Department of Pathophysiology, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang, Hebei, 050017, People's Republic of China.
Hebei Key Laboratory of Critical Disease Mechanism and Intervention, Shijiazhuang, People's Republic of China.
Mol Neurobiol. 2024 Apr;61(4):2336-2356. doi: 10.1007/s12035-023-03703-2. Epub 2023 Oct 24.
Our previous study has proved that the Klotho up-regulation participated in cerebral ischemic preconditioning (CIP)-induced brain ischemic tolerance. However, the exact neuroprotective mechanism of Klotho in CIP remains unclear. We explored the hypothesis that STAT4-mediated Klotho up-regulation contributes to the CIP-induced brain ischemic tolerance via inhibiting neuronal pyroptosis. Firstly, the expressions of pyroptosis-associated proteins (i.e., NLRP3, GSDMD, pro-caspase-1, and cleaved caspase-1) in hippocampal CA1 region were determined during the process of brain ischemic tolerance. We found the expression of pyroptosis-associated proteins was significantly up-regulated in the ischemic insult (II) group, and showed no significant changes in the CIP group. The expression level of each pyroptosis-associated proteins was lower in the CIP + II group than that in the II group. Inhibition of Klotho expression increased the expression of pyroptosis-associated proteins in the CIP + II group and blocked the CIP-induced brain ischemic tolerance. Injection of Klotho protein decreased the expression of pyroptosis-associated proteins in the II group, and protected neurons from ischemic injury. Secondly, the transcription factor STAT4 of Klotho was identified by bioinformatic analysis. Double luciferase reporter gene assay and chromatin immunoprecipitation assay showed STAT4 can bind to the site between nt - 881 and - 868 on the Klotho promoter region and positively regulates Klotho expression. Moreover, we found CIP significantly enhanced the expression of STAT4. Knockdown STAT4 suppressed Klotho up-regulation after CIP and blocked the CIP-induced brain ischemic tolerance. Collectively, it can be concluded that STAT4-mediated the up-regulation of Klotho contributed to the brain ischemic tolerance induced by CIP via inhibiting pyroptosis.
我们之前的研究已经证明，Klotho 的上调参与了脑缺血预处理（CIP）诱导的脑缺血耐受。然而，Klotho 在 CIP 中的确切神经保护机制尚不清楚。我们提出了一个假设，即 STAT4 介导的 Klotho 上调通过抑制神经元细胞焦亡来促进 CIP 诱导的脑缺血耐受。首先，在脑缺血耐受过程中，确定海马 CA1 区中与细胞焦亡相关的蛋白（即 NLRP3、GSDMD、pro-caspase-1 和 cleaved caspase-1）的表达情况。我们发现，与细胞焦亡相关的蛋白表达在缺血性损伤（II）组中显著上调，而在 CIP 组中无明显变化。与细胞焦亡相关的蛋白表达水平在 CIP+II 组中低于 II 组。抑制 Klotho 表达会增加 CIP+II 组中与细胞焦亡相关的蛋白表达，并阻断 CIP 诱导的脑缺血耐受。Klotho 蛋白的注射降低了 II 组中与细胞焦亡相关的蛋白表达，从而保护神经元免受缺血性损伤。其次，通过生物信息学分析鉴定了 Klotho 的转录因子 STAT4。双荧光素酶报告基因检测和染色质免疫沉淀检测表明，STAT4 可以与 Klotho 启动子区域 nt-881 到-868 之间的位点结合，并正向调节 Klotho 的表达。此外，我们发现 CIP 显著增强了 STAT4 的表达。沉默 STAT4 抑制了 CIP 后 Klotho 的上调，并阻断了 CIP 诱导的脑缺血耐受。综上所述，可以得出结论，STAT4 介导的 Klotho 上调通过抑制细胞焦亡来促进 CIP 诱导的脑缺血耐受。
