高磷对维持性血液透析透患者微炎性反应和氧化应激的影响及机制

The effect and mechanism of high phosphorus concentration on microinflammatory and oxidative stress response in maintenance hemodialysis patients

目的探讨高磷对维持性血透患者微炎性反应及氧化应激的影响及机制。方法实验分A组(正常人对照组),B组(维持性血透患者组即Maintenance hemodialysis,MHD),C组(不同磷酸二氢钠浓度剂量组1.5 mmol/L、C2 2.5 mmol/L及C3 5.5 mmol/L),D组(5.5 mmol/L磷酸二氢钠作用不同时间组D1 6 h、D2 12 h及D3 24 h)和E组(无磷酸二氢钠作用空白组E1 6 h、E2 12 h及E3 24 h组)。用real-time PCR及Western blot分别检测外周血单个核细胞NF-κB P65及NOX2/gp91的表达。ELISA检测血浆及细胞培养液上清IL-6含量,硫氮巴比妥酸法检测MDA含量。结果 MHD组PBMC中NF-κBP65、NOX2 mRNA及phospho-NF-κBP65、NOX2蛋白表达及血浆IL-6、MDA水平明显高于正常组;磷酸二氢钠作用下,培养液上清IL-6、MDA产生呈剂量、时间依赖性增加(P<0.05)。NF-κB P65 mRNA、phospho-NF-κBP65蛋白和NOX2/gp91 mRNAN及蛋白水平表达呈剂量、时间依赖性上调(P<0.05);NF-κB P65mRNA及phospho-NF-κB P65蛋白表达与培养液上清IL-6产生呈正相关,NOX2/gp91mRNA及蛋白表达与培养液上清MDA产生呈正相关。结论高磷可能通过活化NF-κBP65、NOX2/gp91信号通路介导炎性介质、氧化应激产物产生增加,高磷血症可能为MHD患者微炎性反应状态和氧化应激状态的危险因素。

Objective To investigate the effect of high phosphate on mRNA and protein expression of intracellular NF-KB and NOX2/gp91 in PBMC of MHD patients and serum inflammation mediators and MDA production, so as to understand the possible relationship among hyperphosphatemia, micro-inflammatory states and oxidative stress. Methods They were divided into five groups： control, MHD, dose, time and blank groups. Western blot and Re al-time PCR were respectively used to determine the mRNA and protein expression of NF-KB P65, NOX2. The con centration of IL-6 in the culture supernatant was quantified by the ELISA kit and the TBA assay kit. Results NF-KB and NOX2 signal pathway in PBMCs show abnormal activation status in MHD patients. Both expression of NOX2/gp91 mRNA and protein levels increased dose-and time-dependent manners totally. IL-6 and MDA produc tion in supernatant up-regulate dose-and time-dependent manners totally. NF-KB 1765 mRNA and phospho-NF-KB 65 protein were positively correlated with IL-6 production. NOX2/gp91 mRNA and protein expression were posi- tively correlated with MDA production. Conclusions High phosphate may increase the production of supernatantIL-6 and MDA by activating NF-KB and NOX2 signaling pathway in PBMCs, thus could be a risk factor for microin flammatory and oxidative stress status of MHD patients.