miR-34a 通过 Notch1 信号通路对脑缺血再灌注大鼠脑神经元凋亡的影响。
Influence of miR-34a on cerebral neuronal apoptosis in rats with cerebral ischemia reperfusion through the Notch1 signaling pathway.
机构信息
Department of Neurological Rehabilitation, the Third People's Hospital of Qingdao, Qingdao, China.
出版信息
Eur Rev Med Pharmacol Sci. 2019 Sep;23(18):8049-8057. doi: 10.26355/eurrev_201909_19021.
OBJECTIVE
To explore the influence and the underlying mechanism of micro-ribonucleic acid (miR)-34a on cerebral neuronal apoptosis in rats with cerebral ischemia-reperfusion (CIR).
MATERIALS AND METHODS
60 adult male Wistar rats were randomly divided into 3 groups: Sham group, CIR group and miR-34a knockdown group. The rat model of CIR was established using the suture occlusion method. The expression level of miR-34a in lesion tissues in the three groups was determined via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The pathological injury of brain tissues was detected via hematoxylin-eosin (HE) staining and the infarction region in each group was evaluated via triphenyl tetrazolium chloride (TTC) staining. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed to measure the cerebral neuronal apoptosis level. The level of Nissl's body in each group was detected via Nissl's staining. The expression level of the platelet-derived neurotrophic factor (PDNF) and Notch1/hypoxia-inducible factor-1α (HIF-1α) signaling pathway-related proteins in brain tissues were detected via immunohistochemistry and Western blotting, respectively.
RESULTS
The expression level of miR-34a in brain tissues of the CIR group was significantly increased compared to that of the Sham group (p < 0.05). After the intervention with miR-34a, the infarction area of brain tissues was markedly reduced when comparing to the CIR group (p < 0.05). In addition, the results of HE staining and Nissl staining revealed that CIR treatment led to edema in cerebral neurons, disorderly arranged neurons, and remarkably decreased number of Nissl's bodies. However, miR-34a knockdown following CIR significantly alleviated the brain tissue injury and markedly increased the number of Nissl's bodies (p < 0.05). The results of TUNEL staining also indicated that miR-34a siRNA could remarkably reverse the cerebral neuronal apoptosis caused by CIR in rats (p < 0.05). According to the immunohistochemical staining results, the expression of PDNF in brain tissues declined in the CIR group, while enhanced in the miR-34a siRNA group (p < 0.05). Furthermore, the Western blotting results manifested that miR-34a siRNA could up-regulate the Notch1 and HIF-1α protein expressions in brain tissues of CIR rats.
CONCLUSIONS
Our data demonstrated that the miR-34a knockdown could alleviate the brain tissue injury and neuronal apoptosis by activating the Notch1/HIF-1α signaling pathway CIR-treated rats.
目的
探讨微小 RNA-34a（miR-34a）对脑缺血再灌注（CIR）大鼠脑神经元凋亡的影响及其潜在机制。
材料与方法
将 60 只成年雄性 Wistar 大鼠随机分为 3 组：假手术组、CIR 组和 miR-34a 敲低组。采用缝线阻塞法建立 CIR 大鼠模型。采用逆转录-聚合酶链反应（RT-PCR）检测 3 组大鼠病变组织中 miR-34a 的表达水平。苏木精-伊红（HE）染色检测脑组织病理损伤，氯化三苯基四氮唑（TTC）染色评估各组梗死区。末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）染色法检测脑神经元凋亡水平。尼氏染色检测各组 Nissl 体。免疫组化法和 Western blot 法分别检测脑组织中血小板源性神经营养因子（PDNF）和 Notch1/缺氧诱导因子-1α（HIF-1α）信号通路相关蛋白的表达水平。
结果
与假手术组相比，CIR 组大鼠脑组织中 miR-34a 的表达水平明显升高（p<0.05）。miR-34a 干预后，与 CIR 组相比，脑梗死面积明显减小（p<0.05）。此外，HE 染色和尼氏染色结果显示，CIR 处理导致脑神经元水肿、神经元排列紊乱、Nissl 体数量明显减少，而 CIR 后 miR-34a 敲低明显减轻了脑组织损伤，显著增加了 Nissl 体数量（p<0.05）。TUNEL 染色结果也表明，miR-34a siRNA 可显著逆转 CIR 诱导的大鼠脑神经元凋亡（p<0.05）。免疫组化染色结果显示，CIR 组大鼠脑组织中 PDNF 表达降低，miR-34a siRNA 组表达增强（p<0.05）。此外，Western blot 结果表明，miR-34a siRNA 可上调 CIR 大鼠脑组织中 Notch1 和 HIF-1α 蛋白的表达。
结论
本研究数据表明，miR-34a 敲低通过激活 Notch1/HIF-1α 信号通路减轻 CIR 大鼠的脑组织损伤和神经元凋亡。
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