The Expression And Their Role Of Notch Pathway Related Molecules In The Glomerular Mesangial Cells Induced By High Glucose

Objective:Diabetic nephropathy is one of the most common microvascular complications of the diabetes,and its pathogenesis has not elucidated yet.Most scholars believe that the hemodynamic changes and glucose metabolism disorders which caused by genetic,hyperglycemia,angiotensin ?(Ang ?)and other cytokines,can promote the development of diabetic nephropathy.In the early developmental stages,Notch signaling was involved in the differentiation of normal structure.In recent years,a large number of studies found that the abnormal expression of Notch is occurred in acute and chronic renal injury and fibrosis model or diabetic kidney biopsy.The block of y-secretase enzyme,which is the key point of Notch signaling pathways,can induce Notch signaling inactivation.Even more,in chronic kidney disease,the expression of Jagged 1,which is a ligand of the Notch signaling pathway,also reported can be increased by TGF-? dependent manners.In recent years,TGF-?a meeting point of the various risk factors of diabetic nephropathy and one of the most studied cytokines which induced renal fibrosis,has a vital role in the development of renal fibrosis.Although the studies in vivo have shown that activation of Notch signaling is existing in diabetic nephropathy specimens,but it has not been reported whether it was involved in the incidence of diabetic nephropathy in vitro.Whether the activition of TGF-(3 pathway is involved in the activition of Notch signaling pathway in the pathogenesis of diabetic nephropathy is also unclear.Therefore,in this study we use y-secretase inhibitors(DAPT)and high glucose as an intervention to observe the change of Notch signaling related molecules and fibrosis molecules in cultured rat glomerualr mesangial cells to explore the role and possible mechanism of Notch signaling in the pathogenesis of diabetic nephropathy.Method:rat GMC were cultured in vitro,DAPT as a Notch signaling pathway inhibitor and high sugar as a stimulating factor were used.Detect the expression of Notch signaling molecules and fibrosis molecules by western-blot,RT-PCR and ilmmunofluorescence after different treatment.Results:1.expression of protein:Compare with 5.6mmol/L group,the expression of Notch signaling molecules protein was significantly increased in high glucose goup(P<0.05),and was no difference in mannitol Group(P>0.05).After treat cell with 25mmol/L high glucose for different time,the expression of Notch1 protein was enhanced both in the 12h and 24h.The expression of Jagged1/Hes1 protein in 12h,24h,48h was all enhanced.Compare with the 25mmol/L group,the expression of Notch signaling molecule protein were diminished in the High Glucose+ DAPT group(P<0.05);2.expression of mRNA:compared with 5.6mmol/L group,the mRNA expression of the Notch signaling related molecules in 25mmol/L group was increased significantly(P<0.05).Wheraese,the mRNA expression of Notch signaling molecules was no difference in the mannitol group(P>0.05).After 25mmol/L high glucose treatment for different times,the expression of Notch 1 was increased in the 12h time point significantly(P<0.05)and the expression of Jaggedl was increased in 12h,24h,48h three time points(P<0.05)and the expression of Hes1 mRNA was increased in 12h and 24h.Compared with 25mmol/L group,the mRNA expression of Notch signaling related molecules and TGF-p,FN is diminished significantly in the High gluose + DAPT group(P<0.05);however,compare with the 5.6mmol/L group,the mRNA expression of TGF-?,FN in 25mmol/L group was increased evidently(P<0.05).3 expression of protein observed with Immunofluorescence microscopy:in the 5.6mmol/L group,the expression of the Notch signaling molecules was low expression.Notch1 and Hesl protein was expressed in cytoplasm only,Jagged1 protein is expressed in cytoplasm and nucleus;Enhanced expression of Notch signaling molecules was observed in 25mmol/L group,and transposition of Hesl protein was appear in nucleus;they are also significantly decreased after treat by DAPT for 24h,and the expression of Jagged1 and Hes1 protein was decreased in nucleus apparently.Conclusion:1?The high glucose can increase the expression of Notch signaling related molecules in glomerular mesangial cells;2?High glucose can induce the upregulation of TGF-? and FN;3?The activation of the Notch signaling pathway can upregulate the expression of TGF-?,FN and probalblely play a role in the pathogenesis of kidney fibrosis and diabetic nephropathy.