抑制线粒体活性氧自由基可减轻高糖诱导的心肌细胞焦亡和铁死亡

目的探讨抑制线粒体氧化应激和炎症小体减轻高糖诱导的H9C2心肌细胞焦亡和铁死亡的作用,分析线粒体活性氧自由基(ROS)和炎症小体之间可能的上下游关系。方法将H9C2心肌细胞按照随机数字表法随机分为5组。正常对照组(CON):含25mmol/L浓度葡萄糖的DMEM完全培养基;高糖损伤组(HG):含35 mmol/L浓度的高糖完全培养基;高糖+线粒体抗氧化剂Mitoquinone(MitoQ)组(HG+MitoQ):35 mmol/L高糖完全培养基中加入终浓度为0.5μmol/mL的MitoQ;高糖+NLRP3抑制剂MCC950组(HG+MCC950):35 mmol/L高糖完全培养基中加入终浓度为1μmol/mL的MCC950;高糖组+MCC950+线粒体电子传递抑制剂Rotenone(ROT)组(HG+MCC950+ROT):35 mmol/L高糖完全培养基中加入终浓度为1μmol/mL的MCC950和0.5μmol/mL的ROT。各组心肌细胞干预24 h后,CCK-8法测定细胞活性;CellRox和MitoSox荧光探针分别测定心肌细胞内和线粒体氧化应激水平变化;免疫荧光法检测细胞内NLRP3炎症小体水平;Western blot检测心肌细胞中NLRP3、GSDMD和CleavedGSDMD(GSDMD-NT)等焦亡相关重要因子的蛋白表达和铁死亡相关因子GPX4蛋白表达。结果与CON组相比,HG组细胞活性明显下降(P<0.01),心肌细胞和线粒体内ROS荧光强度(P<0.01)、NLRP3免疫荧光强度(P<0.01)以及焦亡相关因子NLRP3,GSDMD和GSDMD-NT的蛋白表达(P<0.01)均明显升高,铁死亡相关因子GPX4蛋白表达下降(P<0.01)。与HG组相比,HG+MitoQ和HG+MCC950组细胞活性明显升高(P<0.01),细胞和线粒体内ROS荧光强度(P<0.01)、NLRP3免疫荧光强度(P<0.01)以及NLRP3、GSDMD和GSDMD-NT的蛋白表达明显降低(P<0.05),GPX4蛋白表达增高(P<0.01)。与HG组相比,HG+MCC950+ROT组细胞活性和NLRP3、GSDMD-NT的蛋白表达无明显差异(P>0.05);但与HG+MCC950组相比,HG+MCC950+ROT组细胞活性明显降低(P<0.01),ROS荧光强度、NLRP3炎症小体免疫荧光以及NLRP3、GSDMD-NT的蛋白表达均明显升高,GPX4蛋白表达降低(P<0.05)。结论MitoQ直接抑制线粒体ROS的产生减轻了高糖诱导的心肌细胞NLRP3炎症小体的生成,减少焦亡和铁死亡的发生;抑制NLRP3炎症小体减少线粒体ROS的产生;线粒体ROS和NLRP3之间可能存在着相互影响。

脑创伤后单唾液酸四己糖神经节苷脂对氧自由基线粒体信号通路的影响

目的探讨脑创伤后单唾液酸四己糖神经节苷脂对氧自由基线粒体信号通路的影响。方法采用改进的Marmarou's法建立大鼠中度闭合性弥漫性颅脑损伤模型。应用免疫组化、TUNEL法对大鼠伤后皮质细胞色素c、凋亡细胞数进行动态观察。硫代巴比妥酸法测量丙二醛。结果伤后给予Edaravone进行干预自由基水平明显减少,细胞色素c表达和神经元细胞凋亡数降低;依达拉奉与单唾液酸四己糖神经节苷脂联合干预效果更为明显。结论颅脑创伤后单唾液酸神经节苷脂具有脑保护作用,其可能的机制之一是调节氧自由基线粒体信号通路而发挥作用。

Mechanisms of Inhibitory Effects of Breviscapine on Lipid Peroxidation in Rat Brain Mitochondria

The mechanisms by which breviscapine (Bre) inhibits the lipid preoxidation in rat brain mitochondria were investigated. The mitochondrial lipid peroxidation of rat brain induced by oxygen free radical was measured by thiobarbituric acid spectrophotometry. The chelating activities of Bre for Fe 2+ were tested by differential spectrum. Superoxide anion (O 2)from xanthine xanthine oxidase (Xan XO) system and hydroxyl radical (·OH) from FeSO 4 H 2O 2 system were determined with spectrophotometry. It was found that Bre could effectively inhibit the lipid peroxidation of brain mitochondria induced by free radicals driven from Xan XO and FeSO 4 H 2O 2 system. The IC 50 of Bre were 93 01 μmol·L -1 for Xan XO system and 62 18 μmol·L -1 for FeSO 4 H 2O 2 system. Bre also scavenged O 2 and ·OH produced by Xan XO and FeSO 4 H 2O 2 systems. The IC 50 of Bre were 32 63 μmol·L -1 for O - 2 and 20 22 μmol·L -1 for ·OH. Furthermore, the chelating Fe 2+ activity of Bre was shown. It may be concluded that Bre inhibited lipid peroxidation at different stages of the reaction of oxygen free redial with the mitochondria membrane: (1) the formation of ·OH; (2) the initiation of the lipid peroxidation, by chelating Fe 2+ and scavenging O 2 as well as ·OH. The scavenging oxygen free radicals and chelating iron are the mechanisms of inhibitory effect of Bre on lipid peroxidation.