川芎嗪通过激活SIRT1信号抑制铁死亡减轻脂多糖致内皮细胞炎症损伤

    Tetramethylpyrazine attenuates ferroptosis by activating SIRT1 signal pathway and ameliorates lipopolysaccharide-induced inflammation and injury in endothelial cells

      摘要
    • 摘要:
      目的 观察川芎嗪（tetramethylpyrazine，TMP）对脂多糖（lipopolysaccharide，LPS）诱导的人冠脉内皮细胞（human coronary artery endothelial cells，HCAEC）损伤的作用及机制。
      方法 将HCAEC随机分为4组：Control组、LPS（1 µg/mL）组、LPS+TMP（10 μmol/L）组和LPS+TMP+SIRT1抑制剂EX527组，HCAEC先使用TMP和EX527预处理12 h，然后再用LPS处理24 h。倒置显微镜下观察各组细胞形态变化，CCK-8法检测各组HCAEC活力，乳酸脱氢酶(lactate dehydrogenase，LDH)试剂盒检测各组培养基中LDH的活性，分别检测各组细胞中丙二醛（malondialdehyde，MDA）、谷胱甘肽（glutathion，GSH）、亚铁离子（Fe2+）、活性氧（reactive oxygen species，ROS）水平，Western blot法检测沉默信息调节因子1（silent information regulator 1，SIRT1）、谷胱甘肽过氧化物酶4（glutathuone peroxidase 4，GPX4）的表达、溶质载体家族7成员11（solute carrier family 7 member 11，SLC7A11）、转铁蛋白受体1(transferrin receptor 1，TFR-1）。DCFH-DA 染色观察各组细胞ROS含量的变化，免疫荧光染色检测各组细胞SIRT1表达。
      结果 与Control组相比，LPS组细胞活力降低（P<0.01），且培养基中LDH活性明显升高（P<0.05），细胞中GSH含量及GPX4、SLC7A11的表达降低（P<0.05），MDA、Fe2+和ROS水平及TFR-1的表达显著升高（P<0.05），SIRT1表达下降（P<0.05）；TMP干预后可以提高细胞活力（P<0.01），降低培养基中LDH的活性（P<0.05），减少MDA、Fe2+和ROS水平及TFR-1的表达（P<0.05），增加GSH含量及GPX4、SLC7A11的表达（P<0.05），同时上调SIRT1表达（P<0.05）；而给予SIRT1抑制剂EX527处理后，可明显阻断TMP对HCAEC的保护作用和对SIRT1信号的调控作用（P<0.05）。
      结论 TMP可以改善 LPS诱导的HCAEC损伤，其机制与抑制铁死亡、激活SIRT1信号有关。

       

      Abstract:
      AIM  To investigate the impact of tetramethylpyrazine (TMP) on lipopolysaccharide (LPS)-induced injury in human coronary artery endothelial cells (HCAEC) and elucidate its role and the involved underlying mechanism.
      METHODS HCAEC were randomly divided into 4 groups: control group, LPS (1 µg/mL) group, LPS+TMP (10 µmol/L) group and LPS+TMP+SIRT1 inhibitor EX527 group. Prior to treatment with LPS for 24 hours, HCAEC were pre-treated with TMP and EX527 for 12 hours. Cell morphology was observed using inverted microscope, HCAEC activity was assessed using CCK-8 method and lactate dehydrogenase (LDH) activity was measured using commercial LDH kit. Additionally, levels of malondialdehyde (MDA), glutathion (GSH), ferrous ion (Fe2+) and reactive oxygen species (ROS) were quantified through cellular analysis. Western bolt was performed to analyze the expressions of silent information regulator 1 (SIRT1), glutathuone peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11) and transferrin receptor 1 (TFR-1). DCFH-DA staining was utilized to monitor the changes in ROS content in different groups. Concurrently, immunofluorescence staining was employed to assess the expression of SIRT1.
      RESULTS Compared with the control group, the LPS group exhibited reduced cell viability (P<0.01), a significant increase in LDH activity (P<0.05), decreased GSH content, and down-regulation expressions of GPX4 and SLC7A11 (P<0.01). The levels of MDA, Fe2+ and ROS and the expression of TFR-1 were significantly increased (P<0.05), while SIRT1 expression was decreased (P<0.05). After TMP intervention (P<0.01), there was an improvement in cell viability accompanied by a decrease in LDH activity (P<0.05). Furthermore, there was a reduction in MDA, Fe2+ and ROS levels, along with decreased TFR-1 expression, whereas the GSH content and the expression levels of GPX4 and SLC7A11 were increased (P<0.05). Moreover, SIRT1 expression was up-regulated (P<0.05). However, following treatment with the SIRT1 inhibitor EX527, the protective impact of TMP on HCAEC and the regulation of SIRT1 signal were significantly blocked (P<0.05).
      CONCLUSION TMP exhibits a protective effect against LPS-induced damage in HCAEC by attenuating ferroptosis and activating the SIRT1 signaling pathway.

       

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