Metabolite Of Leflunomide A771726 Inhibited High Glucose-Induced Podocyte Apoptosis And Its Mechanism

Objective1.To observe the biological characteristics of cultured human podocyte,and master the basic process of cell cultuer.2. To explore the therapeutic effects of leflunomide metabolite A771726 on high glucose-induced podocyte injury and understand its mechanism.MethodsExperiment using a temperature-sensitive immortalized human podocytes.Recover the frozen podocyte in 1%ITS,containing 10% fetal calf serum 1640 medium and be placed in 33℃,5%CO2 incubator,it show cobblestone-appearance,then transferred to 37℃,5%C02 incubator two weeks for differentiate maturation.When adherent cells up to 70%-80%, replace to serum-free 1640 medium for 24 hours, Cells after synchronization group.The conditionally immortal human glomerulus podocytes were divided into normal glucose group (NG), high glucose group (HG), mannitol group (MA), high glucose with SB202190 (a p38MAPK inhibitor) group and high glucose with active leflunomide metabolite A771726 group.The levels of p38MAPK and p-p38MAPK protein were determined by western blot and the rate of podocyte apoptosis was evaluated by flow cytometry.Results1.Podocytes with high glucose could activate the p38MAPK signaling pathway. The p38MAPK signal pathway of Group HG began to be activated 10 min after incubation, peaked 30 min after, and returned almost to the baseline level 6 hours after. Little activation of the pathways was observed in Groups NG and MA. SB202190 can significantly reduce high glucose induced the expression of p-p38 protein. Leflunomide active metabolite A771726 also reduce the same time expression of p-p38 protein due to high glucose.2.Flow cytometry detected Podocytes with high glucose increased apoptosis seen after 24h, 24h apoptosis rate was (12.3±0.96)%, 48h was (18.6±0.70)%, the most obvious to 48h,significantly higher than that of Group NG and MA.48h apoptosis of SB202190 group was (13.4±0.85)%, compared with the HG group was decreased 26%.High glucose with Active leflunomide metabolite A771726 can reduce podocyte apoptosis, 20μmol / L A771726 intervene podocyte after 48 h was (15.5±1.19)%, compared with the HG group was decreased 17%,the difference is statistically significant(p<0.05).ConclusionMetabolite of leflunomide A771726 can reduce high glucose-induced podocyte apoptosis and this effect may be involved in its inhibition of activation of p38MAPK.