptolide on ECM accumulation in HRMCs(P < 0.05),and decreased the inhibitory effect of triptolide on Notch1 pathway in HRMCs(P < 0.05).Conclusion:1.Triptolide can improve glomerulosclerosis in diabetic rats and reduce 24-hour urine micro albumin levels independently of hypoglycemic effects.2.Triptolide may inhibit the proliferation of diabetic mesangial cells by inhibiting the PDK1/Akt/mTOR pathway,thereby improving diabetic glomerulosclerosis.3.Triptolide may inhibit the expression of Notch1 pathway by up-regulating the level of miR-137,thereby inhibiting the accumulation of ECM proteins in the mesangial cells under hyperglycemia,thereby improving diabetic glomerulosclerosis.