| Objective:To explore the mechanism of danzhijiangtang capsule on diabetic nephropathy based on TGF-?1 / Samd signaling pathway, to provide experimental basis for its clinical application, by taking diabetic model rats and human renal tubular epithelial cells?HK- 2? as the research objects.Methods:97 SD rats were fed adaptation for a week. 12 randomly selected for normal control group, normal diet. The rest rats were given high fat diet feeding.After 4weeks, fasting 12 h, i.p 34mg/kg 1%STZ.Fasting blood glucose more than or equal to7.0 mmol / L was the diabetic model standard while we eliminated hyperglycemia rats?blood glucose meter display for high?.65 rats which were molded success were randomly divided into model group, high Danzhi Jiangtang Capsule 1080mg/kg*d,medial 540mg/kg*d, low?270mg/kg*d? dose group, pioglitazone group?pioglitazone10mg/kg*d?, normal group and model group were given equivalent solvent,intragastric administration, twice a day, continuously for 8 weeks of treatment. Before administration,we need to sample blood to detect the total cholesterol, triglyceride,blood glucose. During the drug administration, the body weight of rats were measured every week, the food intake, water intake were measured every 2 weeks, random blood glucose were measured every 4 weeks.At the eighth week, 24 h urine was collected to record the amount of urine, urine protein, albumin. Last after administration, rats were given for abdominal aortic blood to measure TC, TG,glycated hemoglobin, blood urea nitrogen, blood creatinine. We took one side of the rat kidney, 4% neutral poly formaldehyde solution fixed, embedded in paraffin wax.Sections were stained with He, Masson and PAS method to observa rats' kidney glomerular structure, basement membrane thickening and tissue fibrosis.of TGF beta1, Smad2, Smad3, Smad7, CTGF protein expression in kidney tissue were detected by immunohistochemical method.We choose HK- 2 cells as the research object. Different glucose concentration groups: normal group?5.5 mm D-Glucose?, high glucose group?30mm D-Glucose?and low sugar group?15mm D-Glucose? cultured for 24 hours. Then we extraced totalprotein to detect HK-2 cell TGF beta 1 protein expression with Western blot.Drug intervention groups: normal group?5.5 mm D-Glucose?, mannitol and model group?30mm D-Glucose? control group(5.5 mm D-Glucose+24.5m M mannitol, blank serum group(30mm D-Glucose+10% normal in the rat serum and high, in, low dose?10% of 30 mm D-Glucose+, in, low dose Medicated Rat Serum? group, positive control group?30mm D-Glucose+10 m of valsartan?,cultured for 72 h, detected apoptosis with AO-EB method. After culturing for 24 h, we extraced total protein to detect HK-2 cell TGF beta 1, Smad2, Smad3, Smad7, CTGF protein expression with Western blot.Results:1.Effect of Danzhijiangtang Capsule on the general signs of DM rats.After 4 weeks of high fat diet, compared with normal control group, the body weight of rats increased significantly. Small dose injection of STZ after modeling,body weight of rats in model group decreased from the fourth week, the body weight of model group was significantly lower than that in normal control group.After modeling, the rats in the model group the food intake and water intake were significantly increased compared with normal group?P < 0.01?. After 8 weeks of treatment, the treatment groups, food intake and water intake than those in the model group decreased in different extent?P < 0.01?.2.Effect of Danzhijiangtang Capsule on blood glucose & blood lipidlevel in DM ratsAfter modeling, BG, Hb Alc, TG and TC of the rats in model group was significantly higher than the normal group, and after has maintained this level?P<0.01?. After 4W administration, rats in each treatment group BG, Hb Alc, TG and TC decreased significantly, significantly lower than the model group?P<0.01,P<0.05?.3.Effect of Danzhijiangtang Capsule on renal function of DM ratsCompared with the normal control group, model group rats of TGF-?1,BUN,SCr, m Alb, urine protein levels were significantly increased?P < 0.01?; after 8 weeks of treatment, the rats in the treatment groups compared with model group, TGF-?1,BUN, SCr, m Alb, urine protein levels were decreased significantly?P < 0.01, P <0.05?.5.Effects of Danzhijiangtang Capsule on renal pathology in DM ratsThree kinds of pathological staining showed that normal glomerular structural integrity, Bauman's pouch is clearly visible, no inflammatory cell infiltration. Model group: glomerular hypertrophy, change Bauman bursa shape, the glomerular and tubular basement membrane thickening, a large area of fibrous tissue hyperplasia,extracellular matrix increased, inflammatory cell infiltration. Compared with the model group, the treatment group had different degrees of improvement, the glomerular and tubular basement membrane thickening decreased, extracellular matrix increased decreased, hyperplasia of fibrous tissue to reduce, reduce the infiltration of inflammatory cells.Semi quantitative pathological score also showed the same results. Model group three staining pathological score and normal control group compared to the increased significantly?P < 0.01?; rats in the treatment groups compared with model group,three kinds of staining scores appeared different degree decreased?P < 0.01, P <0.05?.6.Danzhijiangtang Capsule on DM in renal tissue of rat TGF- beta 1/Smad signaling pathwayTGF beta 1, Smad3, CTGF in normal group, glomerular tissue expression is very weak; model group of TGF beta 1, Smad3, CTGF protein expression was strong positive, significantly higher than the normal control group?P < 0.01?; rats in the treatment groups compared with model group, TGF beta 1, Smad3, CTGF protein expression appeared different degree decreased?P < 0.01, P < 0.05?.Smad2 and Smad7 in normal group, glomerular tissue expression is strong; in the model group of Smad2 and Smad7 protein expression decreased significantly?P < 0.01??P < 0.01?compared with the normal control group, rats in the treatment groups compared with model group, Smad2 and Smad7 protein expression appeared different degree of increase?P < 0.01?.7.Effect of high glucose on the expression of TGF- beta 1 in HK-2 cellsHigh glucose induced 24 hours, the high glucose group and low glucose group TGF- beta 1 was significantly higher than that of normal control group?P<0.01?. High glucose induced HK-2 cells increased significantly TGF- beta 1 protein expression.8.Effects of Danzhijiangtang Capsule on high glucose induced apoptosis in HK-2cellsAfter 24 h culturing, HK-2 cells in model group than normal control group significantly increased apoptosis; mannitol group compared with normal control group the apoptosis has not increased; the blank serum group compared with the model group did not reduce apoptosis. The treatment group compared with model group, the apoptosis appeared different degree of decline.9.Effect of Danzhijiangtang Capsule on expression of TGF-?1/Smad pathway protein in HK-2 cells induced by high glucoseAfter 24 h culturing, HK-2 cells in model group compared with normal control group, TGF- beta 1, Smad3, CTGF protein expression increased significantly?P<0.01?, the expression of Smad2 and Smad7 protein decreased significantly?P<0.01?. Mannitol group compared with normal control group TGF- beta 1, Smad3,CTGF, Smad2, Smad7 protein expression showed no significant difference?P > 0.05?.The blank serum group TGF- beta 1, Smad3, CTGF, Smad2, Smad7 protein expression was also compared with the model group had no significant difference?P >0.05?. The treatment groups compared with model group TGF- beta 1, Smad3, CTGF protein expression decreased?P<0.01?, the expression of Smad2 and Smad7 proteins of varying degrees of increase?P<0.01?.Conclusion:Danzhijiangtang Capsule has significant hypoglycemic and hypolipidemiceffects of DM rats. It can significantly improve the index of kidney function in DM rats, renal pathological lesions, reduce target organ damage, and its mechanism may be related to effecting TGF- beta 1/Smad signaling pathway and its downstream CTGF expression, inhibiting extracellular matrix generation,accumulation, improving renal tissue fibrosis, reducing apoptosis. |
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