| In recent years, with people's lifestyle and diet changes, the incidence of diabetes has increased year by year. Diabetic nephropathy is one of serious complications of diabetes. It is a main cause of end-stage renal failure patients. But its pathogenesis is not entirely clear so far. With the deepening of research, it is considered that the typical pathological changes of DN are kidney hypertrophy, basement membrane thickening, and glomerular accumulation of extracellular matrix, which is mainly related to glucose and lipid metabolism disorder, renal hemodynamic changes in local, cytokines, family and other hereditary factors. The renal protective mechanism of statin, Thiazolidinediones has also been given more and more and more attention. This provided useful tips and important theoretical basis for early treatment for DN.Objective: To investigate the impact of atorvastatin, rosiglitazone and atorvastatin combine rosiglitazone joint intervention about TGF-β1, TIMP-1, MMP-2, MMP-9 and c-fos mRNA and protein expression level in diabetic rats'renal tissue. Explore the relationship between the expression of cytokine such as TGF-β1, TIMP-1, c-fos, MMP-2, MMP-9 with renal tissue microstructure changes. Study the advantages of atorvastatin combine rosiglitazone joint intervention on the kidney tissue of diabetic rats.Methods:1 Trial animal group:75 male SD rats were randomly divided into the following five groups:A group: normal control group; B group: DM group; C group: Atorvastatin intervention group; D group: Rosiglitazone intervention group; E group: Atorvastatin combine Rosiglitazone joint intervention group. Every group has 15 rats. At the end of the experiment, the diabetic rats died 5, atorvastatin intervention group died 2, rosiglitazone intervention group died 1, and joint intervention group had no deaths.2. Research methods and observation indicators:Blood glucose was measured by American Johnson SureStep Glucometer; Lipid was measured by Humalyzer 2000 semi-automatic biochemical analyzer conventionally; AngiotensinⅡ, Crea was measured by homogeneous competition RIA. 24h urine was collected by Metabolic cage, then preserved in -20℃refrigerator. Coomassie brilliant blue method was used to determining the urinary protein. RT-PCR was used to study the above-mentioned gene expression .The protein expression of TGF-β1, mmp2, mmp9, TIMP-1,c-fos were measured by immunohistochemical method. The expression of Collagen changes were examined with Masson staining, and HE staining to observe morphological changes of kidney tissue. Results:1 Changes in the kidney and the impact of drug intervention:Normal control group:Diet and growth were normal. Rats were in good mental status, shiny fur, and acted freely with quick reaction. Diabetic rats showed polydipsia, polyuria, many food, fur rough impatient, unresponsive, reduced activities and other symptoms, which requiring every day for one or two litter. The Serum creatinine, 24h urine protein and kidney weight / body weight ratio in diabetic rats was significantly higher than the normal control group (147.20±1.91 vs 54.89±1.03 ;0.0446±0.0010 vs 0.0165±0.0005 ; 0.0088±0.0003 vs 0.0039±0.0006, P<0.01).Compared with diabetic group, atorvastatin, rosiglitazone and joint intervention group serum creatinine levels were: 94.78±6.13, 86.89±3.05, 72.44±2.15; 24h urine protein were: 0.0356±0.0009, 0.0300±0.0003, 0.0264±0.006; kidney weight / body weight ratio were: 0.0049±0.003, 0.0046±0.003, 0.0042±0.003. There was some improvement than DM group (P<0.05), particularly rosiglitazone combine atorvastatin joint intervention reduce the most significantly.2 The change of serum biochemical indicators:The blood glucose, triglycerides, total cholesterol and angiotensinⅡin Diabetic group were significantly higher than that in the normal control group (20.16±2.35vs5.57±0.87; 2.94±0.16vs0.80±0.19;4.59±0.69vs1.18±0.50;1803.39±144.20v s67.70±15.27, P<0.01). Results after 12 weeks different intervention: the group receiving atorvastatin exhibited a reduction in triglycerides and total cholesterol: 1.76±0.31,3.10±0.30(P<0.01); the group receiving rosiglitazone exhibited a reduction in the blood glucose,angiogenesisⅡ: 12.81±0.72, 724.27±57.18 (P<0.01); Blood glucose, triglycerides, total cholesterol and angiotensinⅡin joint intervention group were: 10.48±1.78, 1.31±0.31, 2.49±0.32, 320.67±33.90, significantly improved than diabetic group(P<0.01).3 Pathologic changes:The changes of histopathology were observed by the HE staining method. There is no obvious pathological change of renal tissue in the control group. The pathological changes of kidney in 12W DM model rats includes glomerular volume increased, in part proximal tubular epithelial cells showing vacuolated degeneration, mesangial areas of pink stained expanded and mesangial matrix markedly increased. It also includes glomerular basement membrane thickening, stenosis or even occlusion of capillaries, mesangial cell proliferation, and scattered infiltration of mononuclear cell infiltration in tubulointerstitium. On the basis of these, it is noted that the model has emerged the typical pathological changes of kidney damage. Rosiglitazone combine Atorvastatin joint intervention group shows that the above-mentioned pathological changes have improved significantly. Rosiglitazone group, atorvastatin group also shows that the above-mentioned pathological changes in varying degrees to alleviate.4 Kidney tissue collagen metabolism changes:The change of collagen metabolism was observed by Masson staining. There was little collagen fiber deposited glomerular basement membrane (GBM) in the control group, which areal density of collagen is 0.0123±0.0006. Compared with normal control group, Glomerular capillary basement membrane and renal interstitial collagen fibers increased and were much disorder in diabetic group, which the areal density of collagen is 0.2672±0.0081. Renal tissue staining in Rosiglitazone combine Atorvastatin joint intervention group is obviously on sparse. The areal density of collagen is 0.0448±0.0005(P<0.01), which is significantly lower than the diabetic group. At the same time, the accumulation of collagen fibers around Glomerular capillary basement membrane and interstitial significantly reduced, with the rules tend to. The areal density of collagen of atorvastatin and rosiglitazone single intervention group is respectively 0.0956±0.0026,0.0907±0.0044,which is no statistical significance ( p>0.05).5 Gene and protein expression:5.1 TGF-β15.1.1 TGF-β1 protein expression:Immuohistochemical study showed that: In the control group, there was little TGF-β1 Protein presented in glomerular as well as tubular (0.0641±0.0020).However, the expression of TGF-β1 protein was remarkably raised in diabetic rats. It shows a clearly visible brown strong positive staining in glomerular, tubular and interstitial, which the positive staining optical density was significantly higher than the normal control group (0.1683±0.0070, p<0.01). Compared with the DM group, TGF-β1 protein level in joint intervention group decreased significantly (0.0992±0.0067, p<0.01), with these sites staining intensity and dyeing area decreased. Compared with the diabetic group , TGF-β1 protein levels in atorvastatin, rosiglitazone intervention groups were 0.1345±0.0039, 0.0992±0.0067, which shows a downward trend (p<0.05).But there is no statistical difference between the two groups (p>0.05).5.1.2 TGF-β1 mRNA gene expression:Gene expression showed: In normal control group, TGF-β1 mRNA relative expression level is 0.34±0.03. Compared with normal control group, the relative expression levels of TGF-β1 in diabetic group increases: 1.67±0.07(p<0.01). After 12 weeks intervention, TGF-β1 relative expression levels in atorvastatin, rosiglitazone and rosiglitazone combine atorvastatin united group were: 1.02±0.05,1.11±0.05,0.66±0.04, which shows a downward trend, especially joint group most lower (p <0.01).But there has not been marked difference between the Rosiglitazone and Atorvastatin groups (p> 0.05).5.2 c-fos5.2.1 c-fos protein expression:Immuohistochemical study: In the control group, there was little c-fos protein presented in the renal tissue (0.0748±0.0025). The expression of c-fos protein was remarkably risen in diabetic rats: This shows a clearly visible brown strong positive staining in glomerular, tubular and interstitial, which the positive staining optical density was significantly higher than the normal control group ( 0.1533±0.0062, p<0.01). Compared with the DM group, c-fos protein level in Rosiglitazone combine Atorvastatin joint intervention group decreased significantly (0.1093±0.0060, p<0.01), with these sites staining intensity and dyeing area decreased. Compared with the diabetic group, c-fos protein levels in atorvastatin and rosiglitazone intervention groups were 0.1286±0.0034, 0.1362±0.0035, which shows a downward trend in the volume (p<0.05).But there is no statistical difference between the two groups (p> 0.05).5.2.2 c-fos mRNA gene expression:Gene expression showed: In normal control group, c-fos mRNA relative expression level is 0.12±0.01. The relative expression levels of c-fos mRNA in diabetic rats renal tissue increases: 1.04±0.06 (p<0.01), which is significantly higher than normal control group. After 12 weeks intervention, c-fos relative expression levels in atorvastatin, rosiglitazone and rosiglitazone combine rtorvastatin united group were: 0.38±0.01,0.77±0.04,0.28±0.01, which is significantly lower than diabetic group (p<0.05). It shows a downward trend .But there has not been marked difference between the rosiglitazone and atorvastatin groups (p>0.05).5.3 TIMP-15.3.1 TIMP-1 protein expression:Immuohistochemical study showed: In the control group, there was little TIMP-1 protein presented in the renal tissue (0.0847±0.0051). The expression of TIMP-1 protein was remarkably raised in diabetic rats. It shows a clearly visible brown strong positive staining in glomerular, tubular and interstitial, which the positive staining optical density was significantly higher than the normal control group (0.1883±0.0082, p<0.01). Compared with the DM group, TIMP-1 protein level in rosiglitazone combine atorvastatin joint intervention group decreased significantly (0.1346±0.0060, p<0.01). Compared with the diabetic group, TIMP-1 protein levels in atorvastatin and rosiglitazone intervention of 12 weeks groups were 0.1582±0.0049,0.1539±0.0079, which shows the expression of a downward trend in the volume (p<0.05). There has not been marked difference between the rosiglitazone and atorvastatin groups (p>0.05).5.3.2 TIMP-1 mRNA gene expression:Gene expression showed: In normal control group, TIMP-1 mRNA relative expression level is 0.50±0.03. Compared with normal control group, the relative expression levels of c-fos in diabetic group increases: 1.45±0.07 (p<0.01). TIMP-1 relative expression levels in atorvastatin, rosiglitazone, and rosiglitazone combine atorvastatin united intervention group were: 0.85±0.05, 0.95±0.07, 0.71±0.06. It shows a downward trend than diabetic group, with joint intervention group most significantly lower (p <0.01). There has not been marked difference between the rosiglitazone and atorvastatin groups (p>0.05).5.4 MMP-25.4.1 MMP-2 protein expression:Immuohistochemical study showed: MMP-2 in the normal control group has shown the expression in glomerular, tubular and interstitial, which with an obvious visible brown strong positive stains (0.1406±0.0046). However, MMP-2 stains in DM group are of a significant decrease in that area, with only weakly positive expression (0.0430±0.0025). Compared with the DM group, there is a restorative increase expression in rosiglitazone + atorvastatin joint intervention group (0.1005±0.0055, p<0.01). Compared with the diabetic group, MMP-2 protein levels in atorvastatin and rosiglitazone intervention groups were: 0.0922±0.0053, 0.0816±0.0049. It shows a restorative raised trend in the volume (p<0.05). There is no marked difference between the rosiglitazone and atorvastatin groups (p>0.05).5.4.2 MMP-2 mRNA gene expression:Gene expression showed: In normal control group, MMP-2 mRNA relative expression level is 0.89±0.05. Compared with normal control group, the relative expression levels of c-fos in diabetic rats renal tissue reduces: 0.16±0.01 (p<0.01). MMP-2 relative expression levels in atorvastatin, rosiglitazone, rosiglitazone + atorvastatin united group were : 0.49±0.05,0.42±0.05,0.73±0.07. It shows a restorative upward trend than diabetic group, with joint intervention group most significantly lower (p<0.01). There has not been marked difference between the rosiglitazone and atorvastatin groups (p>0.05).5.5 MMP-95.5.1 MMP-9 protein expression:Immuohistochemical study showed: In the control group, there was little MMP-9 protein presented in the renal tissue 0.0720±0.0033. The expression of MMP-9 protein was remarkably raised in diabetic rats. It shows a clearly visible brown strong positive staining in glomerular, tubular and interstitial, which the positive staining optical density was significantly higher 0.1752±0.0153 (p<0.01). Compared with the DM group, there is a restorative down in rosiglitazone + atorvastatin joint intervention group 0.1180±0.0070 (p<0.01). Compared with the diabetic group, MMP-9 protein levels in atorvastatin and rosiglitazone intervention groups were 0.1516±0.0097, 0.1401±0.0081, which also shows a downward trend in the volume (p<0.05). But there has not been marked difference between the rosiglitazone and atorvastatin groups (p> 0.05).5.5.2 MMP-9 mRNA gene expression:Gene expression showed: In normal control group, MMP-9 mRNA relative expression level is 0.10±0.01. Compared with normal control group, the relative expression levels of MMP-9 in diabetic group increases: 0.94±0.09 (p<0.01). In atorvastatin, rosiglitazone, rosiglitazone + atorvastatin united group: MMP-9 relative expression levels were: 0.62±0.04, 0.65±0.04, 0.37±0.01, which shows a downward trend than diabetic group, with joint intervention group most significantly lower (p<0.01). There has not been marked difference between the rosiglitazone and atorvastatin groups (p>0.05). 6 Correlation Analyses:Collagen content of kidney tissue was used as the dependent variable, and Crea, TGF-β1, TIMP-1, c-fos, MMP-2, MMP-9 as independent variables, for linear correlation analysis. The results showed that there was a positive correlation between the Kidney tissue collagen content and Crea, TGF-β1, TIMP-1, c-fos, MMP-9 levels. (r=0.98,0.89,0.86,0.88,0.82,p<0.01);there was a negative correlation between the Kidney tissue collagen content and MMP-2 ( r=-0.89,p<0.01).Conclusion:1 The abnormal changes of the expression of TGF-β1,TIMP-1,c-fos,MMP-2,MMP-9 occurs in kidney tissues during the development of type 2 diabetic nephropathy ,which plays an important role in processing of Glomerular sclerosis. It could delay the occurrence and development of DN pathological process.2 The abnormal changes of the expression of TGF-β1, TIMP-1, c-fos, MMP-2, MMP-9 occur in kidney tissues during the development of diabetic nephropathy, which plays an important role in processing of Glomerular lesions. Rosiglitazone + atorvastatin joint intervention may realize its protective effect of kidney by regulating the expression of the above-mentioned cytokines. Joint intervention is superior to single intervention. |
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