The Effect And Mechanism Of Pinocembrin On The Expression Of Inflammatory Factors In Renal Tubular Epithelial Cells Induced By High Glucose

objective:To study the effect of pinocembrin(Pb)on the expression of inflammatory factors in renal tubular epithelial cells induced by high glucose,and to explore its mechanism,so as to provide new ideas for the prevention and treatment of diabetic nephropath(DN).Methods :(1)Screening of the best time for high glucose: Rat renal tubular epithelial cells(NRK-52E)were cultured in vitro and treated with 30 mmol/L glucose for different time(0 hours,6 hours,12 hours,24 hours,36 hours,48 hours,72 hours).The levels of TNF-a,IL-6 and IL-1beta in the supernatant of cell culture were detected by ELISA.(2)Anti-inflammatory effect and mechanism of pinocembrin : NRK-52 E cells were cultured in low-sugar DMEM containing 10% fetal bovine serum.NRK-52 E cells were cultured in low-sugar DMEM containing 10% fetal bovine serum.The cells were randomly divided into normal control(NC)group,mannitol(MA)group,high glucose(HG)group,HG+DMSO group,HG+Pb30 group,HG+Pb60 group(pre-test screened low-dose intervention concentration of 30 umol/L,high-dose intervention concentration of 60 umol/L),HG+Val group:the expression of p-P38 MAPK was detected by immunofluorescence assay,and the levels of P38 MAPK,p-P38 MAPK,NF-kappa B,p-NF-kappa B,JNK,p-JNK,ERK and p-ERK were detected by Western-blot.The levels of TNF-alpha,IL-6,IL-1beta in supernatant of cell culture were detected by ELISA,and the levels of TNF-alpha,IL-6 and IL-1beta in cells were detected by qPCR.(3)Anisomycin(AM),an agonist of P38 MAPK,reversed the anti-inflammatory effect of Pb and ascertained whether the anti-inflammatory effect of Pb depended on the P38 MAPK pathway.NRK-52 E cells were randomly divided into NC group,HG group,HG+AM group,HG+Pb30+AM group.The levels of intracellular P38 MAPK,p-P38 MAPK were detected by Western-blot,and the secretion levels of TNF-alpha,IL-6 and IL-1beta were detected by ELISA.Results: 1.Screening of the optimal time for high glucose to induce cell synthesis and secretion of inflammatory factors: With the prolongation of high glucose intervention time,the expression levels of TNF-a,IL-6 and IL-1beta increased.Compared with 0 hours,TNF-a and IL-6 reached their peak at 48 hours(P < 0.05),and IL-1beta reached their peak at 72 hours(P < 0.05),and the final stimulation time was 48 hours.2.Pinocembrin inhibits the synthesis and secretion of inflammatory factors in HG-induced NRK-52 E cells: ELISA results showed that the levels of IL-6,TNF-alpha and IL-1beta in HG group were significantly higher than those in NC group(P < 0.05),while the levels of IL-6,TNF-alpha and IL-1beta in HG+Pb30,HG+Pb60 group were significantly lower than those in HG group(P < 0.05).The results of qPCR showed that the levels of IL-6,TNF-a and IL-1beta in HG group were significantly higher than those in NC group(P < 0.05),while the levels of IL-6,TNF-a and IL-1beta in HG + Pb30 and HG + Pb60 group were significantly lower than those in HG group(P < 0.05).3.Pinocembrin inhibits the activation of MAPK signaling pathway in HG-induced NRK-52 E cells:(1)At protein level,immunofluorescence results showed that the green fluorescence of HG group was significantly stronger than that of NC group,while the green fluorescence of HG+Pb30 and HG+Pb60 group was significantly weaker than that of HG group;Western blot results showed that the level of p-P38MAPK/P38 MAPK in HG group was significantly higher than that in NC group(P<0.05),while the levels of HG+Pb30 and HG+Pb60 were significantly lower than that in HG group(P<0.05).(2)Western blot results showed that the level of p-JNK/JNK in HG group was significantly higher than that in NC group(P < 0.05),while the expression level of p-JNK/JNK in HG + Pb30 and HG + Pb60 groups was not significantly higher than that in HG group(P > 0.05).(3)Western blot results showed that the level of p-ERK/ERK in HG group was significantly higher than that in NC group(P < 0.05),while the expression level of p-ERK/ERK in HG + Pb30,HG + Pb60 group was not significantly higher than that in HG group(P > 0.05).4.Pinocembrin inhibits the activation of NF-kappa B in NRK-52 E cells induced by high glucose: Western blot results showed that the level of p-NF-kappa B/NF-kappa B in HG group was significantly higher than that in NC group(P < 0.05),while the level of p-NF-kappa B/NF-kappa B in HG+Pb30 and HG+Pb60 groups was significantly lower than that in HG group(P < 0.05).5.AM reverses the inhibitory effect of pinocembrin on the expression of P38 MAPK in NRK-52 E cells induced by high glucose: Western blot results showed that the levels of p-P38MAPK/P38 MAPK in HG,HG+AM and HG+Pb30+AM groups were significantly higher than those in NC group(P < 0.05).Compared with HG group,the levels of p-P38MAPK/P38 MAPK in HG+AM group were further higher(P < 0.05),while the levels of p-P38MAPK/P38 MAPK in HG+Pb30 group were significantly lower(P < 0.05).respectively.There was no significant difference in intracellular p-P38MAPK/P38 MAPK level between HG + Pb30 +AM group and HG group(P > 0.05).6.AM reverses the inhibitory effect of pinocembrin on inflammatory cytokines in NRK-52 E cells induced by high glucose: ELISA results showed that the levels of IL-6,TNF-a and IL-1beta in HG,HG+AM and HG+Pb30+AM groups were significantly higher than those in NC(P < 0.05).Compared with HG group,the levels of IL-6,TNF-a and IL-1beta in HG+AM group were further higher(P < 0.05),while the levels of IL-6,TNF-a and IL-1beta in HG+Pb30 group were significantly lower(P < 0.05).The levels of IL-6,TNF-alpha and IL-1beta in the supernatant of HG+Pb30+AM group had no significant difference compared with HG group(P > 0.05).Conclusion: 1.Pb can inhibit the expression of inflammatory factors in renal tubular epithelial cells induced by HG.2.Lead inhibits HG-induced expression of inflammatory factors in renal tubular epithelial cells by inhibiting the activation of P38 MAPK signaling pathway.