Department of Emergency Medicine, The Second Affiliated Hospital of Guangxi Medical University, Nanning, 530007, Guangxi, People's Republic of China.
Intensive Care Unit, The Second Affiliated Hospital of Guangxi Medical University, 166 Daxuedong Road, Guangxi, 530007, Nanning, People's Republic of China.
Cell Commun Signal. 2023 Aug 14;21(1):204. doi: 10.1186/s12964-023-01211-3.
Cerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Our previous study found that extracellular signal-regulated kinase (ERK) activation, dynamin-related protein1 (Drp1)/Mitofusin2 (Mfn2)-dependent mitochondrial dynamics imbalance, and excessive autophagy were involved in the mechanism of nerve injury after CA/CPR. However, the specific pathological signaling pathway is still unknown. This study aimed to explore the molecular function changes of ERK-Drp1/Mfn2-autophagy signaling pathway in SH-SY5Y cell oxygen-glucose deprivation/reoxygenation (OGD/R) model, to further clarify the pathophysiological mechanism of CIRI, and to provide a new strategy for cerebral protection after CIRI.
SH-SY5Y cells were pretreated with drugs 24 h before OGD/R. The Drp1 and Mfn2 knockdown were adopted small interfering RNAs. The overexpression of p-Drp1S616 and Mfn2 were used recombinant plasmids. The expression levels of mitochondrial dynamics proteins (p-Drp1, Drp1, Mfn2, Mfn1 and Opa1) and autophagy markers (LC3, Beclin1 and p62) were measured with the Western blotting. The mRNA levels after transfection were determined by PCR. Cell injury and viability were evaluated with released LDH activity and CCK8 assay kits. Mitochondria morphology and autophagosome were observed under transmission electron microscopy. Mitochondrial function was detected by the mitochondrial permeability transition pore assay kit. The co-expression of p-ERK, p-Drp1 and LC3 was assessed with multiple immunofluorescences. One-way analysis of variance followed by least significance difference post hoc analysis (for equal homogeneity) or Dunnett's T3 test (for unequal homogeneity) were used for statistical tests.
ERK inhibitor-PD98059 (PD) protects SH-SY5Y cells from OGD/R-induced injury; while ERK activator-TPA had the opposite effect. Similar to autophagy inhibitor 3-MA, PD downregulated autophagy to improve cell viability; while autophagy activator-rapamycin further aggravated cell death. PD and Drp1-knockdown synergistically attenuated OGD/R-induced Drp1 activation, mPTP opening and cell injury; overexpression of Drp1 or ablating Mfn2 partly abolished the protective effects of PD. Multiple immunofluorescences showed that p-ERK, p-Drp1 and LC3 were co-expressed.
Inhibition of ERK downregulates autophagy via reducing Drp1/Mfn2-dependent mitochondrial fragmentation to antagonize mitochondrial dysfunction and promotes cell survival in the SH-SY5Y cells OGD/R model. Video Abstract.
脑缺血再灌注损伤（CIRI）是导致心搏骤停/心肺复苏（CA/CPR）患者高死亡率和神经功能障碍的主要原因。我们之前的研究发现细胞外信号调节激酶（ERK）的激活、动力相关蛋白 1（Drp1）/线粒体融合蛋白 2（Mfn2）依赖性线粒体动力学失衡以及过度自噬参与了 CA/CPR 后神经损伤的机制。然而，具体的病理信号通路仍不清楚。本研究旨在探讨 ERK-Drp1/Mfn2-自噬信号通路在 SH-SY5Y 细胞氧葡萄糖剥夺/复氧（OGD/R）模型中的分子功能变化，进一步阐明 CIRI 的病理生理机制，并为 CIRI 后脑保护提供新策略。
SH-SY5Y 细胞在 OGD/R 前 24 小时用药物预处理。采用小干扰 RNA 转染沉默 Drp1 和 Mfn2。使用重组质粒过表达 p-Drp1S616 和 Mfn2。用 Western blot 检测线粒体动力学蛋白（p-Drp1、Drp1、Mfn2、Mfn1 和 Opa1）和自噬标志物（LC3、Beclin1 和 p62）的表达水平。转染后的 mRNA 水平通过 PCR 测定。通过释放 LDH 活性和 CCK8 试剂盒评估细胞损伤和活力。用透射电子显微镜观察线粒体形态和自噬体。用线粒体通透性转换孔测定试剂盒检测线粒体功能。用多重免疫荧光法评估 p-ERK、p-Drp1 和 LC3 的共表达。采用单因素方差分析，随后进行最小显著差异事后检验（对于同质性）或 Dunnett's T3 检验（对于非同质性）进行统计检验。
ERK 抑制剂-PD98059（PD）可保护 SH-SY5Y 细胞免受 OGD/R 诱导的损伤；而 ERK 激活剂-TPA 则有相反的效果。类似于自噬抑制剂 3-MA，PD 通过下调自噬来提高细胞活力；而自噬激活剂-雷帕霉素则进一步加重细胞死亡。PD 和 Drp1 敲低协同减弱 OGD/R 诱导的 Drp1 激活、mPTP 开放和细胞损伤；过表达 Drp1 或破坏 Mfn2 部分消除了 PD 的保护作用。多重免疫荧光显示 p-ERK、p-Drp1 和 LC3 共表达。
ERK 的抑制通过减少 Drp1/Mfn2 依赖性线粒体片段化来下调自噬，从而拮抗线粒体功能障碍，促进 SH-SY5Y 细胞 OGD/R 模型中的细胞存活。
