The Experiment Research Of Supplementing Qi-Nourishing Yin-Dispersing Blood Stasis-Dredging Collateral Herbs On Preenting And Treating The Early Diabitic Nephropathy

Objective:Diabetic nephropathy (DN) is also known as diabetic glome-rulosclerosis and is one of diabetic microvascular complications in the body. Accordingto General Statistics, DM can damage the kidney through a variety of ways, and all structures kidney are involved, from the glomerulus, renal tubules and blood vessels until the interstitial. DM is the main reason leading to end-stage renal disease(ESRD) and is predicted to become the major cause of ESRD in developing countries. Therefore, the effective prevention of the occurrence and development of DN is important. Over the years, kidney scholars at home and abroad have continued to explore the pathogenesis and treatment of diabetic kidney disease, but the pathogenesis remains unclear and they didn't find effective treatment.so looking for to prevent or delay the development of diabetic nephropathy an effective way to and the method has become a major issue worldwide. Over the last decade, the Chinese medicine sector in China has carried out a wide range of Chinese medicine prevention of diabetic nephropathy, and gradually show greater potential and broad prospects for application. We invented an effective traditional medicine, according to the pathogenesis which is at the early stage of DN and clinical experience for treating DN. This medicine has the functions of promoting circulation of qi and yin, dispersing blood stasis and dredging collateral which was composed of astragali, figwort root, carapax amydae, angle worm, ect. The clinical data showed that the prescription had good effect on the early stage of DN. This study aim to observe the curative effect of Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs and investigate the mechanism in glomerular podocytes, slit membrane and mesangial cells in DN rats. Motheds:1 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on the treatment in DN rats:110 male Sprague-Dauley(SD) rats weighing 250±20 g (SPF level) were carried out left-nephrectomy after making sure the rats'urine protein negative when fed for one week. Two weeks later, dividing the rats randomly into the simple nephrectomy group and the model group which were injected intraperitonally with 40mg·kg-1 streptozotocin. Blood glucose levels were measured for three times continuously(≥16.7 mmol·L-1) on the third day after streptozotocin injection to confirm the development of diabetes. Dividing the model rats randomly into 5 groups:model group, irbesartan group, TCM low dose group, TCM middle dose group and TCM high dose group. Rats in irbesartan group were orally administered with 15 mg·kg-1·d-1 irbesartan, three TCM groups with 5.29 g·kg-1·d-1,10.58 g·kg·d-1,21.16 g·kg-1·d-1 TCM respectively, and the left groups with the same dose of Sodium Chloride everyday for six weeks. At the end of 6th week, collecting 24h urine, body-weighing, obtaining blood samples by puncturing femoral artery, killing rats, right-kidney-weighing, obtaining renal cortex, detecting 24h urine protein (U Pro), blood glucose, glycohemoglobin(HbA1C), total cholesterol(TC), triglyceride (TG), serum creatinine (Scr) and urea nitrogen (BUN) level.2 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on renal histopathology and ultrastructure in DN rats:DM model was duplicated the same as the above. After 6 week of STZ injected, the renal tissue were obtained and observed them by light microscopy and conventional transmission electron microscopy examination. By observing morphological changes before and after treatment, to explore the role of ZY on DN.3 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs on glomerular podocytes and slit membrane in DN rats:DM model was duplicated the same as the above. After 6 week of STZ injected, the renal tissue were obtained. Nephrin, Podocin, CD2AP and WT-1 were measured by immunohistochemical method and RT-PCR.4 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on mesangial cells secreteCTGF,TGF-β1 and ECM in rats of high glucose jointing AngⅡ:In this study by vitro cell culture technology of high glucose environment, to make McS proliferation induced by AngⅡ. After the rats' serum intervention, to the mounting of mesangial cells and extraction of cytoplasmic proteins, By Elisa method to detect mesangial cells, TGF-β1, fibronectin (FN), laminin (LN) of the protein and by western blotting to detect CTGF.From cell level to explore the mechanism of delayed glomerulosclerosis and provide evidence for further study the mechanism of prevention of glomerular sclerosis.Results:1 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on the treatment in DN rats:1.1 The 24 hour urinary protein quantitative in each groupUPro levels of model group and treated group increased(P<0.05), compared with that of simple nephrectomy group. U Pro levels of irbesartan group, TCM low group and TCM middle dose group decreased significantly (P<0.05), compared with that of the model group. But there was no significance statistically between TCM high dose group and model group (P>0.05). There was no significance statistically among irbesartan group, TCM low group and TCM middle dose group(P>0.05).1.2 Comparation HbA1C in each groupBlood glucose and HbA1C levels of model group and treated group increased(P<0.05), compared with that of simple nephrectomy group. There was no significance statistically between the treated groups and the model group(P>0.05).1.3 Comparation of TC and TG in each group TC levels of the model group and the treated groups increased(P<0.01), and TG levels of the model group and the TCM treated groups also increased (P<0.05 or P<0.01), compared with that of simple nephrectomy group. But there was no significance statistically between irbesartan group and simple nephrectomy group about TG (P>0.05). TC and TG levels of all treated groups decreased significantly(P<0.05 or P<0.01), compared with that of the model group. TC levels of irbesartan group, TCM middle group, and TCM high dose group increased(P<0.01), and TG levels of TCM middle group and TCM high dose group increased(P<0.01), compared with that of the TCM low dose group. There was no significance statistically between irbesartan group and TCM low dose group about TG (P>0.05).1.4 Comparation Scr and BUN in each groupScr and BUN levels of the model group and BUN levels of the treated groups increased(P<0.01), compared with that of simple nephrectomy group. There was no significance statistically between the treated groups and the simple nephrectomy group about Scr(P>0.05). Scr levels of the treated groups and BUN levels of TCM low group and TCM high dose group decreased significantly (P<0.05), compared with that of the model group. There was no significance statistically between the other treated groups and model group(P>0.05).2 Pathomorphology observations on kidney of each groupLight microscope:In simple nephrectomy group, there were no obvious hypertrophy of renal glomerulus, thickening of GBM and the change of the MCs quantity, mesangial area and renal tubulointerstitium. In model group, there were obvious hypertrophy of renal glomerulus, thickening of GBM, expansion of mesangial matrix and vacuolar/granular degeneration of renal tubular cell. In irbesartan group and TCM treated groups, the pathomorphology changes above were improved compared with that of the model group. There was no obvious difference among the treated groups.Transmission electron microscopy:In simple nephrectomy group, there were no obvious thickening of GBM, confluence of podocyte foot process and expansion of mesangial matrix. In model group, there were obvious homogeneous thickening of GBM, confluence of podocyte foot process and expansion of mesangial matrix. In irbesartan group and TCM treated groups, the pathomorphology changes above were improved compared with that of the model group. There was no obvious difference among the treated groups.3 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs on glomerular podocytes and slit membrane in DN rats3.1 The mRNA expression of Nephrin, CD2AP, Podocin in renal cortex3.1.1 The mRNA expression of NephrinCompared with nephrectomy group, the mRNA expression of Nephrin in model group and treatment groups was significantly lower (P<0.05); compared with model group, the mRNA expression of Nephrin in treatment groups was significantly higher (P<0.05); there was no significant difference among treatment groups (P> 0.05).3.1.2 The mRNA expression of CD2AP Compared with nephrectomy group, the mRNA expression of CD2AP in model group and treatment groups was significantly lower (P<0.05); compared with model group, the mRNA expression of CD2AP in treatment groups was significantly higher (P<0.01 or P<0.05); there was no significant difference among treatment groups (P> 0.05).3.1.3 The mRNA expression of PodocinCompared with nephrectomy group, the mRNA expression of Podocin in model group was significantly lower (P<0.05); there was no significant difference among other groups (P> 0.05). Compared with model group, the mRNA expression of Podocin in Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose and middle-dose groups was significantly higher (P<0.01 or P<0.05); there was no significant difference among other treatment groups (P> 0.05).3.2 The protein expression of Nephrin, CD2AP, WT-1 in renal tissue3.2.1 The protein expression of Nephrin Compared with nephrectomy group, the protein expression of Podocin inmodel group was significantly lower (P<0.05). Compared with model group, the protein expression of Podocin Irbesartan group in and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose groups was significantly higher (P<0.05). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P> 0.05).3.2.2 The protein expression of CD2APCompared with nephrectomy group, the protein expression of CD2AP in model group was significantly lower (P<0.05). Compared with model group, the protein expression of CD2AP Irbesartan group in and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose groups was significantly higher (P<0.05). There was no significantly difference between Irbesartan group and ZY group (P> 0.05).3.2.3 The protein expression of WT-1Compared with nephrectomy group, the protein expression of WT-1 in model group was significantly lower (P<0.05). Compared with model group, the protein expression of WT-1 Irbesartan group in and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs low-dose groups was significantly higher (P<0.05). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P> 0.05).4 Effect of Supplementing qi-nourishing yin-dispersing blood Stasis dredging collateral herbs on mesangial cells secrete CTGF,TGF-β1 in rats of high glucose jointing AngⅡ4.1 Detection of TGF-p 1 levelsHigh glucose can stimulate secretion of TGF-β1, compared with the control group, there was a significant difference (P<0.01); compared with high glucose, TGF-β1 levels of high glucose+AngⅡgroup were significantly higher; irbesartan group and the Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group could reduce the secretion of TGF-β1, and compared with high glucose+AngⅡgroup, there was a significant difference (P<0.01). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P> 0.05).4.2 Effect of supplementing qi-nourishing yin-dispersing blood stasis-dredging collateral herbs blood-serum on the expression of MCs CTGF proteinThe CTGF protein expression of the other groups increased significantly(P<0.01), compared with that of the control group. The CTGF protein expression of the high glucose+AngⅡgroup increased significantly (P<0.01), compared with that of the high glucose group. The CTGF protein expression of the irbesartan blood-serum group and TCM blood-serum group decreased significantly(P<0.01), compared with that of the high glucose+Angll group. There was no significance statistically between the two treated blood-serum groups(P>0.05).4.3 Detection of FN and LN levelsCompared with the control group, FN and LN levels of high glucose group were significantly higher (P<0.01); compared with high glucose group, FN and LN levels of high glucose+AngⅡgroup were significantly higher; irbesartan group and the Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group could reduce the secretion of FN and LN, and compared with high glucose+AngⅡgroup, there was a significant difference (P<0.01). There was no significantly difference between Irbesartan group and Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs group (P>0.05).Conclusions:1. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could decreased the DN rat's proteinuria,improve the lipid metabolism,decrease Scr and BUN2. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could lessen the damage of nephridial tissue. 3. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could protect the glomerular filtration barrier hole in the outer membrane and podocyte structure and function, reduce proteinuria and delay the development of DN.4. Supplementing qi-nourishing yin-dispersing blood Stasis-dredging collateral herbs could decreased CTGF,TGF-β1 of mesangial cells and reduce extracellular matrix accumulation. This result suggests that mesangial cells can innhibit Mesangial cells to secreteCTGF,TGF-β1 and reducing the accumulation of ECM may be one of the important mechanisms that the herbs delayed glomerulosclerosis.