丹参酮ⅡA对腹膜透析液诱导人腹膜间皮细胞氧化应激及其损伤的影响

Effect of Tanshinone ⅡA on Oxidative Stress Damage in Cultured Human Peritoneal Mesothelial Cells Induced by Peritoneal Dialysis Fluid

【目的】研究丹参酮ⅡA对腹膜透析液（PDF）诱导人腹膜间皮细胞（HPMCs）氧化应激及其损伤的影响。【方法】体外培养的HPMCs同步后分为五组：A组即对照组（DMEM培养基＋完全培养基）；B组即腹膜透析液组（含4．25％PDF的完全培养基）；C组即丹参酮组（含100μmmol／L丹参酮的完全培养基）；C1组即丹参酮1组（含50μmol／L丹参酮ⅡA的PDF）、C2组即丹参酮2组（含100μmol／I。丹参酮ⅡA的PDF），干预72h后（其中B组、C1、C2组为加热的PDF），流式细胞仪及荧光显微镜检测线粒体膜电位的变化；荧光显微镜检测胞内钙离子浓度的变化。采用活性氧捕获剂二氯二氢荧光素-乙酰乙酸酯（DCFH—DA）孵育细胞，通过流式细胞仪检测细胞内的荧光强度而测得细胞内活性氧水平。并检测上清液中超氧化物歧化酶、谷胱甘肽过氧化物酶、和丙二醛含量；不同浓度丹参酮ⅡA干预48h后，MTT比色法检测不同浓度丹参酮ⅡA干预下细胞增殖活力。【结果】B组上清液丙二醛含量、胞内钙离子、细胞内活性氧水平较对照组及C1、C2组显著增加（P〈0．01），超氧化物歧化酶、谷胱甘肽过氧化物酶及线粒体膜电位水平显著降低（P〈0．01）。不同浓度丹参酮ⅡA组HPMCs的增殖活性显著高于B组（P〈0．01），丹参酮ⅡA不同浓度间无显著差异（P〉0．05）。【结论】丹参酮ⅡA可以通过降低PDF干预下丙二醛含量、减轻钙离子负荷、维持线粒体膜电位的水平、保护抗氧化还原酶的活性来拮抗商业性PDF对HPMCs的氧化应激及其损伤。

[Objective]To investigate the effect of tanshinone IIA on oxidative stress damage in cultured human peritoneal mesothelial cells（HPMCs） induced by peritoneal dialysis fluid（PDF）. [Methods] HPMCs cultured in vitro were used for our experiment. After being incubated with DMEM for 24 hours, HPMCs were divided into eontrol（DMEM medium plus complete medium） group（group A）, PDF（complete medium with 4.25 % PDF） group（group 13）, tanshinone ⅡA（eomplete medium with 100/μmol/L tanshinone Ⅱ A） group（group C）, tanshinone Ⅱ A1 （PDF with 50μmol/L tanshinone Ⅱ A） group（group C1） and tanshinone Ⅱ A2（PDF with 100μmol/L tanshinone Ⅱ A） group（group C2）. After 72 hours of intervention（heated PDF in group B, C1 and C2）, flow cytometry and fluorescence microscope were used to detect the change of mitochondria membrane potentials（MMP） and intracellular calcium concentration. Reactive oxygen species（ROS） trapping agent was used to incubate the cells. Flow cytometry was used to detect the intracellular fluorescence intensity in order to measure ROS level. Superoxide dismutase（SOD）, glutathione peroxidase（GSH） and malondialdehyde（MDA） were measured. After 48 hours of intervention with different concentration of tanshinone Ⅱ A, MTT method was used to detect the cell proliferation. [Results] Compared with the control group and group C1 and C2, the levels o MDA, intracellular calcium and ROS in the supernatant of group B in- creased significantly（ P 〈0.01）, but the levels of SOD, GSH and MMP decreased significantly（ P 〈0.01）. The proliferation activity of HPMCs in groups with different concentration of tanshinone IIA decreased markedly（ P 〈0.01）. There was no sig- nificant difference among different concentration tanshinone ⅡA （ P 〉0. 05）. [Conclusion]TanshinoneⅡ A may protect the HPMCs from oxidative stress damage induced by PDF through decreasing MDA content, reducing calcium load, maintaining MMP level and preserving the activity of oxido-reductase.