Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China; Department of Neurosurgery, First Affiliated Hospital of Wannan Medical College, 2 West Zheshan Road, Wuhu, Anhui Province, China; Non-coding RNA Research Center of Wannan Medical College, Wuhu, Anhui Province, China.
Department of Neurosurgery & Brain and Nerve Research Laboratory, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu Province, China; Department of Neurosurgery, The people's Hospital of Bozhou, Bozhou, Anhui Province, China.
Exp Neurol. 2020 Oct;332:113386. doi: 10.1016/j.expneurol.2020.113386. Epub 2020 Jun 23.
Intracerebral hemorrhage (ICH) is the common brain diseases in middle-aged and elderly people, with high disability and/or mortality rate, and is a serious public health concern. Both WNK3 kinase and the WNK3/SPAK/NKCC1 signaling pathway play an integral role in maintaining normal cell homeostasis. However, their role and underlying mechanisms in ICH-induced secondary brain injury (SBI) have yet to be elucidated.
We established an ICH model using male Sprague-Dawley (SD) rats by injecting autologous arterial blood into the unilateral basal ganglia. To establish ICH model in vitro, oxyhemoglobin (OxyHb; 20 μM) and neurons were cultured for 6 h at 37 °C, 5% CO atmosphere. To investigate the role of WNK3 and the WNK3/SPAK/NKCC1 signaling pathway in SBI, after genetic interventions, rotation and water maze test, brain edema and neuroinflammation were detected, and terminal-deoxynucleoitidyl transferase mediated dUTP nick end labeling (TUNEL), Fluoro-Jade C (FJC), and Nissl staining were performed.
Our data showed that WNK3 expression in brain tissue were upregulated after ICH induction. In addition, silencing of WNK3 reduced neuronal apoptosis, and inflammatory responses in rats that underwent ICH. Inhibition of WNK3 expression reduced the damaged blood-brain barrier (BBB), alleviated the impaired degree of cerebral edema, and improved disruptive neurobehavioral cognition caused by ICH. Moreover, overexpression of WNK3 had the opposite effects. Finally, WNK3/SPAK/NKCC1 signaling pathway may be involved in the above-mentioned processes.
In conclusion, our findings showed that WNK3 and WNK3/SPAK/NKCC1 signaling pathway play a vital biological function in ICH-induced SBI. Depletion of WNK3 attenuated brain injury after ICH both in vivo and in vitro. Thus, WNK3 and WNK3/SPAK/NKCC1 signaling pathway are potential targets for treating SBI after ICH.
脑出血（ICH）是中老年人常见的脑部疾病，具有高残疾率和/或死亡率，是严重的公共卫生问题。WNK3 激酶和 WNK3/SPAK/NKCC1 信号通路在维持正常细胞内稳态方面都起着重要作用。然而，它们在 ICH 诱导的继发性脑损伤（SBI）中的作用和潜在机制尚不清楚。
我们通过向单侧基底节注射自体动脉血，建立了雄性 Sprague-Dawley（SD）大鼠的 ICH 模型。在体外，将氧合血红蛋白（OxyHb；20 μM）和神经元在 37°C、5% CO 气氛下孵育 6 小时，建立 ICH 模型。为了研究 WNK3 和 WNK3/SPAK/NKCC1 信号通路在 SBI 中的作用，通过基因干预后进行旋转和水迷宫测试，检测脑水肿和神经炎症，并进行末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）、氟-杰氏 C（FJC）和尼氏染色。
我们的数据显示，ICH 诱导后脑组织中 WNK3 的表达上调。此外，沉默 WNK3 可减少 ICH 大鼠的神经元凋亡和炎症反应。WNK3 表达抑制可减少受损的血脑屏障（BBB），减轻脑水肿的损害程度，并改善 ICH 引起的神经行为认知障碍。相反，WNK3 的过表达则产生相反的效果。最后，WNK3/SPAK/NKCC1 信号通路可能参与了上述过程。
总之，我们的研究结果表明，WNK3 和 WNK3/SPAK/NKCC1 信号通路在 ICH 诱导的 SBI 中发挥着重要的生物学功能。在体内和体外，WNK3 的耗竭均可减轻 ICH 后的脑损伤。因此，WNK3 和 WNK3/SPAK/NKCC1 信号通路可能是治疗 ICH 后 SBI 的潜在靶点。
