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目的:观察糖基化终末产物(AGEs)对肾间质成纤维细胞中血管新生关键调节因子血管生成素(Ang)1、2及其受体酪氨酸激酶受体2(Tie-2)表达的影响,探讨其可能的机制以及抗氧化剂银杏叶提取物(EGb)的干预作用。方法:选择正常大鼠肾间质成纤维细胞系(NRK-49F)为研究对象,以低糖DMEM培养液培养细胞作对照,分别用含AGEs(400mg/L)和高糖(25mmol/L)的DMEM培养液体外培养24h,用抗氧化剂EGb(100mg/L)预处理后再分别加入AGEs和高糖继续培养。采用实时定量PCR检测Ang-1、Ang-2、Tie-2,核因子κB(NF-κB)及其抑制物(I-κB)mRNA表达水平,Westernblot检测蛋白表达水平。二乙酰二氯荧光素(DCFH)染色后荧光倒置显微镜及流式细胞仪检测细胞内活性氧(ROS)水平。结果:与对照组相比,AGEs和高糖组细胞内ROS水平明显升高;且Ang-1及NF-κBmRNA和蛋白表达显著增高,I-κBmRNA和蛋白表达显著下降,Ang-2和Tie-2表达则无明显变化。EGb预处理后可降低细胞内ROS水平及Ang-1和NF-κB表达水平,提高I-κB表达水平,而对Ang-2和Tie-2表达无影响。结论:AGEs可通过增强细胞内氧化应激,抑制I-κB表达而激活NF-κB,上调Ang-1表达,参与糖尿病肾病(DN)血管新生的调控。EGb可通过减少氧化应激,上调I-κB表达,抑制NF-κB途径而下调Ang-1表达,在防治DN方面有良好的应用前景。
OBJECTIVE: To observe the effects of advanced glycation end products (AGEs) on angiogenin 1, 2 and its receptor tyrosine kinase receptor 2 (Ang-2) in angiogenesis of renal interstitial fibroblasts The possible mechanism as well as the intervention of antioxidant Ginkgo biloba extract (EGb) were explored. Methods: Normal rat renal interstitial fibroblast cell line (NRK-49F) was selected as the research object and cultured in low glucose DMEM culture medium as control. The cells were treated with AGEs (400 mg / L) and high glucose The DMEM culture medium was cultured in vitro for 24 h and then pretreated with antioxidant EGb (100 mg / L) and then added with AGEs and high glucose respectively for further culture. The mRNA expression of Ang-1, Ang-2, Tie-2, NF-κB and I-κB were detected by real-time quantitative PCR. The protein expression was detected by Western blot. Fluorescence inverted microscope and flow cytometry were used to detect intracellular reactive oxygen species (ROS) levels after staining with diacetyl dichlorofluorescein (DCFH). Results: Compared with the control group, the level of ROS in AGEs and high glucose group was significantly increased. The mRNA and protein expressions of Ang-1 and NF-κB were significantly increased, while the expressions of I-κB mRNA and protein were significantly decreased. Ang-2 and Tie- 2 expression was no significant change. After pretreatment with EGb, the intracellular ROS level and the expression of Ang-1 and NF-κB were decreased and the expression of I-κB was increased, while the expression of Ang-2 and Tie-2 was not affected. CONCLUSION: AGEs are involved in the regulation of angiogenesis in diabetic nephropathy (DN) by increasing intracellular oxidative stress, inhibiting the expression of I-κB, activating NF-κB and up-regulating the expression of Ang-1. EGb has a good application prospect in the prevention and treatment of DN by reducing the oxidative stress, up-regulating the expression of I-κB, inhibiting the NF-κB pathway and down-regulating the expression of Ang-1.