The Effect Of Regulating P53 On The Proliferation And Activation Of Glomerular Mesangial Cells Cultured In High Glucose

Background and Objective Diabetes mellitus(DM)is a systemic metabolic disease with increasing incidence world wildly and has high morbidity and mortality.DN is one of the most common microvascular complication of diabetes.It is a leading cause of end-stage renal failure.The high mortality of DN makes many patients to accept the blood purification treatment lifetime that brings heavy economic burden to the society and family.At present,the pathogenesis of DN is not completely clear.It is generally considered to be the result of a variety of factors such as changes in glomerular hemodynamics,glucose metabolism disorder,oxidative stress,cytokine and genetic susceptibility.In the high glucose environment,especially excessive intake of glucose in glomerular mesangial cells stimulates sorbitol metabolism,PKC,advanced glycosylation end products,TGF-?,angiotensin and endothelin and other active substances that are the pathogenesis of DN.Mesangial cell proliferation and activation play an important role in the pathogenesis of DN.High glucose can promote mesangial cell proliferation and extracellular matrix(ECM)synthesis.p53 has the functions of DNA repair,inhibition of cell proliferation and proapoptosis.There is no report on the role of p53 in mesangial cell proliferation and activation.In this study,we first detected the expression of p53 in the kidney of diabetic mice,and then we used the cell culture technique to observe whether there is an inhibitory effect of nutlin-3 on the mesangial cells cultured in high glucose.It lays the foundation for the treatment of diabetic nephropathy from the level of gene or protein.Methods1.Establishment of DN model: 26 healthy male Kunming mice were randomly divided into 3 groups: negative control group(6 mice injected with of equal amount of citrate buffer intraperitoneally),single high-dose group(10 mice injected intrapertoneally with150mg/Kg STZ only once)and multiple low-dose group(10 mice injected intraperitoneally with 40mg/kg STZ for 5 consecutive days).After injection,random blood glucose was detected continuously for 3 days,then the blood glucose was measured on the 1st,2nd,4th and 10 th week.If the random blood glucose was more than 16.7 mmol/L,diabetes was successfully established.the daily water intake was recorded.In week 4 and week 10 after STZ treatment,half of the mice were executed,mice blood,urine and kidney tissue were collected.The random blood glucose,body weight,24-hour urine volume were determined.The kidney pathological changes were observed by light microscope.The expression of p53 in the kidneys were evaluated by immunohistochemistry.2.Cell culture: The mesangial cells SV40(MCs)were divided into 4 groups: mannitol group(25 mmol/L),control group(5.5 mmol/L),high glucose group(25 mmol/L),high glucose + nutlin-3 group(nutum-3 concentration was 40 umol/L)and nutlin-3 group(40 umol/L).The CCK-8 assay was used to measure the activity of mesangial cells proliferation.The flow cytometry was used to detect the apoptosis of mesangial cells.The expression of p53,p-p53,bax,bcl-2 protein was measured by Western blot.Results1.The early DN model of Kunming mice can be successfully established with two ways of intraperitoneal injection with STZ.The single high-dose was superior to multiple low-dose STZ in diabetic nephropathy model construction with lower mortality.Compared with the negative control group,24-hour urinary protein in the 4th week was increased,and it was significantly increased in the 10 th week(P <0.05).The renal pathological changes in the 4th week were not obvious,but they exhibitedglomerular hypertrophy,mesangial cell proliferation and increased extracellular matrix in the 10 th week.The results of immunohistochemistry showed that the expression of p53 protein in diabetic kidney tissue was significantly increased compared with the control group(P <0.05).1.40 uM nutlin-3 inhibited the mesangial cells proliferation in HG environment.Western blot showed that HG could decrease the expression of p53,bax and p-p53 protein compared with control group(P<0.05),and increase the expression of bcl-2(P<0.05).Compared with HG group,nutlin-3 could increase the expression of p53,bax,p-p53 protein(P< 0.05),and decrease the expression of bcl-2(P<0.05),the ratio of bax/bcl-2 is greater than 1.Flow cytometry displayed that the apoptosis rate of mesangial cells in HG group was not significantly difference compared with the control group(P>0.05).The agonist could promote the apoptosis of mesangial cells with high glucose environment(P<0.05).Conclusions1.Animal experiments showed that STZ(40 mg/kg,consecutive injection for 5 days)can establish a stable model of early diabetic nephropathy and the expression of p53 in the kidney of diabetic mice increased.2.The vitro experiments showed that high glucose environment can promote mesangial cell proliferation,but p53 agonist can inhibit the proliferation of mesangial cells under high glucose and promote apoptosis.