| Objective: Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes mellitus (DM). Long-term hyperglycaemia and metabolic disorders activates many pathways and causes renal pathological irreversible changes. DN, a principal cause of end-stage renal diseases, is characterized by glomerular sclerosis, thickening of glomerular basement membrane and extracellular matrix deposition. The pathogenesis of DN is not clear at present. Pigment epithelium-derived factor (PEDF) is a potent angiogenic inhibitor, that can protect neuronal nutrition, regulate vascular permeability and antiangiogiogenic activity. The synthesize and/or degradation of extracellular matrix is abnormal in the process of occurrence and development of DN. Matrix metalloproteinases (MMPs) play an important role in the metabolism of extracellular matrix. MMPs are classified into five groups based on substrate. MMP-2 belongs to gelatinases and is expressed in Mesangial cell, endotheliocyte, fibroblast and so on. It degradations typeⅣcollagen majorly. The alterations of expression and activity of MMP-2 may participate in genesis and development of diabetic nephropathy. Overexpression of TGF-β1 in diabetic glomeruli is believed to contribute to the glomerular hypertrophy, matrix accumulation, filtration barrier lesion, meanwhile inhibiting MMPs synthesis, diminishing ECM accumulation, accelerates the development of DN. Recent studies show that PEDF inhibits oxidant stress reactions, fiber production and inflammatory reaction, thus ameliorates renal hemodynamics and maintains renal homeostasis, and may be responsible for its salutary effect in DN. Cordyceps sinensis (Berk.)Sacc is a valuable traditional chinese medicine thainrich in polyoses, amino acids, fatty acid, mannitol and many kinds of microelements. Researches show that its protection may function by lowering inflammation reaction, inhibiting Mesangial cell proliferation and so on. Rosiglitazone is thiazolidinediones (TZDs) euglycemic agent and is believed to work through binding and modulating the activity of a family of nuelear transcription fators termed peorxisome proliefrator-activated receptorγ(PAPRγ). It is associated with slow improvement in glycemic control, antiinflammatory, regulation of lipid metabolism, inhibition of cell growth fators, reduction of urine protein excretion, etc.In the study, rat model of type 1 diabetes was set up by an high-dose intraperitoneal injection of streptozotocin (STZ), and Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone was applied for intervention. Applying the methods of biochemistry, pathomorphology for detecting the changes of renal morphous and function and the expression of PEDF, MMP-2 and TGF-β1, explore the effect of PEDF and MMP-2 in the genesis and development of diabetic nephropathy and protection of Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone.Methods: 60 eight weeks male SD rats were divided into two groups randomly: 15 normal control rats (group A) and 45 test rats. STZ (55mg/kg) was injected intraperitoneally to destroy some pancreas in order to induce hyperglycemia for test rats. 72 hours later, the rats whose blood glucose level was higher than 16.7 mmol/L in the subsequent 3 days were considered as diabetes. The diabetic rats were randomly divided into following groups: diabetic model rats (group B), diabetic model rats treated with 2.4g/kg/d Cordyceps Sinensis cultivated by Artificial Fermentation (group C), diabetic model rats treated with 4mg/kg/d rosiglitazone (group D). The experiment lasted 12 weeks. Blood glucose and body mass were measured at the beginning of the experiment and at the end of 0 weeks, 4 weeks, 8 weeks and 12 weeks respectively. After 12 weeks, 24 hour urine volume was collected with metabolic cage. Serum total cholesterol (TC), serum triglyceride (TG), blood urea nitrogen (BUN), serum creatinine (Scr), alanine aminotransferase (ALT), kindey weight index (KWI) and 24 hour urinary albumin excretion (UAE) were detected. Morphological changes were observed by microscope; The protein expression and location of PEDF, MMP-2 and TGF-β1 were examined by immunohistochemistry. Moreover, PEDF mRNA expression in kidney was analysed with reverse transcription polymerase chain reaction (RT-PCR). All the experimental data were dealt with SPSS13.0. Group comparisons adopt analysis of variance, the comparison of two sets adopt LSD test, level data adopt Kruskal-Wallis test.Results:1 72h after injecting STZ, blood glucose in B, C, D group, all over 16.7 mmol/L, was markedly increased compared with A group (P all<0.01). It suggested the model of type 1 diabetic mellitus was set up. At the end of 4, 8 and 12 weeks, blood glucose in B, C, D group all significantly higher than A group (P all<0.01).2 At the end of 72h after injecting STZ and 4, 8 and 12 weeks, body mass in B, C, D group were markedly decreased compared with A group (P all<0.01). KWI in B, C, D group were markedly increased compared with A group (P all<0.01). KWI in C, D group were markedly improved compared with B group.3 At the end of 12 weeks, urinary albumin excretion (UAE) in B,C,D group were significantly increased compared with A group (P all<0.01). UAE in both C and D group were significantly decreased compared with B group (P all<0.01).4 Compared with A group, TG and TC in B, C, D group were increased (P all<0.01), TG and TC in C group were decreased compared with B group (P all<0.01); TG in D group were decreased compared with B group (P<0.01), while TC had no difference (P>0.05). Compared with A group, Scr, BUN and ALT in B, C, D group were increased (P<0.01, P<0.01, P<0.01; P<0.05, P<0.01, P<0.01; P<0.01, P<0.01, P<0.01); Scr, BUN in C, D group were decreased compared with B group (P all<0.01), while TC had no difference (P>0.05).5 Compared with A, serum level of PEDF and TGF-β1 was significantly increased in B, C, D group (P all<0.05). Compared with B group, serum level of PEDF and TGF-β1 was decreased in C, D group (P all<0.05).6 H.E. stain and PAS stain showed that compared with A, glomerular hypetrophy, thickening of glomerular basement membrance, mesangial expansion and cell proliferation in B group. A few glomerulars appeared sclerosis lightly, some renal tubules showed vacuolar degeneration. C and D group were ameliorated than B group.7 Immunohistochemistry suggested that compared with A, expression of PEDF and MMP-2 in B group was obviously decreased, while that of TGF-β1 was significantly increased (P all<0.05). Expression of PEDF and MMP-2 in C, D group were meliorated compared with B group (P all<0.05), the expression of TGF-β1 downregulated (P<0.05).8 Reverse transcription-polymerase chain reaction (RT-PCR) results showed that: compared with A group, the expression of PEDF mRNA was decreased significantly in B, C and D group (P<0.01, P<0.05, P<0.05); the expression of PEDF mRNA was markly upregulated in C and D group (P all<0.01).Conclusion:1 Rat model of type 1 diabetic mellitus can be set up by high-dose intrapenitoneal injection STZ. The model characterized by significantly diabetic symptoms analog to human, such as continuous hyperglycemia, polyuria, weight reduction and so on. So this model can satisfy the need of diabetic complication researches.2 With the persistence of high blood glucose, irreversible damages appeared in diabetic rat kidneys, pathological structure damaged and renal function declined. Morphology and laboratory tests are in line with changes in diabetic kidney damage. Dysglycemia can cause dyslipidemia and hepatic dysfunction. These disturbances may play an adverse role in organism.3 Interaction between PEDF, MMP-2 and TGF-β1 may exist in diabetic rat kidneys. Changes in the expression levels of the three may promote the progression of diabetic kidney damage. 4 Serum content of PEDF and TGF-β1 elevated in diabetic rats, indicating that PEDF may be compensated increased by other means and interacted with TGF-β1 in the pathogenesis of diabetic kidney damage.5 After treated by Cordyceps sinensis cultivated by artificial fermentation and rosiglitazone, kindey function and pathologic changes were improved noticeably. The results showed it could protect the kindey through ameliorating renal function, reducing the level of urine albuminuria, regulating the levels of serum lipids and the expression of PEDF, MMP-2 and TGF-β1. |
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