KLF4 通过 Nrf2/Trx1 通路对脑缺血再灌注后大鼠血脑屏障及氧化应激的保护作用。
Protective effects of KLF4 on blood-brain barrier and oxidative stress after cerebral ischemia-reperfusion in rats through the Nrf2/Trx1 pathway.
机构信息
Neurology Department, Laizhou City People's Hospital, Laizhou, Shandong 261400, China.
Neurology Department, Laizhou City People's Hospital, Laizhou, Shandong 261400, China.
出版信息
Cytokine. 2023 Sep;169:156288. doi: 10.1016/j.cyto.2023.156288. Epub 2023 Jul 11.
PURPOSE
To investigate the role of KLF4 in CI/R injury and whether Nrf2/Trx1 axis acted as a downstream pathway of KLF4 to exert the protective role in blood-brain barrier destruction after CI/R.
METHODS
The tMCAO rat model in vivo was constructed and received the intracerebroventricular injection of 5 μg/kg and 10 μg/kg rhKLF4 before operation. TTC, brain water content, neurological function, ELISA, behavioral tests, HE, TUNEL, and qRT-PCR were performed to detect the protective role of KLF4 on CIR. Double-fluorescence staining and western blot were performed to determine the localization and spatiotemporal expression in brain tissues. Furthermore, we also analyzed the effect of KLF4 on the blood-brain barrier (BBB) and related mechanisms in vivo and in vitro. Nrf2 inhibitor tretinoin was applied, which was intraperitoneally injected into CIR rat. Evans blue staining was conducted. In vitro OGD/R models of bEnd.3 cells were also established, and received KLF4 overexpressed transfection and 12.5 µM tretinoin incubation. The permeability of bEnd.3 cells was evaluated by TEER and FITC-dextran leakage. BBB-related factors and oxidative stress were also analyzed, respectively. The tubular ability of KLF4 on OGD/R bEnd3 cells was also evaluated.
RESULTS
In vivo study confirmed that KLF4 was expressed in astrocyte, and its content increased with time. KLF4 protected against brain injury caused by cerebral ischemia-reperfusion, reduced cerebral infarction area and oxidative stress levels, and promoted the recovery of behavioral ability in rats. Simultaneously, mechanism experiments confirmed that the repair effect of KLF4 on cerebral ischemia-reperfusion injury was closely related to the Nrf2/Trx1 pathway. KLF4 exerted the neuroprotective effect through upregulating Nrf2/Trx1 pathway. Consistent with in vivo animal study, in vitro study also confirmed the effect of KLF4 on the permeability of bEnd.3 cells after OGD/R injury through Nrf2/Trx1 pathway.
CONCLUSION
Collectively, KLF4 played neuroprotective role in CIR induced MCAO and OGD/R, and the beneficial effects of KLF4 was partly linked to Nrf2/Trx1 pathway.
目的
探讨 KLF4 在脑缺血再灌注（CI/R）损伤中的作用，以及 Nrf2/Trx1 轴是否作为 KLF4 的下游途径在 CI/R 后血脑屏障破坏中发挥保护作用。
方法
构建体内大脑中动脉阻塞（tMCAO）大鼠模型，在手术前分别接受 5μg/kg 和 10μg/kg rhKLF4 的侧脑室注射。TTC、脑水含量、神经功能、ELISA、行为测试、HE、TUNEL 和 qRT-PCR 用于检测 KLF4 对 CIR 的保护作用。双荧光染色和 Western blot 用于确定脑内组织中的定位和时空表达。此外，我们还分析了 KLF4 在体内和体外对血脑屏障（BBB）的影响及其相关机制。用 Nrf2 抑制剂维 A 酸（tretinoin）对 CIR 大鼠进行腹腔注射，进行伊文思蓝染色。还建立了 bEnd.3 细胞的体外 OGD/R 模型，并进行了 KLF4 过表达转染和 12.5µM 维 A 酸孵育。通过 TEER 和 FITC-葡聚糖渗漏评估 bEnd.3 细胞的通透性。分别分析 BBB 相关因子和氧化应激。还评估了 KLF4 对 OGD/R bEnd3 细胞的管状形成能力。
结果
体内研究证实 KLF4 在星形胶质细胞中表达，其含量随时间增加。KLF4 可防止脑缺血再灌注引起的脑损伤，降低脑梗死面积和氧化应激水平，促进大鼠行为能力的恢复。同时，机制实验证实 KLF4 对脑缺血再灌注损伤的修复作用与 Nrf2/Trx1 通路密切相关。KLF4 通过上调 Nrf2/Trx1 通路发挥神经保护作用。与体内动物研究一致，体外研究还通过 Nrf2/Trx1 通路证实了 KLF4 对 OGD/R 损伤后 bEnd.3 细胞通透性的影响。
结论
综上所述，KLF4 在 MCAO 诱导的 CIR 和 OGD/R 中发挥神经保护作用，KLF4 的有益作用部分与 Nrf2/Trx1 通路有关。
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