| Objective:Diabetes can cause the retina, kidneys, nerves and other organs, complications, including diabetic nephropathy. Diabetic nephropathy(DN) is one of the most serious chronic complications of diabetes. According to the United States, Japan and many European countries'statistics show that diabetic nephropathy has risen to become the first cause of end-stage renal failure,which also contributed to the main cause of death in diabetes mellitus. Organism exists in patients with diabetic nephropathy is extremely metabolic disorders.Once developed to the end-stage renal failure, treatment of kidney disease is more difficult than any other disease. The main pathological features of early diabetic nephropathy are glomerular basement membranet thickening, mesangial cell proliferation, glomerular mesangial expansion, which are due to renal hypertrophy.The clinical manifestations are the high filtration and microalbuminuria. Glomerular sclerosis and tubulo-interstitial fibrosis are promoted with the progression of glomerular mesangial matrix increase.Renal function gradually decreased, eventually leading to end-stage renal failure. At present, the experiments show that extracellular signal regulated kinase(ERK) signal transduction pathway plays an important role in the occurrence and development of diabetic nephropathy.ERK can mediate cell growth, proliferation and differentiation activated by a variety of growth factors and cytokines. AngiotensinⅡ(AngⅡ) can stimulate vascular smooth muscle cells and renal mesangial cells, renal tubular epithelial cells hypertrophy. Vascular endothelial growth factor(VEGF) is a cytokine which can promote endothelial cell proliferation and migration and can increase vascular permeability. Connective tissue growth factor(CTGF) is a fibrogenic cytokine.The combination of advanced glycation end products (AGEs) and advanced glycation end products receptor (RAGE) can release a variety of cytokines, causing vascular endothelial injury, extracellular matrix proliferation and other pathological changes. The main components of Tongluo recipe (TLR) are ginseng, leech, scorpion, eupolyphaga, centipedes, Paeonia veitchii Lynch and some other drugs.Experiments with TLR have already found that glomerular basement membrane and high-sugar induced oxidative damage repaired.TLR can increase the role of antioxidant capacity of the body, but whether TLR can influence intracellular signal transduction pathway is currently unclear.This experiment intended to study the effects of TLR on the expression of AngⅡ, VEGF, CTGF, RAGE, ERK1/2, phosphorylated ERK1/2 (p-ERK) in STZ-induced diabetic rats,as well as the possible renal protective mechanism of TLR.Methods:SPF-class adult male Sprague-Dawley(SD) rats weighing 200g±20g were randomly divided into 2 groups:control group(A,n=10) and Experimental group.Diabetic rats in experimental group were induced by intraperitoneal injection of Streptozotocin(60mg/kg body weight),and randomly subdivided into B group(diabetic control,n=10), C group(diabetic rats treated with low dose of Tongluo Recipe,0.5 g/(kg-d),n= 10), D group(diabetic rats treated with medium dose of TongluoRecipe,1.0g/(kg-d),n=10),E group (diabetic rats treated with high dose of Tongluo Recipe,2.0g/(kg-d),n=10) and F group(diabetic rats treated with Losartan, 30mg/(kg-d),n=10).At the end of 12th week,serum glucose,triglyceride(TG),total cholesterol(CHOL),serum creatine(Scr),ureanitrogen(BUN)in blood,24h albumin excretion rate(UPRO24),microalbuminura (MAU),and creatinine in urine(Ucr) were analyzed by bio-chemical methods.Kidney weight/body weight(KW/BW) and creatinine clearance rate(Ccr) were calculated.Concentration of angiotensinⅡ(AngⅡ) in blood serum and kidney were measured by Elisa.The kidney tissues obtained from each group were used for observing pathologic changes under light microscope and transmission electron microscopy,assessing the expression of p-ERK 1/2 by immunohistochemical staining and detecting vascular VEGF,RAGE,CTGF,p-ERKl/2 and total ERK1/2 by western-blot. SYBR Green real-time PCR is used to detect expression of CTGF mRNA, RAGE mRNA in renal tissues.Results:1.Under ordinary light microscope, no pathological changes were observed in A group.Glomerular basement membrane thickening, mesangial cell proliferation, cloudy swelling of renal tubular degeneration and mild renal interstitial fibrosis were observed in B group. Pathological damages in diabetes treatment groups were improved compared with B group. B group were observed basement membrane thickening and foot process fusion, glomerular endothelial cells rupture, renal tubular epithelial cell degeneration, necrosis under transmission electron microscope.Lesions in C, D, E, F groups were lighter than in B group.2. The weight of rats in each diabetic group compared with the normal rats were significantly reduced (P<0.05).The rats in C, D, E, F treatment groups, body weight increased compared with rats in non-treated group.Compared with rats in B group,body weight of rats in D group was significantly increased (P<0.05). Renal hypertrophy index of diabetic rats was significantly higher than the rats in normal control group (P <0.05).Renal hypertrophy index of rats in diabetic treatment groups decreased compared with rats in diabetic non-treated group.Compared with rats in diabetic non-treated group,renal hypertrophy index of rats in D, E and F groups decreased significantly (P< 0.05).3. The levels of GLU and TG of rats in each diabetic group were significantly higher than those in normal group (P<0.05).HDLC and LDLC in blood of diabetic rats was significantly lower than those in normal group(P<0.05). The levels of GLU, TG, HDLC, LDLC in rats of diabetic treated groups and non-treated group were not statistically different (P> 0.05). There was no statistical significance in the level of CHOL among each group.4. BUN, Ccr, UPRO24, UMA was significantly higher in rats in diabetic groups than those in normal group (P<0.05).BUN, Ccr, UMA in rats of diabetes treatment groups was significantly lower than those of non-treatment group (P<0.05).24-hour urine protein in D, E and F treatment groups decreased significantly compared with non-treated group (P<0.05).5.Concentration of AngⅡin serum of rats in B, C, E and F groups was significantly higher than rats in A group (P<0.05),There was no statistical difference between A group and D group. Serum levels of AngⅡin diabetic treatment groups were significantly lower than diabetic non-treatment group(P<0.05).Serum levels of AngⅡin C and E groups were significantly higher than in F group(P<0.05).There was no was no statistical difference among serum levels of AngⅡin A,D and F groups.The levels of AngⅡin renal tissues of diabetic rats were significantly higher than normal rats(P<0.05).Renal content of AngⅡin diabetic treatment groups decreased significantly compared with diabetic untreated group(P<0.05).There was no statistically significant differense in renal content amongD, E and F groups (P> 0.05).6.VEGF, RAGE, CTGF and p-ERK1/2 protein in B, C, D, E and F groups increased significantly than those in A group (P <0.05).Expression of VEGF, RAGE, CTGF and p-ERK1/2 protein in D, E and F groups was significantly lower than in B group(P<0.05).Total-ERK1/2 protein in each group was not significantly different.7.Levels of RAGE mRNA in D,E and F groups decreased significantly compared with B groups(P<0.05). D,E group rat kidney tissue levels of CTGF mRNA in D and E groups were significantly lower than in B and F groups(P <0.05).Conclusion:1.Tongluo recipe has a protective effect on kidney of diabtic rats of type1.The effection does not rely on the levels of blood glucose and blood lipid.2.The protection mechanisms of Tongluo recipe on renal may be related to blocking ERK1/2 signal transduction pathway by inhibiting the expression of AngⅡ,RAGE,CTGF and VEGF. |
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