| Diabetic nephropathy (DN) is an important microvascular complication of diabetic mellitus (DM) which caused the injury of the renal. As DM is growing quickly in the world wide rage, DN has been the first reason result in end-stage renal disease (ESRD) in the western country. The prevalence of DM is also growing fast in our country, according to one estimate there are more than 40,000,000 DM patients, so the ESRD induced by DN is growing continuously.There are characteristic pathological changes in the early stages of DN, such as the hypertrophy of kidney, the thickening of glomerular and tubular basement, the high perfusion and cross membrane pressure in the glomerulus. As DN progresses, there will be extracellular matrix accumulation , such as glomerulosclerosis and interstitial fibrosis.A central concept in understanding the pathogenesis of DN is that it results from a series of specific progressive renal pathological changes that have their onset early in the course of diabetes. These changes develop during along silent period, before features of clinical renal disease-including proteinuria, hypertension, and declining glomerular filtration rate (GFR)-are evident. Central to the nephropathological changes is the accumulation of extracellular matrix,leading to expansion of glomerular mesangial regions at the expense of filtration surface area, and thickening of the glomerular and tubular basement membranes.Evidence is accumulating that macrophages and their products play a significant role in those pathological changes. Increased glomerular macrophages have been observed in rats with experimental diabetes, as well as in biopsy samples of kidney from DM patients. There are associations prove that macrophages accumulate in kidney glomerulus and interstitium of patients with diabetic nephropathy is due to monocyte chemoattractant protein-1 (MCP-1), which is a chemokine produced by both tubular epithelial and glomerular mesangial cells (GMCs). The prolonged hyperglycemia, excessive production of Advanced Glycation End Products, overactivation of Protein kinase C, vasoactive substances like Angiotensin II and increased kidney chemokine production all can induce the production of MCP-1, and then the MCP-1 will recruiting and activating monocytes/macrophages from the circulation to kidney, activating the adhesion molecules and induceing the release of lysosomes, inducing TGF- P 1 and promoting the generation of superoxide anion and Collagens, thus play a key role in the progress of DN.There are clinical findings that urinary MCP-1 levels were significantly elevated in patients with diabetic nephrotic syndrome. And urinary MCP-1 excretion is proportional to the number of MCP-1 positive cells in the renal biopsy specimens. And patients used HMG-coA reducing enzyme inhibitor, angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor (ARB), and euglycemic agent like rosiglitazone all can reduce the levels of urinary MCP-1 and albuminuria excretion. The present study demonstrated that up-regulation of locally produced MCP-1 involved in advanced renal lesions in DN patients possibly through macrophages recruitment.The ginsenoside Rbl and Rgl used in this experiment are extracted from the yunna wenshan notoginseng radix, which are the principal active monomers in Panaxnotoginoside (PNS). Notoginseng is the radix of Araliaceae perennial herb Panax notoginseng ( Burk) F. H. Chen (PNG). The characteristic of PNG according to Chinese medicine is warm, sweet flavor, mini-bitter and entering liver, stomach meridians, with the effect of stop bleeding by removing stasis and arrest pain by promoting blood circulation, reinforcing deficiency and invigorating. With the two effects of promoting blood flow and nourish blood. The ginsenoside Rbl and Rgl are the major active components of PNS and ginseng, interestingly the content of the former is higher than the latter. Rbl, Rgl have many biological functions, the study of their pharmacologic action is range from expanding blood vessel, inhibiting platelet agglutination, prolonging coag time, anti-inflammatory, antioxygen, and to Inducing apoptosis. According to Chinese medicine theory, the change occur in DN is due to blood stasis and zheng qi deficiency, thus medicine with the effects of invigorating blood circulation and reinforcing deficiency have great effect. Notoginseng, radix with the advantage of nourishing the blood without aggravating haemostasis and promoting blood flow without injuring blood is very suitable for DN. The animal experiment and clinical research also indicate that either PNS or its key components Rgl and Rbl all have renal protective effect.In this thesis we investigate the mechanism of Rbl and Rgl in relief DN from levels of tissue, cell, protein and gene.Objective:In order to investigate the mechanism of Rbl and Rgl in relief DN from levels of organ, tissue, cell, protein and gene, we established DN rat model and high glucose cell model then observed the efficiency of Rbl and Rgl in influence MCP-1 gene and protein's expression.Methods:Diabetes induced in SD rats by intraperitoneal injection of streptozotocin (STZ) was used in vivo experiments. 75 SD rats were used in the study, 10 of the rats in normal group except that all the other were injected by STZ, after that the mice whose blood glucose were over 16.7mmol/l and urinary glucose were always over ++++ were recognized as DM animal model. After randomly divided into 5 groups, model group, irbesartan group, Rgl high dose group, Rgl low dose group and Rbl middle dose group, each group were treated accordingly.1.The urine were collected respectively at the end of the 4, 8, 12th week, and24-hour urinary protein were determined by coomassie briliant blue method.12 weeks after intraperitoneal injection of streptozotocin (STZ), all the ratswere sacrificed, the serum was collected for serum biochemical indexesdetermination, such as blood urea nitrogen (BUN), serum creatinine (Scr)and Triglyceride(TG). Kidneys were taken for pathological examination(HE, HE-PASM, and Mallory).2.Immunohistochemistry was used to examine the kidney's expression of MCP-1 protein. 3. In situ Hybridization was used to examine the kidney's expression of MCP-lmRNA.In the vitro study, we investigated the influence of Rbl and Rgl in GMCs' proliferation and expression of MCP-1 protein stimulated by high glucose. The cells were divided into control group, high glucose group, irbesartan group, and Rgl high dose group, Rgl low dose group and Rbl high dose group, Rbl low dose group.1.The rat mesangial cells were cultured successfully in vitro. High glucosewas used to induce proliferation of mesangial cells. Changes of mesangial cells'proliferation were detected by MTT reduction assay at 12 hours, 24 hours, 48hours and 60 hours.2.Western-blot was used to detecting the expression of MCP-1 protein in theintracytoplasm of GMCs stimulated by high glucose.Result:1.Compared with model group, the body weight increased in all the othergroups, but there is no significant statistics difference; The kidney weight/body weight ratio of Rbl group were significantly decreased(P<0.01), Rgl high andlow dose group were also decreased (P<0.05); the Urine protein of Rgl high and low dose group and Rbl group were significantly decreased (P<0.01) at the end of the 4 and 8 week, and alsodecreased (P<0.05) at the end of the 12 week. And Rgl, Rbl also decreasethe biochemical parameters such as BUN, Scr and TG. Compared withmodel group, the BUN of all the other groups were significantly decreased(P<0.01); Scr of Rgl high dose and Rbl group were significantly decreased(P<0.01), and the Rgl low dose group were also decreased (P<0.05); the TGof all the other groups were decreased, but there is no significant statisticsdifference.2.The severe pathological changes of kidney structure in rats with DN wereobserved, while the Ginsenoside-Rgl, Rbl groups were obviously lessened. 3.The expression of MCP-imRNA and protein in the normal group is lowwhile markedly increased in the model group. In compare with model group,the expression of MCP-1 protein of Ginsenoside-Rgl, Rbl groups showedsignificant decrease (P<0.01); And the expression of MCP-lmRNA of Ginsenoside-Rgl, Rbl groups all showed decrease, but only the Rgl high dose group showed statistics difference (P<0.05),4.Rgl and Rbl both have the inhibiting effect on the proliferation of GMCsstimulated by high glucose, while the Rgl high dose group is the best.5.Rgl and Rbl can significantly decrease the over production of MCP-1protein in high glucose stimulated GMCs and with the dose dependenteffect.Conclusion:1.Pharmacodynamic Study found that Rg1, Rb1 may regulate the serum biochemical indexes, reduce the urine protein excretion, and alleviate severe pathological changes of kidney in rats with DN.2.Further study found that Rg1, Rb1 can reduce proliferation of GMCs, anddecrease the over expression of MCP-1 protein and mRNA in both renaltissue and the high glucose stimulated GMCs in vitro. This maybe one ofthe mechanisms in retarding DN renal injury by Rgl, Rbl. |
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