The Expression Of NF-κB, MCP-1, FN And The Intervention Of Cordyceps Sinensis Cultivated By Artificial Fermentation And Rosiglitazone In Kidneys Of Diabetic Rats

Objective: Diabetic nephropathy (DN) is the most important microangiopathies of diabetes mellitus (DM). The pathogenesis of DN includes abnormal polyol pathway, formation of advanced glycation end products (AGEs), change of glomerular hemodynamics, oxidative stress, dyslipidemia. In addition, a low degree of inflammation is also increasingly considered to contribute to the development of glomerular damage in diabetes. Nuclear factor-kappa B (NF-κB) is a nuclear transcription factor widely presenting in various cell, which is involved in intracellular signaling and regulation of expression of many genes. The activation of NF-κB can lead to the release of a variety of inflammatory mediators. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine secreted by actived macrophages and mesenchymal cells, which plays a significant role in the recruitment of monocyte and T lymphocytes. MCP-1 can induce monocyte and endothelial cells to express adhesion molecules, it also can induce the aggregation of various inflammatory cells especially monocyte. MCP-1 promoter and enhancer regions contain NF-κB binding sequences. NF-κB is one of the important transcription factors that regulates the gene expression of MCP-1. As the major component of glomerular extracellular matrix (ECM), fibronection (FN) has apparent ability to combine fibrin, fibrinogen and collagen. The persistent increase of FN can promote renal fibrosis and sclerosis. Cordyceps sinensis (Berk.) Sacc is a kind of valuable, traditional and invigorating herbs in our country. It confirms that Cordyceps sinensis (Berk.)Sacc has effects of reinforcing kidney and lung, improving fragile conditions, and assisting pneuma. Rosiglitazone, one of the thiazolidinediones (TZDs), is an effective selective agonist of the nuclear receptor peroxisome proliferator-activated receptor-γ(PPAR-γ), which can play a role in renoprotection through a variety of ways, including regulating glucose and lipid metabolism, improving insulin resistance, antihypertensive, improving inflammation and atherosclerosis. In this study, Type 1 diabetic rat model was established by intraperitoneal injection of streptozocin (STZ). Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone were applied to the diabetic rat. The expression of NF-κB, MCP-1 and FN in the rat kidney was detected by the use of pathological morphology and molecular biology to explore the possible effect of NF-κB, MCP-1 and FN in the occurrence and development of diabetic nephropathy. At the same time, the renal protective mechanism of Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone were investigated in this study.Methods: 60 eight weeks male SD rats were divided into two groups randomly: 15 normal control rats (group A) and 45 test rats. Test rats were injected with STZ (55mg/kg) intraperitoneally. 72 hours later, the rats whose blood glucose (BG) levels were higher than 16.7mmol/L in the subsequent 3 days were considered as diabetic rats. The diabetic rats were randomly divided into three groups: diabetic control rats (group B), diabetic rats treated with 2.4g/kg/d Cordyceps sinensis cultivated by artificial fermentation (group C) and diabetic rats treated with 4mg/kg/d rosiglitazone (group D). The experiment lasted 12 weeks. BG and body weight were measured at the beginning of the experiment and the end of 0 weeks, 4 weeks, 8 weeks, 12 weeks. At the end of the experiment, 24-hour urine was collected using metabolic cages and 24-hour urine albumin excretion (UAE) was measured by radioimmmunoassay. At the end of 12 weeks, serum creatinine (Scr), blood urea nitrogen (BUN), serum triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT) were detected. Kidney specimens were prepared for H.E stain and PAS stain to observe the changes of renal morphous. The protein expression and location of NF-κB, MCP-1 and FN in rat kidney were examined by immunohistochemistry. The expression of NF-κB in renal cortex was determined by Western-blot. All the experimental data were dealt with SPSS 13.0. If multiple sets of variables were consistent with homogeneity of variance, analysis of variance (ANOVA) was used to compare multi-group variables, LSD test was used to compare inter-group variables. Kruskal-Wallis H test was used to compare level variables, Nemenyi test was used to compare inter-group variables.Results:1 At the end of 4 weeks, 8 weeks, 12 weeks, the body weight of group B, C and D were significantly lighter than group A (all P<0.01). The BG of group B, C and D were significantly higher than group A (all P<0.01). There was no significant difference in body weight and BG of group B, C and D (all (P>0.05).2 At the end of 12 weeks, the renal hypertrophy index of group B was significantly higher than group A (P<0.01), while the parameters decreased obviously in group C and D compared with group B (all P<0.01).3 The biochemical parameters of rats at the end of 12 weeks: BUN, Scr and UAE of group B were significantly higher than group A (all P<0.01), while all the above parameters decreased obviously in group C compared with group B (P<0.01, P<0.05, P<0.01) and all the above parameters decreased obviously in group D compared with group B (all P<0.01). Serum TC of group B was significantly higher than group A (P<0.01), while serum TC of group C decreased obviously compared with group B (P<0.01). There was no significant difference in group D and B. Serum TG of group B was significantly higher than group A (P<0.01), while serum TG of group C and D decreased obviously compared with group B (all P<0.01). ALT of group B, C and D were significantly higher than group A (all P<0.01), and there was no significant difference in group B, C and D (all P>0.05).4 H.E stain shows, in group B, the glomerular became larger and there were significant mesangial matrix hyperplasia. The glomerular basement membrane (GBM) and glomerular capsule walls were thickened. There were vacuolar degeneration in renal tubular cells and the tubular lumens were narrowed. The glomerular lesions of group C and D were significantly lighter than group B.5 PAS stain shows, in group B, there were significant glomerular mesangial extracellular matrix hyperplasia, and PAS stain was strongly positive. There were some improvements in group C and D compared with group B.6 Immunohistochemistry shows, the positive expression of renal NF-κB, MCP-1 and FN in group A were not significant, while the positive expression of the factors were significantly increased in group B compared with group A (all P<0.01), and there were strong expression in the glomerular basement membrane, renal tubule and interstitial. The positive expression of the factors in group C and D decreased compared with group B (all P<0.05).7 Western blot shows, compared with group A, the protein expression of NF-κB were significantly increased in group B (P<0.01). The protein expression of NF-κB in group C and D were significantly decreased compared with group B (all P<0.01).Conclusions:1 The rats were intrapenitoneally injected a high-dose STZ to severely destroyedβcells of pancreas islet and lead to an absolute lack of insulin secretion, by which the rat model similar to the clinical of type 1 diabetes can be established.2 The protein expression of NF-κB, MCP-1 and FN in diabetic model rats were significantly higher than normal contral group, which shown that the inflammatory responses involved NF-κB, MCP-1 and FN were closely related to the diabetes.3 Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone had renal protective effects through inhibiting NF-κB-mediated inflammtory response and reducing renal monocyte-macrophage inflitration and fibronectin deposition. The renal protective effects of two drugs were independent of these hypoglycemic effects.