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Objective: Diabetic nephropathy (diabetic nephropathy, DN) diabetes is the most common and serious complications. Recent years, with the improvement of living standards, lifestyle changes, showed an increasing trend in the incidence of DN becoming the lead to end-stage renal failure (end-stage renal failure, ESRF), the first or the main cause to the family and society a heavy economic burden. DN major pathological findings of kidney volume increased glomerular basement membrane thickening, mesangial cell proliferation, hypertrophy and mesangial extracellular matrix (of extracellular matrix, ECM) progressive build-up, and gradually developed into a glomerular sclerosis. Mesangial cells is an intrinsic glomerular cells, plays an important role in the maintenance of normal structure and function of the glomerulus, while inevitably involved in the pathogenesis of DN. Megsin advantage of a mesangial cells express the gene, located on chromosome 18 q21.3 homology analysis showed that the gene a are seprin superfamily. Serpin a wide range of functions, many members, mainly involved in blood coagulation, fibrinolysis, complement activation, inflammation, and tissue remodeling process of cell proliferation, apoptosis, signal transduction, extracellular matrix metabolism play an important influence. Certain physiological and pathological activities the Megsin as one of the members of the serpin bound to participate in the mesangial cells, so explore role of megsin mesangial cells is of great significance to reveal the mechanism of DN. DN occurrence, development is the result of a combination of factors, such as biochemical metabolic disorders, glomerular hemodynamic abnormalities, abnormal secretion of cytokines and oxidative stress, which of PDGF, TGF-β are two types of mesangial cell proliferation and ECM accumulation cytokines have an important impact. Studies have shown that high glucose, mesangial cells, transforming growth factor β1 (transforming growthfactor-β1, TGF-β1), platelet-derived growth factor-BB (platelet derived growth factor-BB, PDGF-BB) were increased mRNA expression of ERK (of extracellular signal-regulated protein kinase) signaling pathway is activated. PDGF-BB may mesangial cells via ERK pathway may raise the expression of TGF-β1mRNA promote ECM fibronectin protein (FN) synthesis and inhibit the degradation of ECM to participate in the process of glomerulosclerosis. ERK is a principal member of the mitogen-activated protein kinase (MAPK) family, is one of the important signaling system mediated intracellular to extracellular stimuli intracellular, including two highly homologous subclasses ERK1/ERK2 phosphorylation activated. The activated ERK external signals transmitted to the nucleus, involved in cell growth, development, division, death and other physiological reaction process. Another study found that, in rat mesangial cells the transfection megsin gene, the visible expression of PDGF-BBmRNA also significantly up-regulated the expression of TGF-β1mRNA, the two transfection megsin gene was time-dependent manner. That rat mesangial cells after the transfection megsin gene, can stimulate cells of PDGF-BB secretion of TGF-β1, which speculated that occurred in the DN, the development process, megsin PDGF-BB, TGF-β1 in between there is bound to in close contact, but whether there is a link between ERK and megsin is not yet known. This study mesangial cells cultured in vitro, conducted intervention on of mesangial cells megsin gene expression, explore the high-sugar environment overexpression megsin in gene changes in mouse mesangial cells to synthesize the ECM, as well as by the high over express megsin gene glucose-stimulated mesangial cells may signal transduction pathway. Further research in high-sugar environments, megsin gene would affect PDGF-BB ERK pathway and thus promote mesangial cell proliferation and ECM accumulation want to be able to further reveal the pathogenesis of DN, provides a new theoretical basis for the prevention and treatment of DN. Method: cultured mouse mesangial cells were divided into six groups: the normal control group (group A, D-glucose 5.5mmol / L), high glucose group (group B, D-glucose 30mmol / L), empty plasmid group (C group, D-glucose 30mmol / L) of the high-sugar, high the sugar megsin plasmid group (group D, D-glucose 30mmol / L), high sugar megsin plasmid ERK pathway inhibitor group (E group, D-glucose 30mmol / L), high sugar siRNA group (group F, D-glucose 30mmol / L). Massive withdrawals the built megsin expression plasmid, the empty plasmid megsin siRNA expression plasmid, grouped according to experimental design application liposomes transiently transfected with the appropriate plasmid (western blot assay groups cells megsin of protein expression to detect transfection efficiency) given the appropriate stimulus, respectively, 12, 24, 48 hours in culture, immunocytochemistry and western blot method was measured in each group mesangial cells megsin PDGF-BB phosphorylated ERK1 / 2 (pERK1 / 2) of TGF-β1 FN expression, RIA assay concentration of type IV collagen in cell supernatants (results with correction of total protein). Results: Immunocytochemistry: normal glucose environment, pERK1 / 2 exist in the cytoplasm of mesangial cells was light brown yellow, weak expression; visible pERK1 / 2 from the cytoplasm to the extracellular high sugar stimulate 12h nuclear transfer, and with time, expressed in the nucleus gradually increased, was dark brown yellow strong expression, and cytoplasmic expression was also significantly higher than the normal group (P lt; 0.05); environment of high glucose, mesangial cells transfected megsin gene this trend is more pronounced. ERK pathway inhibitors after application, pERK1 / 2 in the cytoplasm, the expression in the nucleus are significantly inhibited. From 12h B group compared with the group A mouse mesangial cells megsin, PDGF-BB, pERK1 / 2, TGF-β1 and FN protein levels began to rise, 48h peaked (P lt; 0.05); C group and B group compared to the same period the difference was not significant sex (P gt; 0.05); D group and B group the same period compared megsin, PDGF-BB, pERK1 / 2, elevated TGF-β1 and FN protein levels, there were significant differences (P lt; 0.05); E group compared with group D over the same period, megsin and PDGF-BB expression, no significant change (P gt; 0.05), pERK1 / 2, TGF-β1 and FN protein levels decreased significant difference ( P lt; 0.05); F group and B group the same period compared megsin, PDGF-BB, pERK1 / 2, reduce the level of TGF-β1 and FN protein, there are significant differences (P lt; 0.05). Western blot analysis showed: from 12h B group compared with the group A mouse mesangial cells megsin, PDGF-BB, pERK1 / 2 and TGF-β1 protein levels began to increase, 48h reached a peak (P lt; 0.05); C group and no significant difference in sex (P gt; 0.05) compared to group B, over the same period; D group and B group the same period compared to megsin, PDGF-BB, pERK1 / 2 and TGF-β1 protein levels, and the difference was statistically significant ( P lt; 0.05); E group compared with group D over the same period, megsin and PDGF-BB expression, no significant change (P gt; 0.05), pERK1 / 2 and TGF-β1 protein levels, there were significant differences (P lt; 0.05); F group and B group the same period compared to megsin, PDGF-BB, pERK1 / 2 and TGF-β1 protein levels decreased, there were significant differences (P lt; 0.05). The radioimmunoassay display: from 12h, the concentration of type IV collagen in the B mice mesangial cell supernatant (total cell protein concentration correction) began to increase compared with group A, 48h reached its peak (P lt; 0.05); C group no significant difference in sex (P gt; 0.05) over the same period, compared with group B; D group over the same period, compared with group B, line type IV collagen concentration in the membrane cell supernatant, the difference was significant significance (P lt; 0.05 ); E group compared with group D over the same period, the Department of the concentration of type IV collagen membrane cell supernatants reduce, the difference was statistically significant (P lt; 0.05). Compared to the same period of the Group F and Group B, mesangial cell supernatants reduce the concentration of type IV collagen, the difference was statistically significant (P lt; 0.05). Conclusion: In vitro experiments confirmed that the activation of the ERK pathway in mouse mesangial cells in high glucose, megsin, PDGF-BB, TGF-β1, type IV collagen and FN expression. Megsin gene can be further raised the the mesangial fine PDGF-BB overexpression pERK1 / 2, of TGF-β1, type IV collagen, and FN expression under high glucose environment. ERK pathway inhibitor application on this basis, can significantly inhibit mesangial cells pERK1 / 2, the expression of TGF-β1, collagen type IV and FN, but no inhibitory effect on the expression of the mesangial megsin and PDGF-BB. High glucose, interference megsin gene, the factor downregulated, suggesting that high-sugar environment, the transfection megsin gene mesangial cells, PDGF-BB may be partly caused by ERK pathway elevated TGF-β1 and ECM synthesis .
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