| Objective: Diabetic kidney disease is glomeular sclerosis caused by diabetic metabolic abnormalities. Once diabetic nephropathy patients developed into overt nephropathy, they will finally progress to the end-stage kidney disease. Podocy injury during the diabetic nephropathy is the main pathogenesis of microalbuminuria?glomerulomegaly and so on, applying to glomerulosclerosis and decresed renal function. The first find of diabetic kidney disease pathologic change is kidney mesangial cell matrix accumulation and thickening of glomerular basement. According to the study find that high glucose could cause the more positive expression of mesangial matrix protein and matrix accumulation which can result in glomeular dysfunction by and by. Podocyte injury plays a key role in the development of diabetic nephropathy, endothelial cell with window stucture makes up the major defensive line of glomeular filtration barrier. Endothelial could secrete various vasoactive substance consisting of vascular endothelial growth factor. The unusual incresed secretion will lead to glomeular filtrtion membrane up regulating the permeability to plasma protein which can give rise to proteinuria. We study the influence of yiqi-yangyin-xiaozheng-tongluo of traditional Chinese medicine on VEGF and Flt-1 to observe its clinical therapeutic effect.Methods:1 The preparation for yiqi-yangyin-xiaozheng-tongluo of traditional Chinese medicine serum and normal serum12 healthy male SD rats, SPF, weight 200-220 g, provided by experimental animal center of Hebei Medical University, animal certificate No. 1404079.The animals were randomly divided into 3 groups, the normal group, low-dose group, middle-dose serum drug group, high-dose serum drug group. Normal groups were given the corresponding dose of saline, the rest three groups at a low-dose group, according to the formula Rubner-Meeh for clinical half quantity for clinical dosage, middle-dose group, high-dose group is two times of clinical dosage convert lavage doses, boil-free granule of traditional Chinese medicine, purchased from Hebei Medical University affiliated hospital of TCM pharmacy in Hebei province. Each group was given 2 ml dosage, 12 hours apart, the third day 1~2 hours after the last drug,we took blood from heart aseptically 4500 rpm/min centrifuge for 10 minutes to separate serum, 56? constant temperature water bath living for 30 minutes, 0.22?m microporous membrane filtration,-20? refrigerated. Set aside.2 The culture of mice podocyte and the influence of vascular endothelail growth factor?Flt-1 for yiqi-yangyin-xiaozheng-tongluo of traditional Chinese medicine serumConditional immortalized mouse podocytes in 33?, gamma-under the licensing conditions for the existence of ?-IFN training after the wedding, at 37?, the absence of ?-IFN subculture for 10-14 days, promote cell differentiation and mature. Differentiation of mature enough cells were randomly divided into 6 groups: Normal Control group(Normal Control, NC), High glucose group(High Glucose, HG) and Normal Rat serum group(Normal Rat, NR) group, Low-dose drug serum(Low dosed of drug, LD), the dose of drug serum group(Normal dosed of drug, ND) group, High-dose of drug serum(High dosed of drug, HD), NC group cells were given culture medium containing D- Glucose tendency for 5.5 L+5% fetal bovine serum, HG group was given culture medium containing 30 tendency/L +D-Glucose 5% fetal bovine serum, NR group was given culture medium containing 30 tendency/L+D-Glucose 5% fetal bovine serum+5% to Normal Rat serum, LD group was given culture medium containing 30 tendency/L+D-Glucose 5% fetal bovine serum+5% serum group, Low-dose of Chinese traditional medicine ND group was given culture medium containing 30 tendency/L+D-Glucose 5% fetal bovine serum+5% dose of Chinese traditional medicine serum group, HD group was given culture medium containing 30 tendency/L+D-Glucose 5% fetal bovine serum+5% High dose of Chinese traditional medicine serum group. 24 hours later, collecting enough cells for immunochemical method, western blot, Real- time PCR detection of VEGF?Flt-1 gene and protein expression.Results: The expression of VEGF and Flt-1 were significantly up-regulated in normal rat serum group and high-glucose group, while the level of mRNA and protein were down-regulated in Chinese traditional medicine serum group(P<0.05).Conclusions:1 Mice podocytes under high glucose condition would upregulate the level of VEGF and Flt-1.2 Yiqi-yangyin-xiaozheng-tongluo of traditional Chinese medicine can obviously down-regulate VEGF protein and mRNA expression in mice podocytes under high glucose environment, which confirms that yiqi-yangyin-xiaozheng-tongluo of traditional Chinese medicine can contribute to the treat of diabetic nephropathy by reducing VEGF and Flt-1 mRNA and protein expression. |
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