Effect Of Paeoniflorin On High Glucose-induced Proliferation Of Glomerular Mesangial Cells Via P53

Objective The prevalence of diabetes mellitus(DM)in China is rising.It has become the country with the largest number of DM patients in the world.Long-term hyperglycemia in diabetic patients leads to renal microvascular disease,Diabetic nephropathy(DN),which is a common complication of diabetes and one of the main causes of end-stage renal disease(ESRD).The pathogenesis of DN is not fully understood,and its pathological type involves the proliferation of mesangial cells and the accumulation of extracellular matrix.Studies have shown that the proliferation and apoptosis of mesangial cells may be related to p53,which has the functions of DNA repair,inhibition of cell proliferation and promotion of apoptosis,and may be involved in the regulation of proliferation and activation of mesangial cells.Inhibition of mesangial cell proliferation is important for slowing the development of microvascular disease of diabetic nephropathy.At present,the treatment of DN mainly focuses on measures such as early intervention of various risk factors and renal replacement therapy for ESRD,there are no particularly effective treatments.Total glucosides of paeony(TGP),its effective active ingredient is Paeoniflorin(PF),which has pharmacological effects such as anti-inflammatory,anti-oxidation and immune regulation,and may have protective effects on various kidney diseases.Basic research has found that PF can inhibit the proliferation of mesangial cells,and whether it is related to p53 need to be further studied.In this study,PF was used to intervene in mesangial cells cultured under high glucose to observe cell proliferation,apoptosis and p53-related gene expression,preliminary observation of whether PF inhibits mesangial cell proliferation is associated with the p53.Methods 1.The CCK-8 kit was used to detect the effects of different concentrations of paeoniflorin on the viability of mesangial cells cultured under normal glucose concentration,and the appropriate concentration of paeoniflorin was selected for testing.2.The experiment was then divided into six groups: mannitol group(Motl),normal glucose concentration control group(NG),high glucose group(HG),high glucose+20 umol/L PF group(HG+PF20),high glucose+40 umol/L PF group(HG+PF40),high glucose+80umol/L PF group(HG+PF80).CCK-8 kit and EdU kit were used to detect the effect of PF on the viability and proliferation of mesangial cells.Flow cytometry was used to detect the effect of PF on apoptosis of mesangial cells.Western Blot was used to detect the expression of type IV collagen(IV-Col),p53,p-p53,Bcl-2 and Bax.3.The CCK-8 kit was used to select the appropriate concentration of p53 inhibitor.These four groups were as follows: high glucose group(HG),high glucose+0.01 umol/L PFT-? group(HG+PFT),high glucose+80 umol/ L PF group(HG+PF80),high glucose+0.01 umol/L PFT-?+80 umol/L PF group(HG+PFT+PF80).EdU kit and flow cytometry were used to detect the proliferation and apoptosis of mesangial cells under the combined intervention of PF and PFT-?.Results 1.20 umol/L,40 umol/L,80 umol/L PF had no significant effect on the mesangial cell viability under normal glucose concentration(P>0.05).Therefore,PF with a concentration of 20 umol/L,40 umol/L,and 80 umol/L was used for the next test.2.Compared with normal glucose concentration,high glucose environment can promote mesangial cell proliferation.The levels of p53,Bax,and p-p53 proteins were decreased,and the levels of Bcl-2 and IV-Col proteins were increased.3.Compared with the HG group,the addition of 40 umol/L or 80 umol/L PF significantly inhibited the proliferation of mesangial cells induced by high glucose stimulation(P<0.05),and the apoptosis rate increased,the addition of 20 umol/L PF can not significantly inhibit mesangial cell proliferation and promote apoptosis.The results of Western blot showed that the levels of p53,Bax and p-p53 protein in HG+PF20,HG+PF40 and HG+PF80 groups were significantly higher than those in HG group(P<0.05),and the levels of Bcl-2 and IV-Col protein were decreased(P<0.05).4.Compared with the HG+PF80 group,the addition of p53 inhibitor reduced the effect of PF on inhibiting mesangial cell proliferation and promoting apoptosis.ConclusionsMesangial cell proliferation and activation in high glucose environment may be associated with decreased p53 protein levels.PF can inhibit the proliferation of mesangial cells under high glucose culture,which may be related to the increase of p53 protein level.