Department of Neurointerventional, Dongying People's Hospital, No. 317 Dongcheng South Road, Dongying, 257091, Shandong Province, China.
Neurochem Res. 2024 Nov 25;50(1):32. doi: 10.1007/s11064-024-04291-w.
Cerebral ischemia-reperfusion (I/R) injury is a complex pathophysiological process involving multiple mechanisms, including apoptosis and autophagy, which can lead to significant neuronal damage. PIEZO1, a stretch-activated ion channel, has recently emerged as a potential regulator of cellular responses to ischemic conditions. However, its role in neuronal cell survival and death during ischemic events is not well elucidated. This study aimed to ascertain the regulatory function of PIEZO1 in neuronal cell apoptosis and autophagy in an in vitro model of hypoxia-reoxygenation and an in vivo model of brain I/R injury. HT22 hippocampal neuronal cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate ischemic conditions, with subsequent reoxygenation. In vitro, PIEZO1 expression was silenced using small interfering RNA (si-RNA) transfection. The effects on cell viability, apoptosis, and autophagy were assessed using CCK-8 assays, PI-Annexin/V staining combined with flow cytometry, and Western blot analysis. Additionally, intracellular Ca levels in HT22 cells were measured using a Ca probe. The involvement of the AMPK-mTOR pathway was investigated using rapamycin. For in vivo validation, middle cerebral artery occlusion/reperfusion (MCAO/R) in rats was employed. To determine the neuroprotective role of PIEZO1 silencing, sh-PIEZO1 adeno-associated virus was stereotaxically injected into the cerebral ventricle, and neurological and histological outcomes were assessed using neurological scoring, TTC staining, H&E staining, Nissl staining, and immunofluorescence. In HT22 cells, OGD/R injury notably upregulated PIEZO1 expression and intracellular Ca levels. Silencing PIEZO1 significantly diminished OGD/R-induced Ca influx, apoptosis, and autophagy, as indicated by lower levels of pro-apoptotic and autophagy-related proteins and improved cell viability. Additionally, PIEZO1 modulated the AMPK-mTOR signaling pathway, an effect that was counteracted by rapamycin treatment, implying its regulatory role. In vivo, PIEZO1 silencing ameliorated brain I/R injury in MCAO/R rats, demonstrated by improved neurological function scores and reduced neuronal apoptosis and autophagy. However, these neuroprotective effects were reversed through rapamycin treatment. Our findings indicate that PIEZO1 is upregulated following ischemic injury and facilitates Ca influx, apoptosis, and autophagy via the AMPK-mTOR pathway. Silencing PIEZO1 confers neuroprotection against I/R injury both in vitro and in vivo, highlighting its potential as a therapeutic target for stroke management.
脑缺血再灌注（I/R）损伤是一个涉及多种机制的复杂病理生理过程，包括细胞凋亡和自噬，这可能导致显著的神经元损伤。PIEZO1 是一种张力激活的离子通道，最近被认为是细胞对缺血条件反应的潜在调节剂。然而，其在缺血事件中神经元细胞存活和死亡中的作用尚未阐明。本研究旨在确定 PIEZO1 在体外缺氧再氧合模型和体内脑 I/R 损伤模型中对神经元细胞凋亡和自噬的调节功能。HT22 海马神经元细胞进行氧葡萄糖剥夺/再氧合（OGD/R）模拟缺血条件，随后再氧合。在体外，使用小干扰 RNA（siRNA）转染沉默 PIEZO1 表达。通过 CCK-8 测定、PI-Annexin/V 染色结合流式细胞术和 Western blot 分析评估细胞活力、细胞凋亡和自噬的影响。此外，使用钙探针测量 HT22 细胞内的 Ca 水平。使用雷帕霉素研究 AMPK-mTOR 通路的参与情况。体内验证采用大鼠大脑中动脉闭塞/再灌注（MCAO/R）。为了确定 PIEZO1 沉默的神经保护作用，将 sh-PIEZO1 腺相关病毒立体定向注射到脑室中，并使用神经评分、TTC 染色、H&E 染色、尼氏染色和免疫荧光评估神经保护作用。在 HT22 细胞中，OGD/R 损伤显著上调 PIEZO1 表达和细胞内 Ca 水平。沉默 PIEZO1 显著降低 OGD/R 诱导的 Ca 内流、细胞凋亡和自噬，表现为促凋亡和自噬相关蛋白水平降低，细胞活力提高。此外，PIEZO1 调节 AMPK-mTOR 信号通路，雷帕霉素处理可拮抗该作用，表明其具有调节作用。在体内，沉默 PIEZO1 改善了 MCAO/R 大鼠的脑 I/R 损伤，表现为神经功能评分改善，神经元凋亡和自噬减少。然而，这些神经保护作用通过雷帕霉素处理而逆转。我们的研究结果表明，PIEZO1 在缺血损伤后上调，并通过 AMPK-mTOR 通路促进 Ca 内流、细胞凋亡和自噬。沉默 PIEZO1 在体外和体内均对 I/R 损伤具有神经保护作用，提示其作为中风管理治疗靶点的潜力。
