Based On Akt-mTOR Signaling Pathway In Diabetic Rats To Investigate The Mechanism Of Huangqitujian Decoction On Renal Protection

Background:Diabetic nephropathy has become the primary cause of End-stage renal failure. At present, the number of dialysis patients with DN have occupied second place in our country. Diabetic nephropathy greatly affected the quality of patients’life and brought a heavy financial burden.Thus exploring effective measures for prevention and treatment of diabetic nephropathy is an important issue.Abnormally increasing of Mammalian Target of Rapamycin(mTOR) activity in Podocyte is one of the key mechanism about proteinuria occurred. Inhibition of mTOR with Rapamycin may prevent the development of diabetic nephropathy, but excessive inhibition of mTOR will cause podocytes damage and other side effects. Rapamycin might are difficult to applied in the clinical treatment because of their side effects.Our previous study found that Chinese medicine Tujian decoction reduced proteinuria in diabetic rats^ relieved the pathological changes of diabetic nephropathy and inhibited Akt (Thr308) phosphorylation of the glomerular mesangial cells.Objective:Observing effect of HuangqiTujian decoction on P13K-Akt-mTOR signaling pathway to study protection mechanism of HuangqiTujian decoction on Diabetic rats’kidney.Methods:Male Sprague-Dawley (SD) rats were randomly divided into normal control group whitch the rats’number is 10 and diabetic group the rats’number is 50. The rats were made diabetic by intraperitoneal injection of streptozotocin (STZ) at 65mg/Kg body wt in 0.01M citrate buffer(PH4.2) after 12h of food deprivation.Rats receiving an injection of citrate buffer were used as controls. Hyperglycemia was determined 3 days after injection,and rats with blood glucose levels= 16.7mmol/L were considered to have diabetes.Rats were divided in to five groups:normal control rats (group CON,n=10)-diabetic control rats(group DM, n= 10), diabetic rats treated with rapamycin(group RAPA,n=11), diabetic rats treated with HuangqiTujian decoction(group CM, n= 10), diabetic rats treated with both rapamycin and HuangqiTujian decoction(group RAPA+CM, n= 10). Rapamycin (0.5mg/kg/qod) was administered intraperitoneally every other day to rats in group RAPA and in groupRAPA+CM.Huangqi Tujian decoction(10g/kg/day) was administered by intragastric administration daily to rats in group CM and in group RAPA+CM.12 weeks after individual rat were placed in metabolic cages for 24 h urine collection.The blood was collected from the carotid artery of rats into serum tube.The kidneys were decapsulated promptly.Part of the kidney was placed in Formalin,and another part of the kidney was immediately frozen in liquid nitrogen.Blood glucose (Glu), blood creatinine(Scr),blood urea nitrogen(BUN),urinary albumin excretion rate(Albumin creatinine ratio,ACR)were measured when therats were sacrificed. Observe the renal pathological changes in rat by HE staining.The protein levels of nephri^ ZO-1^ fibronectin (FN) were detected by immunohistochemical staining,the protein levels of p-Akt(Thr308、p-S6k1、p-Pten were detected by Weatern blot.The mRNA levels of Akt、S6k1、Pten were detected by reverse-transcriptional-polymerase chain reaction (RT-PCR).Results:After 12 weeks, the blood glucose of DM group was significantly higher than the CON group, The CM group compared with DM group have significantly lower blood glucose, RAPA group and RAPA+CM group have no significant difference in blood glucose levels compared with DM rats;RAPA、RAPA+CM group rats body weight was significantly lower than DM group, while the CM group rats had no significant difference in body weight with the DM group rats. The mortality rate of the RAPA group rats was significantly higher than DM group,while the mortality rate of CM group、RAPA+CM group and DM group showed no significant difference.Glomerular volume increased in DM group rats than in CON group rats, while glomerular hypertrophy of RAPA group rats, CM group rats, RAPA+CM group rats was significantly reduced compared with DM group.Urinary albumin excretion rate in DM group rats was significantly higher than in CON group rats, while urinary albumin excretion rate of RAPA group rats, CM group rats, RAPA+CM group rats was significantly reduced compared with DM group.There is a small amount of FN staining in CON group glomerular of rats,and there are many positive staining FN expression in DM group rats.The FN expression in the other three treatment groups were significantly lower than DM group.The positive staining of ZO-lprotein, nephrin protein in DM group rats’glomerular was significantly lower than CON group’s,however, ZO-1、nephrin expression in the other three treatment groups was significantly higher than DM group.PTEN protein expression in DM group decreased significantly compared with CON group; CM group, RAPA+CM could significantly increase the expression of PTEN, however PTEN expression in RAPA group rats had no significant difference with DM group rats.Protein expression and mRNA expression of S6K1 in DM group rats was significantly higher than in the CON group rats, expression of S6K1 in the RAPA group,CM group,RAPA+CM group was significantly lower than DM group.There is a significant increase in DM group about Akt (Thr308) protein expression and AktmRNA expression compared with CON group; CM group, RAPA+CM group significantly decreased Akt (Thr308) protein expression and AktmRNA, but compared with DM group. Akt (Thr308) protein expression in RAPA group has no significant difference with DM group.Conclusion:Rapamycin and HuangqiTujian decoction can effectively protect the STZ-induced diabetic rat podocyte integrity, protect the kidney structure, reduce urinary albumin excretion rate. But rapamycin single inhibition of mTORC1 activation, causing serious side effects.It can not be directly used in clinical. HuangqiTujian decoction can regulate PI3/Akt/mTOR signaling pathway comprehensively, reduc the toxicity of rapamycin; decreases blood glucose.Its clinical value higher than clinical value of rapamycin...