| Objective: Diabetic nephropathy (DN) is one of the most common and serious complications of diabetes mellitus(DM).For the past few years,with the improvement of living standards and the changes of life style,the incidence of DN showed an upward trend year by year,DN has been gradually becoming the major or first cause of leading to the end-stage renal failure(ESRF),which brought about a heavy financial burden to the family and the community.The histopathological hallmarks of DN are increased volume of kidney,increased thickness of the glomerular basement membrane,the glomerular mesangial cells (GMC)proliferation,hypertrophy, progressive accumulation of extracellular matrix(ECM),occurring glomerular sclerosis step by step.GMC are a kind of inherent cells in kidney and play a central role in maintaining a normal structure and function of the glomerulus. Therefore,they are involved in the pathogenesis of DN inevitablely. Megsin is a mesangium-predominant gene,located in 18q21.3.Amino acid homology search revealed that this gene was highly homologous to members of the serpin superfamily.Serpin superfamily is composed of a large number of members and has a wide range of functions.Its functions are involved in coagulation,fibrinolysis,complement activation,inflammation,development and so on.Serpin superfamily also has important effects on cell proliferation, apoptosis,signal transduction, ECM metabolism and other systems and is closely related to the normal physiological activities of the body.we speculate that megsin,as a member of the serpin ,may be involved in certain physical and pathological activities of MC.Thus,discussing megsin in the role of the GMC has great significance to reveal the mechanism of DN.The occurrence and development of DN are result from many factors,such as biochemical metabolic disorders,glomerular hemodynamic abnormalities,abnormal secretion of cytokines,oxidative stress and so on.PDGF and TGF-βare two crucial pairs of cytokines that have a major impact on mesangial cell proliferation and extracellular matrix accumulation. Studies have shown that the expression of platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) mRNA of GMC was up-ragulated and extracellular signal-regulated protein kinase (ERK) signal pathway was activated in the presence of high glucose, moreover, PDGF-BB might up-regulate the expression of TGF-β1mRNA of GMC, promote ECM (such as FN) synthesis and inhibit the degradation of ECM through ERK signal pathway to speedup the process of glomerular sclerosis. ERK is the key member of the MAPK family and is one of the most important signaling systems that transfer extra-cellular stimulus to cell nucleus.ERK consists of two highly homologous subclass ERK1/ERK2. The activation of ERK will be completed through phosphorylation.Phosphorylated ERK is able to transfer extra-cellular stimulus to cell nucleus participating in a variety of physiological responses such as cell growth,development,division,death and so on.Another study found that the expression of TGF-β1 and PDGF-BB mRNA up-ragulated in rat MC over-expressed megsin gene, which had time dependent effect. Rat MC stably over-expressed megsin gene could be seen the continue expression of PDGF-BB,that is to say rat MC transfected megsin gene were able to strengthen the secretion of PDGF-BB,TGF-β1. Speculated that there is tight connection among PDGF-BB,TGF-β1 and megsin during the occurrence and development of DN,however,whether there is a link between ERK and megsin is not yet known.We will culture mouse GMC in vitro and interfere in the expression of megsin gene in order to investigate the changes of ECM synthesized by GMC over-expressed megsin gene and the possible signal pathway of the cells in the presence of high glucose.To further study whether PDGF-BB can stimulate the proliferation of GMC over-expressed megsin gene and the deposition of ECM through ERK siginal pathway or not. Hope to further reveal the pathogenesis of DN and provide a new theoretical basis to the prevention and treatment of DN.Methods: Cultured cells are divided into six groups:normal glucose group(A group,D-glucose 5.5mmol/L), high glucose group(B group,D-glucose 30mmol/L), high glucose+empty vector group(C group,D-glucose 30mmol/L), high glucose+ megsin expression plasmid group(D group,D-glucose 30mmol/L), high glucose+ megsin expression plasmid + UO126group(E group,D-glucose 30mmol/L), high glucose+megsin siRNA expression plasmid group(F group,D-glucose 30mmol/L). Large collection of constructed megsin expression plasmid,empty vector and megsin siRNA expression plasmid,transfecting transiently with the corresponding plasmid of mouse GMC by Lipofectamine2000 according to experimental design(the efficiency of transfection is determined by the protein expression of megsin in each group detecting by western blot ) and giving the corresponding group of corresponding stimulus are needed.Being cultured for at 12,24 and 48 hours, the protein expression of megsin, PDGF-BB,pERK1/2,TGF-β1,FN of total protein in each group was detected by using immunocytochemistry and western blotting.The concentrations of type IV collagen in cell supernatant were detected by RIA (the results of total protein with correction).Results: Immunocytochemistry results showed that under normal glucose condition,phosphorylated ERK1/2 was present in the cytoplasm of MC,light brown yellow,weak expression.After 12 hours of high glucose stimulation,it transfered from the cytoplasm to the nucleus,hasing a time dependent effect.The protein expression of phosphorylated ERK1/2 in both cytoplasm and nucleus was higher than that of normal glucose group,but the trend in MC over-expressed megsin gene was more obvious in the presence of high glucose. After application of ERK pathway inhibitor,the protein expression of phosphorylated ERK1/2 in both cytoplasm and nucleus are inhibited obviously. Starting from the 12 hours,the protein expression of megsin,PDGF-BB,phosphorylated ERK1/2,TGF-β1,FN in group B enhanced,peaked at 48 hours, there was no significant difference between group C and group B during the same period(P>0.05); The protein expression of megsin, PDGF-BB,phosphorylated ERK1/2,TGF-β1,FN in group D were higher than that of group B,there was significant difference between group D and group B during the same period(P<0.05). The protein expression of megsin and PDGF-BB had no significant difference between group E and group D during the same period(P>0.05),but the protein expression of pERK1/2,TGF-β1 and FN were decreased, there was significant difference between group E and group D(P<0.05).The protein expression of megsin,PDGF-BB,phosphorylated ERK1/2,TGF-β1,FN in group F were lower than that of group B,there was significant difference between group F and group B during the same period(P<0.05).Western blotting results showed that Starting from the 12 hours,the protein expression of megsin,PDGF-BB,phosphorylated ERK1/2 and TGF-β1 in group B enhanced,peaked at 48 hours, there was no significant difference between group C and group B during the same period(P>0.05); The protein expression of megsin,PDGF-BB,phosphorylated ERK1/2 and TGF-β1 in group D were higher than that of group B,there was significant difference between group D and group B during the same period(P<0.05).The protein expression of megsin and PDGF-BB had no significant difference between group E and group D during the same period(P>0.05),but the protein expression of pERK1/2 and TGF-β1 were decreased, there was significant difference between group E and group D(P<0.05).The protein expression of megsin,PDGF-BB,phosphorylated ERK1/2 and TGF-β1 in group F were lower than that of group B,there was significant difference between group F and group B during the same period(P<0.05).RAI results showed that from the beginning of 12 hours,the concentrations of type IV collagen (cell total protein correction) in supernatant of MC became higher in group B than that of group A,peaked at 48hours(P <0.05),there was no significant difference between group C and group B(P> 0.05),but the trend in group D was more obvious, thers was significant difference between group D and group B during the same period(P<0.05). The concentrations of type IV collagen decreased in group E, thers was significant difference between group E and group D during the same period(P<0.05).The concentrations of type IV collagen in group F became lower than that of group B during the same period(P<0.05).Conclusions: Experiments in vitro confirmed that ERK signal pathway was activated , the protein expression of megsin, PDGF-BB,pERK1/2,TGF-β1, FN and the concentrations of type IV collagen in the supernatant of MC increased in the presence of high glucose.After high glucose-stimulated,the trend in MC over-expressed megsin gene was more obvious. After application of ERK pathway inhibitor, the protein expression of pERK1/2,TGF-β1,FN and the concentrations of type IV collagen in the supernatant of MC were obviously inhibited,but the protein expression of megsin and PDGF-BB had no changes.The protein expression of megsin, PDGF-BB,pERK1/2, TGF-β1,FN and the concentrations of type IV collagen in the supernatant of MC decreased in MC low-expressed megsin gene in the presence of high glucose,which proved that the PDGF-BB in MC over-expressed megsin gene might up-regulate the expression of TGF-β1 and increase of ECM synthesis in part through the ERK signal pathway in the presence of high glucose.
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