Aldose Reductase Regulates Tnf-α-induced Inos Production In Human Mesangial Cells

Glomerulonephritis can be induced by several reagents among which immune complexes are the most common reaction. Nitric oxide (NO) radicals generated by inducible NO synthase (iNOS) is the important mediator in inflammatory processes. Aldose reductase (AR), a member of the aldo-keto reductase superfamily, represents the first and rate-limiting step of polyol pathway. To analyze the role of AR in glomerular inflammation, we studied the in vitro expression of iNOS in human mesangial cells (HMC) induced by TNF-a in the condition of reduced activity of AR, and the possible involving signaling pathways.Part I Interfering aldose reductase in the process of iNOS production induced by TNFa in human mesangial cells1.1 The impact of the expression of iNOS and AR induced by TNF-aObjective To observe the effects of AR in the process of iNOS production in human mesangial cells, we compared the iNOS expression in the condition of TNF-a.Methods HMC was stimulated by different does of TNF-a in the condition of reduced activity of AR for different time. iNOS expression was measured by Western blot.Results 6ng/ml of TNF-a could highly induce the production of iNOS in 12 hours.Conclusion iNOS could be highly induced by TNF-a.1.2 TNF-a-induced expression of iNOS of HMC with AR-siRNAObjective To observe the effects of AR in the process of iNOS production in HMC, we compared the quantity of iNOS expression in the condition of TNF-a with or without AR-siRNA.Methods When cell growth to about 70-80%confluence, using AR-siRNA pretreatment HMC for 48h, using TNF-a for 12h, closed-cell cytoplasmic protein extraction. The expression of iNOS and AR at the protein and mRNA levels detected with Western blot and Real-time PCR.Results AR can inhibit iNOS induced by TNF-a in HMC with AR-siRNA, and inhibition works from the nucleic acid level.Conclusion AR decreased the expression of iNOS in the inflammatory process.1.3 The impact of AR on IKK-IκB signal pathway in HMC with AR-siRNAObjective To observe of changes of IKK-IκB signaling pathway promoted by TNF-a promote in HMC with or without AR-siRNA.Methods In cultured HMC, when cell growth to 70-80%, pre-treated cells with AR-siRNA for 48 hours, and then stimulated by TNF-a, cells were collected at 0,30,60,120 min. Phosphorylation of IKK-IκB pathway was detected by Western blot.Results Phosphorylation of IKK a/βand IκB increased in 30min,and got to the top in 60min, while the total IKKa/βshows no significant changes. Corresponding, total of IκB enhanced with the p-κB reduced. Compaired with TNF-a group, phosphorylation of IκBα/βwas reduced in AR-siRNA+TNF-a group.Conclusion Interfering with AR can inhibit TNF-a induced activation of NF-κB signaling pathway upstream IKK phosphorylation.1.4 AR-siRNA regulates the activity of NF-κB in the condition of TNF-a in HMCObjective To observe the activity of NF-κB stimulated by TNF-a with or without AR-siRNA could affect the activation effects. Methods The neucleoprotein was extracted by CelLyticTM NuCLEARTM Extraction Kit. The NF-κB activation degree was evaluated by EMSAResults TNF-a could significantly inhance the NF-κB activation, which could be inhibited byAR-siRNA.Conclusion The activation of AR could regulate the NF-κB activation induced by TNF-a, which may be the mechanism of the AR effection.Part II Transfection of aldose reductase in the process of iNOS production induced by TNFa in human mesangial cells2.1 TNF-a-induced expression of iNOS of HMC with AR-siRNAObjective To observe the effects of AR in the process of iNOS production in human mesangial cells, we compared the quantity iNOS expression in the condition of TNF-a with or without pcDNA3.1-AR transfection.Methods HMC was stimulated by TNF-a in the condition of enhanced activity of AR for 12 hours. iNOS expression was measured by Western blot.Results TNF-a could highly induce the production of iNOS in 12 hours; pcDNA3.1-AR transfecting could stimulate this effect in protein level.Conclusion The enhancement of AR could inhibit the production of iNOS induced by TNF-a, which showed that AR could regulate the iNOS production in the early stage of the inflamation.2.2 The impact of AR on IKK-IκB signal pathway in HMC with transfection of aldose reductaseObjective To observe of changes of IKK-IκB signaling pathway promoted by TNF-a promote in HMC with transfection of aldose reductase.Methods In cultured HMC, when cell growth to 70-80%, pre-treated cells with pcDNA3.1-AR for 48 hours, and then stimulated by TNF-a, cells were collected at 0,30,60,120 min. Phosphorylation of IKK-IκB pathway was detected by Western blot.Results Phosphorylation of IKK a/βand IκB increased in 30min,and got to the top in 60min, while the total IKKa/p shows no significant changes. Corresponding, total of IκB enhanced with the p-κB reduced. Compaired with TNF-αgroup, phosphorylation ofκBa/βwas increased in pcDNA3.1-AR+ TNF-a group.Conclusion Transfecting with AR can increase TNF-a induced activation of NF-κB signaling pathway upstream IKK phosphorylation.2.3 Transfection of aldose reductase regulates the activity of NF-κB in the condition of TNF-a in HMCObjective To observe the activity of NF-κB stimulated by TNF-a with or without pcDNA3.1-AR could affect the activation effects.Methods The neucleoprotein was extracted by CelLyticTM NuCLEARTM Extraction Kit. The NF-κB activation degree was evaluated by EMSAResults TNF-a could significantly inhance the NF-κB activation, which could be increased by pcDNA3.1-AR.Conclusion The activation of AR could regulate the NF-κB activation induced by TNF-a, which may be the mechanism of the AR effection.Conclusions1. The quantity of AR could affect the iNOS expression in early inflammation period.2. AR could regulate the NF-κB activation, which is the main regulator in the iNOS production.