| With the development of economics, people's life style has changed greatly and the prevalence rate of diabetes mellitus has rised so rapidly that it has became one of public problems that severely threaten the health of the people worldwide. Diabetic nephropathy has been the most principal cause of end-stage renal failure. Numerous factors have been recognized as contributing to the pathogenesis and progression of diabetic nephropathy. However, the exact mechanism of action is unclear. It has recently been proposed that oxidative stress plays an important role in the progression of diabetic chronic complications; many relevant experiments and clinical studies have completed to confirm this concept. The current data provide evidence that reactive oxygen species (ROS) generated by glucose metabolism not only do harm to kidney directly, but also play an important role in signaling pathways acting as integral signaling molecules as in other membrane receptor signaling to modulate gene expression. So selecting appropriate antioxidant to alleviate the diabetic glomerular injury has important significance in clinical work.Tea is the most widely consumed beverage in the world. It is believed to have medical efficacy in the prevention and treatment of many diseases, so longevity is often associated with the habit of drinking tea. However, scientific and medical evaluation of tea began only recently according to a literature survey from PubMed. Since 1995, increasing number of publications can be found on green, oolong, and black tea, catechins, and epigallocatechin -3- gallate (EGCG), which is one of the major green tea catechins considered as a cancer, obesity, diabetes, and cardiovascular disease chemopreventative agent. While few epidemiological and clinical studies show the healthy benefits of EGCG on diabetes, the mechanisms may be related to certain pathways, such as through the modulations of energy balance, endocrine system, food intake, lipid and carbohydrate metabolism, the redox status, and activities of different types of cells. Because the EGCG receptor, the so-called 67 kDa laminin receptor (LR), has been discovered with colocalization of other types of LR and cytoskeleton in both cancer cells and normal cells, this may explain that EGCG possesses numerous actions. In the recent years, attention had been concentrated on the antioxidative effects, which are believed to be responsible for its antitumor, anti-aging effect. And it is believed that EGCG is the most effective lipophilic antioxidant so far. However, the exact mechanism of action of this EGCG remains to be elucidated. Present study suggests that it mainly involves two sides: to neutralize free radical directly as electron donor; to reduce oxidative reaction indirectly by activating glutathione peroxidase and inhibiting nitric oxide synthase activity.In this study, we used several study techniques and methods to resolve the following questions: 1. To investigate the effect of EGCG on renal function and morphological changes in kidneys of STZ-induced diabetic rats; 2. To elucidate the existence of oxidative stress in diabetic rats and mesangial cells cultured in media containing high glucose concentration, and to demonstrate the antioxidative effects of EGCG. 3. To investigate the changes of phosphorylated extracellular signal-regulated kinase (pERK) in the kidney of diabetic rats, and the changes of extracellular signal-regulated kinase (ERK), pERK and cyclin kinase inhibitor p27 (p27) in the mesangial cells cultured in high glucose, and to reveal the effects of EGCG on the expression of pERK and p27.Main methods and results:The first part: Methods: diabetes mellitus was induced in male Sprague-Dawley (SD) rats (150-200mg) by an intraperitoneal in injection (65mg/kg) of streptozotocin. Blood glucose levels were measured three days after the injection to ensure a diabetic state (≥16.7mmol/L). Then diabetic rats were assigned to one of the following groups: 1) diabetic control (DM), untreated; 2) diabetic+EGCG treatment ? (the first week after diabetic model); 3) diabetic+EGCG treatment II (the third week after diabetic model). In addition, the normal control group treated with intraperitoneal in injection of citric acid. The treatment started the first and the third week after diabetic model and continued for 12 weeks. Periodically, urine albumen/creatinine value (A/V) was observed in the rats at week 4, 8 and 12 respectively as renal function index. Body weight, blood glucose, plasma cholesterol and creatinine levels were measured as clinical and biochemical parameters; Profile of kidney hypertrophy (KW/BW), mean glomerular area (MGA), mean glomerular volume (MGV) were determined at week 12 when the rats were sacrificed. The histological changes in electron microscope were evaluated as histological damage index. Result: 1. EGCG treatment had significant effects on body weight, blood glucose, plasma cholesterol levels of diabetic rats, especially in EGCG treatment I. 2. EGCG treatment can reduce urinary protein excretion. A/V value was significantly reduced in EGCG-treatment group I from 4 to 12 week, compared with model groups. However, A/V value was decreased in EGCG-treatment group II until 12 week. 3. EGCG treatment decreased kidney weight/ body weight ration, reduced mean glomerular area (MGA), mean glomerular volume (MGV), which were increased in DM rats. The protective effects of EGCG were more remarkable in EGCG treatment I. Electron microscope results also showed EGCG treatment rats had relived basement membrane thickness of diabetic rats.The second part: Methods: 1. Experimental diabetic rats were administered with EGCG for 12 weeks. Blood samples and kidney homogenate were collected for determination of systemic and renal intrinsic anti-oxidant enzyme activities, lipid peroxide (MDA) levels by chromatometry as oxidative stress indies. 2. The rat mesangial cells were cultured in high glucose (30mM). And EGCG at different concentrations was added in the culture medium with high glucose. Anti-oxidant enzyme activities and MDA levels in the culture medium were detected to elucidate the oxidative stress in the high glucose circumstance and the antioxidantive effects of EGCG. Result: 1. Glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities were lower and MDA levels were higher in renal cortex of diabetic rats than those in controls. And erythrocyte SOD activity was decreases while plasma MDA levels increased in the diabetic rats. EGCG treatment increased SOD and GSH-Px activities and reduced lipid peroxide levels in renal cortex of diabetic rats. Moreover, EGCG-treatment group I reversed the decreased of SOD activities in erythrocyte and the increase of MDA levels in plasma of diabetic rats. 2. Rat mesangial cells cultured in high glucose had increased MDA levels and decreased SOD and GSH-Px activities than the normal control group. EGCG at pharmacological concentration reverse all the changes in high glucose group.The third part: Methods: 1.Experimental diabetic rats were administered with EGCG for 12 weeks, then the rats were killed and the kidney were removed, processed for immunohistochemistry staining of pERK to evaluate the protein expression level. 2. The rat mesangial cells were divided into six groups: normal glucose (NG, 5mmol·L-1) control group, high glucose (HG, 30mmol·L-1) control group, HG+EGCG (100μg·L-1), HG+EGCG (200μg·L-1), HG+EGCG (400μg·L-1), Mannitol group. 48h later, the proliferation activity of GMCs was observed by MTT assay, the expression of pERK and p27 protein was examined by Western blot. Results: 1. Compared with CN group, there was significant increase in pERK staining area in the glomerulus of untreated diabetic rats. EGCG treatment significantly suppressed the increased in pERK staining area. 2. Results: Compared with normal glucose group, high glucose significantly increased GMCs proliferation activity and the expression of pERK and p27 protein.Conclusion:1. EGCG treatment can ameliorate the disorders of glucose and lipid metabolism, improve renal function and delay the progression of glomerulosclerosis of diabetic rats.2. EGCG may ameliorate oxidative stress in kidneys of diabetic rats; EGCG at pharmacological concentrations may ameliorate the oxidative stress of rat mesangial cells cultured in the media containing high glucose concentrations; Overproduction of reactive free radicals and the insufficiency of intrinsic anti-oxidant enzymes may contribute to the development of diabetic nephropathy. EGCG treatment can relieve the oxidative stress and proved to be an effective antioxidant.3. EGCG can inhibit pERK protein expression in kidneys of diabetic rats. EGCG may ameliorate the pERK and p27 protein expression of the rat mesangial cells cultured in high glucose. These findings indicate that EGCG exerts obvious renal protective effects and may be used to prevented or cure diabetic nephropathy.
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