小胶质细胞铁死亡在烟雾吸入性脑损伤中的作用机制探讨

Mechanism of microglia ferroptosis in smoke inhalation-induced brain injury

目的探究小胶质细胞铁死亡在烟雾吸入性脑损伤中的作用机制。方法将20只C57BL/6小鼠随机分为对照组(Control组)、烟雾吸入性损伤(SII)组、铁抑素治疗组(Fer-1组,2.5 mmol/kg Fer-1)、甲磺酸去铁胺治疗组(DFO组,200 mg/kg DFO),每组5只。Fer-1组和DFO组于造模后第1、3、5天分别腹腔注射Fer-1和DFO。第6天通过苏木素伊红(HE)染色、普鲁士蓝染色观察各组小鼠脑组织的病理变化。实时荧光定量逆转录PCR(RT-qPCR)检测脑损伤因子、炎性因子、铁死亡因子基因表达水平。双联吡啶比色法测定小鼠脑组织中铁含量。硫代巴比妥酸比色法测定小鼠脑组织脂质过氧化物(LPO)、丙二醛(MDA)含量。黄嘌呤氧化酶法测定小鼠脑组织超氧化物歧化酶(SOD)活性。二硫代二硝基苯甲酸(DTNB)直接法测定小鼠脑组织谷胱甘肽(GSH)含量。DMEM完全培养基培养BV2细胞,分为Control组、Erastin组(10μmol/L Erastin刺激)、Fer-1组(10μmol/L Erastin与5 mmol/L Fer-1同时刺激)、DFO组(10μmol/L Erastin与50 mmol/L DFO同时刺激)。24 h后,CCK-8法检测细胞活力变化,流式细胞术检测细胞凋亡情况,2,7-二氯荧光素二乙酸酯(DCFDA)检测细胞内活性氧(ROS)水平,线粒体红色荧光探针检测线粒体膜电位。结果与Control组相比,SII组小鼠脑组织出现明显铁沉积和炎症反应,脑组织脑损伤因子、炎性因子mRNA表达水平升高,乙酰辅酶A合成酶长链家族4(ACSL4)、核受体共激活因子4(NCOA4)mRNA表达水平升高,谷胱甘肽氧化酶4(GPX4)、溶质载体家族7成员11(SLC7A11)mRNA表达水平降低,小鼠脑组织GSH和SOD含量降低,LPO和MDA含量升高(P<0.05)。与SII组相比,Fer-1组和DFO组小鼠相应指标变化与上述相反(P<0.05),小鼠脑组织损伤减轻。BV2细胞实验结果显示,与Control组相比,Erastin组BV2细胞存活率下降、凋亡率升高(P<0.05),细胞内ROS水平升高,线粒体膜电位下降;与Erastin组相比,Fer-1组、DFO组细胞相应指标变化与上述相反(P<0.05),BV2细胞氧化损伤得到缓解。结论铁死亡抑制剂Fer-1和DFO能够抑制小胶质细胞铁死亡并缓解烟雾吸入性脑损伤。

Objective To investigate the underlying mechanism of microglia ferroptosis in smoke inhalation-induced(SII)brain injury.Methods Twenty SPF-grade male C57BL/6 mice were randomly divided into the control group,the SII group,the ferrostatin-1 group(Fer-1,2.5 mmol/kg)and the deferoxamine group(DFO,200 mg/kg),with 5 mice in each group.Mice in the Fer-1 group and the DFO group were intraperitoneally injected with Fer-1 and DFO 1,3 and 5 day after smoke exposure,respectively.The pathological changes of brain tissue were examined by HE staining and Prussian blue staining on the 6th day after smoke exposure.RT-qPCR was used to detect levels of inflammatory factors,brain tissue damage markers and ferroptosis markers.The contents of iron in mouse brain tissue were determined by double pyridine colorimetric assay.The contents of malondialdehyde(MDA)and lipid peroxide(LPO)in mouse brain tissue were determined by thiobarbituric acid(TBA)colorimetric assay.Superoxide dismutase(SOD)activity in mouse brain tissue was measured by xanthine oxidase assay kit.The contents of glutathione(GSH)in mouse brain tissue were determined by direct method of dithiodinitrobenzoic acid(DTNB)assay.BV2 cells were cultured in complete DMEM medium and divided into the control group,the erastin group(10μmol/L),the Fer-1 group(erastinc stimulation combined with 5 mmol/L Fer-1 treatment)and the DFO group(erastinc stimulation combined with 50 mmol/L DFO treatment).After 24 h,cell viability was detected by CCK-8 assay,cell apoptosis was detected by Annexin V-FITC/PI flow cytometry,intracellular reactive oxygen species(ROS)production was detected by DCFDA staining,and mitochondrial membrane potential was detected by MitoTracker Red CMXRos staining.Results Compared with the control group,enhanced iron deposition and inflammation in brain tissue,elevated mRNA expression of inflammatory markers and damage markers in brain tissue,up-regulated ACSL4 and NCOA4 mRNA levels,down-regulated GPX4 and SLC7A11 mRNA levels,decreased GSH and SOD contents,and increased LPO and MDA contents were observed in brain tissue of the SII group(P<0.05).The mRNA expression level of 7 member of solute carrier family 11(SLC7A11)was decreased in mice of the SII group.The contents of GSH and SOD were decreased,and the contents of LPO and MDA were increased(P<0.05).Compared with the SII group,all the above parameters were reversed in the Fer-1 group and the DFO group,and the damage of mouse brain tissue was alleviated(P<0.05).In BV2 cell experiments,compared with the control group,decreased survival rate of BV2 cells and increased apoptosis rate were found in the erastin group(P<0.05),and increased intracellular ROS level and decreased mitochondrial membrane potential were also observed in the erastin-stimulated BV2 cells.The above parameters were opposite to those of the erastin group in the Fer-1 group and the DFO group(P<0.05),and the oxidative damage of BV2 cells was alleviated.Conclusion The ferroptosis inhibitors Fer-1 and DFO can inhibit microglia ferroptosis and alleviate smoke inhalation-induced brain injury.