P‑hydroxybenzyl alcohol ameliorates neuronal cerebral ischemia‑reperfusion injury by activating mitochondrial autophagy through SIRT1
P‑hydroxybenzyl alcohol ameliorates neuronal cerebral ischemia‑reperfusion injury by activating mitochondrial autophagy through SIRT1
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DOI：
10.3892/mmr.2023.12955
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发表时间：
2023-02
影响因子：
3.4
通讯作者：
Xing Yu;Yuan Luo;Liping Yang;Pu Chen;Xiaohua Duan
中科院分区：
文献类型：
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作者：
Xing Yu;Yuan Luo;Liping Yang;Pu Chen;Xiaohua Duan
线粒体自噬在清除受损线粒体中起关键作用。对羟基苯甲醇（pHBA）可改善脑缺血再灌注（I/R）所致的神经元损伤。然而，pHBA通过线粒体途径改善I/R损伤的机制尚不清楚。本研究采用大鼠大脑中动脉闭塞再灌注（MCAO/R）模型。用sirtuin 1 （SIRT1）抑制剂EX527和pHBA治疗大鼠7 d，然后再灌注。再灌注后24 h计算梗死面积，评估神经损伤程度。苏木精染色、伊红染色和尼氏染色显示缺血半暗区细胞变化。电镜观察线粒体结构的变化。采用市售试剂盒，通过检测线粒体膜电位（MMP）、线粒体通透性过渡孔（mPTP）和ATP水平来评估线粒体功能。此外，对缺血半暗带组织进行p62和LC3蛋白的免疫荧光染色。western blotting检测SIRT1和线粒体自噬相关蛋白、pten诱导的激酶1 （PINK1）和Parkin的表达。最后，TUNEL染色检测细胞凋亡，western blot检测细胞凋亡相关蛋白Bax、Bcl-2、Caspase-3的表达。结果提示，术后给予pHBA治疗可减轻MCAO/R大鼠脑梗死面积和神经系统损伤，改善缺血半暗区细胞损伤。与MCAO/R未处理组相比，phba处理组大鼠线粒体结构改善，MMP和ATP水平升高，mPTP开放程度降低。同时免疫荧光和western blotting结果显示，与MCAO/R组相比，pHBA组LC3-和tunel阳性细胞数量增加，p62阳性细胞数量减少，SIRT1和自噬蛋白（PINK1、Parkin和LC3 II/I）表达水平升高，p62表达水平降低。然而，这些改善被EX527治疗阻断。综上所述，本研究结果提示，pHBA可能通过sirt1激活的线粒体自噬和线粒体介导的神经元凋亡，改善MCAO/R大鼠缺血半暗区神经元损伤。
Mitochondrial autophagy serves a key role in clearing damaged mitochondria. P-hydroxybenzyl alcohol (pHBA) can improve neuronal injury induced by cerebral ischemia-reperfusion (I/R). However, the mechanism of pHBA improving I/R damage through the mitochondrial pathway remains unclear. A rat model of middle cerebral artery occlusion and reperfusion (MCAO/R) was used in the present study. The rats were treated with sirtuin 1 (SIRT1) inhibitor EX527 and pHBA for 7 days, followed by reperfusion. At 24 h after reperfusion, the infarct size was calculated and the severity of nerve damage was evaluated. Hematoxylin and eosin and Nissl staining revealed cellular changes in the ischemic penumbra. Changes in mitochondrial structure were observed using electron microscopy. Mitochondrial function was evaluated by detecting mitochondrial membrane potential (MMP), mitochondrial permeability transition pore (mPTP) and ATP levels using commercially available kits. In addition, the ischemic penumbra tissues were used for immunofluorescence staining for p62 and LC3 proteins. The expression of SIRT1 and mitochondrial autophagy-related proteins, PTEN-induced kinase 1 (PINK1) and Parkin, were detected by western blotting. Finally, apoptosis was analyzed by TUNEL staining and the expression of apoptosis-related proteins (Bax, Bcl-2 and Caspase-3) by western blotting. The results suggested that postoperative pHBA treatment may reduce the size of cerebral infarction and damage to the nervous system, and may improve cell damage in the ischemic penumbra of MCAO/R rats. Compared with rats in the untreated MCAO/R group, the mitochondrial structure of the pHBA-treated group was improved, the levels of MMP and ATP were increased, and the degree of opening of mPTP was decreased. Simultaneously, immunofluorescence and western blotting results showed that compared with the MCAO/R group, the number of LC3- and TUNEL-positive cells increased, the number of p62-positive cells decreased, SIRT1 and autophagy protein (PINK1, Parkin and LC3 II/I) expression levels increased and p62 expression decreased in the pHBA group. However, these improvements were blocked by treatment with EX527. In summary, results from the present study suggested that pHBA may improve neuronal injury in the ischemic penumbra of MCAO/R rats through SIRT1-activated mitochondrial autophagy and mitochondrial-mediated neuronal apoptosis.