黄芪甲苷调控铁死亡途径改善高糖腹透液诱导的人腹膜间皮细胞损伤

Astragaloside IV interferes with ferroptosis to reduce the damage of HMrSV5 induced by peritoneal dialysis solution

目的 关于黄芪甲苷(AS-Ⅳ)能否调控PMCs铁死亡的研究报道少见。文章探究AS-Ⅳ对高糖腹透液(PDS)诱导人腹膜间皮细胞(HMrSV5)铁死亡的干预作用及可能机制。方法 (1)采用4.25%PDS构建PD小鼠模型,予以不同剂量的AS-Ⅳ干预(0.5与1 mg/kg),分组为对照组,PD模型组,低、高剂量干预组。免疫荧光检测腹膜组织GPX4的表达。(2)体外培养HMrSV5细胞,CCK-8检测PDS及AS-IV对HMrSV5细胞活性的影响,根据检测结果确定后续实验的PDS浓度及AS-Ⅳ剂量范围。(3)采用3%PDS构建HMrSV5细胞损伤模型,予以不同剂量AS-Ⅳ干预(50、100及200μmol/L),分组为:空白组,模型组,低、中、高剂量AS-Ⅳ干预组,FerroOrange荧光探针检测Fe^(2+)水平;DCFH-DA荧光探针检测活性氧水平;相应的试剂盒检测GSH和MDA含量;Western blot和RT-PCR分别检测P53、SLC7A11、GPX4和ACSL4等铁死亡相关蛋白及mRNA的表达。结果 与对照组相比,高剂量AS-Ⅳ部分上调PD小鼠模型腹膜组织GPX4的表达;模型组GSH含量[(15.82±1.00)μmol/g]较空白组[(32.63±1.44)μmol/g]减少(P<0.01),MDA含量[(3.87±0.21)nmol/mg]较空白组[(1.48±0.09)nmol/mg]增加(P<0.01),Fe^(2+)、活性氧、P53及ACSL4的表达较空白组上调(P<0.01),GPX4与SLC7A11的表达较空白组下调(P<0.01);与模型组相比,中、高剂量AS-Ⅳ干预组GSH含量[(25.23±2.02、30.03±1.21)μmol/g]增加(P<0.01),MDA含量[(2.92±0.25、1.89±0.15)nmol/mg]减少(P<0.01),Fe^(2+)、活性氧、P53及ACSL4表达下调(P<0.01),GPX4和SLC7A11的表达上调(P<0.01),mRNA的水平与蛋白表达一致。结论 PDS诱导的HMrSV5细胞损伤模型中存在铁死亡,且AS-Ⅳ能通过抑制细胞铁死亡,发挥保护细胞的作用,其机制可能与AS-Ⅳ降低细胞内Fe^(2+)含量、抑制活性氧生成、提高xCT与GPX4活性有关。

Objective Studies have shown that Astragalus membranaceus and its active components can inhibit reaction oxygen species(ROS) production, antagonize PMCs apoptosis, and improve PMCs mesenchymal transformation. Whether astragalus Ⅳ can regulate iron death in PMCs is still unclear. This study investigated the intervention effect and possible mechanism of astragaloside Ⅳ(AS-Ⅳ) in protecting human peritoneal mesothelial cell(HMrSV5) high against glucose peritoneal dialysis solution(PDS)-induced injury. Methods(1)Established PD mouse model and intervened with concentrations of AS-Ⅳ(0.5 and 1 mg/kg). Animal experiment group: blank control group, PD group, AS-Ⅳ low and high concentration group. The expression of GPX4 in peritoneal tissues was detected by immunofluorescence.(2)HMrSV5 cells were cultured in vitro. The effects of PDS and AS-IV on HMrSV5 cell activity were detected by CCK-8. And the concentrations of PDS and AS-Ⅳ in subsequent experiments were determined according to the detection results.(3)HMrSV5 cell injury model was established by 3%PDS, and different concentrations of AS-Ⅳ intervention(50, 100 and 200 μmol/L) were administered. Cell experiment group: blank group, PDS group, AS-Ⅳ low, medium and high concentration group. The content of Fe^(2+) in cells were detected by FerroOrange’s fluorescence imaging. The content of ROS in cells were detected by DCFH-DA. The content of glutathione(GSH) and malonaldehyde(MDA) were detected by kits. The expressions of tumor suppressor gene 53(P53), long-chain acyl-CoA synthetase 4(ACSL4), cationic amino acid transporter 11(SLC7 A11) and glutathione peroxidase 4(GPX4) were detected by Western blot and RT-PCR. Results Compared with the blank control group, the expression of GPX4 in peritoneal tissues of PD mouse model was partially up-regulated by high dose AS-Ⅳ. Compared with blank control group, GSH content in model group was decreased [(15.82±1.00) μmol/g], while MDA content was increased [(3.87±0.21) nmol/mg]. The expressions of Fe^(2+)(P<0.01), ROS(P<0.01), P53(P<0.01) and ACSL4(P<0.01) were up-regulated, while the expressions of GPX4(P<0.01) and SLC7 A11(P<0.01) were down-regulated. Compared with model group, the content of GSH was increased [(25.23±2.02, 30.03±1.21) μmol/g] and the content of MDA was decreased [(2.92±0.25, 1.89±0.15) nmol/mg] in medium-dose and high-dose AS-Ⅳ intervention groups. The expressions of Fe^(2+)(P<0.01), ROS(P<0.01), P53(P<0.01) and ACSL4(P<0.01) were down-regulated, and the expressions of GPX4(P<0.01) and SLC7 A11(P<0.01) were up-regulated. The mRNA level was consistent with the protein expression. Conclusion AS-IV can interfere with the ferroptosis caused by PDS, and have a protective effect on HMrSV5 cells. The mechanism may be related to the ability of AS-Ⅳ to scavenge reaction oxygen species, reduce intracellular Fe^(2+) levels, and enhance the activity of system xc-and GPX4.