Protective effects of KLF4 on blood-brain barrier and oxidative stress after cerebral ischemia-reperfusion in rats through the Nrf2/Trx1 pathway.

ABSTRACT

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.