The Effect Of Sitagliptin On The ERK1/2 Pathway Of Renal Tubular Epithelial Cells In High Glucose Environment

Objective:To observe the protective effect of Sitagliptin on renal tubular epithelial cells in high glucose environment and its effect on the ERK1/2 pathway.Methods:Recover the frozen rat renal tubular epithelial cells(NRK-52E) in containing 10%fetal calf serum DMEM culture medium and be placed in 37℃,5%CO2 incubator. When adherent cells up to 70%-80%, replace to serum-free DMEM culture medium for 24 hours, Cells after synchronization group.NRK-52 E were divided into normal glucose group, hypertonic group,high glucose,high glucose with PD98059(ERK1/2 inhibitor) group and high glucose with Sitaglipti group, high glucose group which was detected at 0.5h, 1h, 6h, 12 h, 24 h after induced.The levels of ERK1/2 and p-ERK1/2 protein were determined by western blot;Apoptosis related protein bax/bcl-2 and the rate of NRK-52 E apoptosis in each group was evaluated by flow cytometry;and the expression level of bax/bcl-2 m RNA were detected via real-time PCR.Results:1.NRK-52 E cells with high glucose could activate the ERK1/2 signaling pathway.The ERK1/2 signal pathway of group with high glucose began to be activated 30 min after incubation, peaked 12 h after, and returned almost to the baseline level 24 hours after.We can not detect the activation of ERK1/2 protein in groups of high glucose andhypertonic.PD98059 could reduce the level of P-ERK1/2 protein induced by high glucose obviously. Sitaglipti can also reduce the expression of p-ERK1/2 protein due to high glucose at the same time point.2.Flow cytometry detected results:(1)Detection results of apoptosis related protein bax/bcl-2 in renal tubular epithelial cells of each group: The expression of bax/bcl-2 in normal glucose group and hypertonic group had no significant difference; The expression of bax/bcl-2 in high glucose group increased significantly than normal glucose group(P<0.05); The level of bax/bcl-2 protein in PD98059 group and sitagliptin group were decreased significantly when compared with high glucose group(P<0.05), and the bax/bcl-2 protein decreased more significantly in the PD98059 group.(2)Detection results of apoptosis rate in each group: The apoptosis rate of renal tubular epithelial cells in normal glucose group was(4.45 + 0.28)%; The apoptosis rate of hypertonic group was(4.63 + 0.58)%, there was no significant difference between the normal group and the hypertonic group; The apoptosis of high glucose group increased obviously(16.28 +1.07)%, and the difference was significant when compared with normal glucose group(P<0.05); The apoptosis rate of renal tubular epithelial cells was(9.65 + 0.69)%after ERK1/2 specific inhibitor(PD98059) treatment, the difference was significant when compared with the high glucose group(P<0.05); The apoptosis rate of renal tubular epithelial cells in Sitagliptin intervention group was(11.67 + 1.13)%, compared with the high glucose group the apoptosis rate decreased by 28%, there was the statistically significant difference between Sitagliptin intervention group and high glucose group(P<0.05).3.The RT-PCR results:(1)The level of Bax m RNA were increased obviously in high glucose group when it compared with normal glucose group; Compared with high glucose group,the level of Bax m RNA in PD98059 group and sitagliptin group were significantly decreased; There was no difference of Bax m RNA level in normal glucose group and hypertonic group, and the level of Bax m RNA were decreased more obvious in the PD98059 group.(2)Compared with normal glucose group, the level of bcl-2 m RNA in hypertonic group had no significant changes; The level of Bcl-2 m RNA was significantlydecreased in high glucose group; Compared with high glucose group,the level of Bcl-2m RNA in PD98059 group and sitagliptin group were increased significantly(P<0.05), and the the level of Bcl-2 m RNA were increased more obvious in the PD98059 group.Conclusion:1.The results confirmed that high glucose could stimulate the activation of ERK1/2signaling pathway in renal tubular epithelial cells.2.Sitagliptin can reduce the apoptosis of rat renal tubular epithelial cells in high glucose environment by partly inhibiting the activation of ERK1/2 signaling pathway.