The Study About The Effect Of Megsin On The Activity Of P38MAPK, ICAM-1 And MCP-1 In Glomerular Mesangial Cells Exposed To High Glucose

Objective: Currently Diabetic Nephropathy (DN) has become the major cause leading to the maintenance dialysis with end-stage renal disease which brought about a heavy financial burden and stress to the family and the community. Glomerular mesangial cell hypertrophy, extracellular matrix accumulation and glomerulosclerosis is the basic pathological feature in the development of DN which is considered to be an irreversible process that eventually leads to end-stage renal failure. Megsin gene whose expression product belongs to serine protease inhibitor expresses superiorly in the mesangial cells. The role of Serpin includs coagulation, fibrinolysis, extracellular matrix metabolism, inflammation, cell differentiation, proliferation and apoptosis and so on. Several studies have found that its gene and protein expression were significantly raised in diabetic nephropathy whose main pathological changes were mesangial cell proliferation and mesangial matrix accumulation. The increasing of megsin gene and protein'expression in the pathogenesis of diabetic nephropathy has been concerned by scholars at home and abroad. Studies have found that p38MAPK signal transduction pathway can be activated in mesangial cells with high glucose, with upregulation of TGF-β1 and inflammatory factors. PDGF-BB participates in the process of glomerular sclerosis through activating p38MAPK pathway in glomerular mesangial cells, inhibiting the degradation of extracellular matrix and promoting the synthesis of extracellular matrix such as typeⅣcollagen and FN. At present, there is few reports that wether megsin promotes the development of DN through activating p38MAPK pathway and upregulating inflammatory factors In high glucose-stimulated gene transfection Megsin mesangial cells. In this study We Constructed megsin cDNA expression plasmid then transfected them to mesangial cells cultured in high glucose, and investigated the expression of megsin, p-p38MAPK, MCP-1, ICAM-1, SOD, GSH-PX, MDA and so on. We also Constructed megsin siRNA expression plasmid and transfected them to mesangial cells cultured in high glucose, examined the above factors. We explored the mechanism of megsin in diabetic mesangial cells in high glucose environment, which may be helpful to find new target and provide the experimental theoretical basis for early intervention of DN.Methods: Mouse mesangial cells were grown in DMEM F12 containing 5%FBS,penicillin(100 U/ml),streptomycin(100μg/ml)at 37°C and 5%CO2. The cells were divided into 5 groups:a normal glucose group(N), a high glucose group(D), high glucose transfected a pBAsimU6 Neo control plasmid group(V), and high glucose transfected a pBAsimU6 Neo megsin siRNA plasmid group(S), and high glucose transfected a pCMVsport6.1 megsin cDNA plasmid group(M). When the cells grown to 80% confluence, transplanted the cells to 6-well culture plates(2×106cells per well). Cells were cultured without antibiotics for 24 hours, then were transfected with pBAsi mU6 Neo megsin siRNA plasmid or pBAsi mU6 Neo control plasmid using lipofectamineTM 2000 reagent. After 24 hours, H group, V group, S group and M group cells were further cultured in DMEM F12 containing high glucose while N group cells were cultured in normal glucose for up to 48 hours. Cells in 6-well culture plates were collected for protein extraction and the culture medium were collected for SOD, GSH-PX, MDA measurement at hour-12, hour-24 and hour-48. The levels of megsin, p-P38, ICAM-1, MCP-1 in GMCs were measured by Western blot and supernatant SOD, GSH-PX, MDA were measured by chromatometry. All of the data was analysed by SPSS15.0 statistics software, P value<0.05 was considered to have a statistical significance.Results: 1 western blot: the megsin, p-P38, ICAM-1, MCP-1 protein expression levels in mesangial cells of N group, S group, D group, M group increased in turn at 12h, 24h, 48h time points. There was no significant difference between D group and V group (P>0.05), the difference was significant compared with the M group, S group and N group(P<0.05).2 supernatant SOD, GSH-PX, MDA level: the concentration of supernatant SOD,GSH-PX of N group, S group, D group, M group became lower in turn at 12h, 24h, 48h time point (P<0.05), the level of MDA in N group, S group, D group, M group was increased in turn at 12h, 24h, 48h time points (P<0.05). There is no significant difference between D group and V group(P>0.05).3 result of immunocytochemistry: Immunocytochemically, the positive staining for megsin, ICAM-1 and MCP-1 was observed in cytoplasm of the mesangial cells cultured in the medium with high glucose. the megsin, ICAM-1, MCP-1 protein expression levels in mesangial cells of N group, S group, D group, M group was increased in turn at 12h, 24h, 48h time points. There was a significant difference compared with the other groups(P<0.05). There was no significant difference between D group and V group (P>0.05). Positive signals for p-p38MAPK were observed in the nucleus of the cells.Compared with those exposed to normal level glucose, positive staining for p-p38 MAPK were strengthened in the mesangial cells exposed to high glucose . p-p38 MAPK protein expression levels in mesangial cells of N group, S group, D group, M group was increased in turn at 12h, 24h, 48h time points. there was a significant difference compared with the other groups(P<0.05). there was no significant difference between D group and V group (P>0.05).Conclusions: 1 The expression of megsin increases in cultured mesangial cells expose to high glucose in time-dependent manner.2 The expression of p-p38, ICAM-1 and MCP-1 is up-regulated in megsin over-expression mesangial cells, suggesting that megsin may contribute to proliferation of mesangial cell in high glucose and the occurrence of oxidative stress, and have a close correlation with p38MAPK signal transduction pathway and its downstream activation of inflammatory factors .3 The expression of p-p38, ICAM-1 and MCP-1 is reduced in the lower-expressed megsin glomerular mesangial cells that be confirmed by the transfection of megsin siRNA, suggesting that inhibiting megsin expression can reduce diabetic mesangial cell injury by inhibiting p38MAPK pathway. Inhibition of megsin may be an effective means of prevention and treatment in early diabetic nephropathy, and providing a experimental theoretic basis for the screening of a new target for diabetic nephropathy.