| Objective: Diabetes mellitus and its complications have become a public health problem. Diabetic nephropathy (DN), as a serious chronic microvascular implication of diabeties, has become one of the main etiological factors resulting in end stage of renal failure with the increasing incidence of diabetics worldwide. In spite of our greater understanding of this complication, the intimate mechanisms leading to the development and progression of renal injury are not well understood. So there is a great need to clarify the pathogenesis as well as to seek the methords of preventing and treating DN for nephrologist nowadays.Currently considered diabetic nephropathy is a multifactorial disease caused by combined effects.Its pathogenesis may be due to glucose metabolism and the resulting non-enzymatic glycation,polyol pathway activation,protein kinase C activation,lipid metabolism disorders,hypertension-induced changes in renal hemodynamics, oxidative stress,vascular active substances and cytokines,genetic factors and other factors. Megsin gene and protein expression increased in the pathogenesis of diabetic nephropathy has been the domestic role of the attention of scholars. Megsin is a dominant gene expression in mesangial cells, test analysis showed that serine protease inhibitor is (seprin) superfamily member, specifically expressed in human, rat and mouse mesangial cells, with important functions. The amount of expression of the human body to maintain normal in vivo physiological and pathological processes and adjust the balance is required network. The pathophysiological process, including coagulation, fibrin degradation, extracellular matrix metabolism, cell differentiation, proliferation and apoptosis, development, blood pressure, cancer and inflammation. Megsin in the renal tissue of diabetic mice to reveal the role of the pathogenesis of diabetic nephropathy has very important significance. Therefore, further study of the role of the relevance of megsin and ERK5MAPK in the kidney for diabetic renal disease may provide for the pathogenesis of diabetic nephropathy and control provides a number of experimental basis.We Constructed megsin siRNA expression plasmid and megsin cDNA expression plasmid,transfected them to STZ-induced diabetic mice, then investigate the relationship between megsin,TGF-β,FN and ERK5 through the immunohistochemical method and Western-Blot to discuss the possible roles of megsin in inflammation of nephropathy, which may provide an important therapeutic targets that can be translated into clinical treatments for diabetic nephropathy.Methods:60 healthy male unilateral nephrectomy CD-1 mice were randomly divided to 5 groups,12 erevy group.One of 5 groups was regarded as normal control group(A),then the others received a single intraperitoneal injection of STZ(dissolved in 0.1mol/L citrate buffer,pH 4.5)at a dose of 150mg/kg Wt.The diabetic model was considered to be successful when the blood glucose was 16.7mmol/L randomly and urinary glucose(+++)～(++++) after 72 hours of STZ injection.Then megsin plasmid,megsin-siRNA plasmid and control plasmid via the tail vein of mice were severally injected to diabetic group(B), diabetic control group(C),megsin plasmid group(D)and megsin-siRNA group(E).All mice were allowed free access to food and water during the experiment. At the end of 2 week,4 week,12 week,collecting the blood,24-hour urine and renal tissue samples,testing serum creatinine;24h urinary protein excretion rate,creatinine;kidney weight/body weight ratio; staining renal tissue by HE,MASSON and periodic acid - Schiff (PAS) , observed the changes in glomerular pathology in ordinary light microscope.The expression of megsin,TGF-β,FN and ERK5 in renal cortex was measured with immunohistochemical method and Western-Blot.The results were semiquantitatively analyzed with an image-processing system. All the data were analysed by SPSS13.0 statistics software,P value<0.05 was considered to have statistical significance. Results:1. The mice of group B,C,D and E all accurred polydipsia, polyuria and polyphagia after 4 or 5 days of STZ injection.There is no significant difference in four groups.Group A does not have Kidney mass/body mass ratio,UP and Scr were higher in group B and C at 12 weeks(P<0.01),group D in those were highest(P<0.01),group E is less than group B and C (P<0.01).2.light microscopy HE:at 2 and 4 weeks,the number of glomerular cells in group B and C was more than in group A (p<0.01),but less than group D (p<0.01),the number of glomerular cells decreased in group B,C and D at 12 weeks ,but still higher than in group A (p<0.01), group E is less than in group B and C (p<0.05);light microscopy HE,PAS and MASSON: glomerular hypertrophy, thickened of GBM and accumulation of ECM in group B and C were significantly higher than those in group E,group D is the most serious.3. The results of immunohistochemistry and Western blot showed that the levels of megsin, TGF-β,FN and ERK5,at the end of 2 weeks,was obviously increased in group B and C and higher than group D but lower than group E (p<0.01).There was a significant difference (p<0.05,p<0.01).Changes of above-mentioned pointors were more significantly different among these groups at 4 weeks (p<0.01).At 12 weeks the expression level of ERK5 droped in group B and C,but still higher than in groupA and E,lower than in group D(p<0.05,p < 0.01),other indicators's expression such as megsin,TGF-βand FN further enhanced , there was significant difference (p<0.01).Conclusion:1 The expression of megsin and ERK5 heightened in diabetic mice,suggesting that megsin and ERK5 may play a role in the development of diabetic nephropathy.2 In the condition of high glucose,Over-expressed megsin may increase the expression levels of TGF-β,FN and ERK5MAPK in renal tissue of diabetic mice which may help explain the synergistic action of megsin with other factors in accelerates diabetic nephropathy.3 Megsin siRNA plasmid can reduce the expression level of megsin, TGF-β,FN and ERK5MAPK,lower the impaired renal function and nephridial tissue pathological,prompting that control the expression levels of megsin can lessen kidney damage of diabetic mice.It provide a new idea in the prevention and treatment of early DN. |
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