目的 探究钙激活氯通道参与七氟醚对大鼠脑缺血后神经血管单元的保护机制研究。方法 将TMEM16A局部脑内基因敲除大鼠分为假手术组、模型组、沉默TMEM-16A(LV-shTMEM16A)组、七氟醚组、七氟醚+LV-shTMEM16A组。除假手术组外,其余各组大鼠建立中动脉脑缺血模型,七氟醚组与七氟醚+LV-shTMEM16A组大鼠中动脉缺血期间,持续吸入3%七氟醚治疗。大鼠脑缺血2h后,拔除线栓,检测神经元中Ca²⁺与Cl^-含量。3 d后评估神经功能。将大鼠处死,取大鼠脑组织分别开展TTC染色分析各组大鼠脑梗死区面积,TUNEL染色观察大鼠脑组织中TUNEL染色阳性细胞率,CD31免疫组化法检测新生血管生成标记物CD31,Western blotting检测凋亡和神经营养相关蛋白表达。结果 与模型组相比,七氟醚组大鼠神经元中Ca²⁺与Cl^-浓度降低,神经功能评分降低且脑梗死面积明显减少。七氟醚组大鼠脑组织中CD31水平与VEGF蛋白表达进一步升高,TUNEL染色阳性细胞率减少,凋亡相关蛋白Bcl-2相关X蛋白(Bax)、活化的半胱氨酸蛋白酶-3(cleaved-Caspase-3)表达量降低,神经营养相关蛋白转化生长因子-β(TGF-β)、脑源性神经营养因子(BDNF)与酪氨酸激酶受体B(TrkB)蛋白表达量升高。与七氟醚组相比,七氟醚+LV-shTMEM16A组大鼠神经元中虽然Cl^-没有变化,但Ca²⁺浓度增加,神经功能评分升高且脑梗死面积增加,大鼠脑组织中CD31水平与VEGF蛋白表达减少,TUNEL染色阳性细胞率增加,Bax、cleaved-Caspase-3蛋白表达量增加,而TGF-β、BDNF与TrkB蛋白表达量减少。结论 七氟醚可能通过抑制TMEM16A蛋白表达,降低Ca²⁺与Cl^-电流,一方面抑制了神经元凋亡,另一方面促进了神经血管单元的新生,最终达到减轻神经功能损伤的作用。

Objective To explore the protective mechanism of calcium-activated chloride channels involved in sevoflurane on neurovascular units after cerebral ischemia in rats. Methods TMEM16A local brain knockout rats were divided into sham operation group, model group, LV-shTMEM16A group, sevoflurane group, sevoflurane + LV-shTMEM16A group. Except for the sham operation group, the middle artery cerebral ischemia model was established in other groups. During the middle artery ischemia, sevoflurane group and sevoflurane +LV-shTMEM16A group were treated with continuous inhalation of 3% sevoflurane. The content of Ca²⁺ and Cl^- in neurons was detected after 2 h of cerebral ischemia. Neurological function was assessed after 3 days. The rats were killed, and the brain tissues of the rats were analyzed by TTC staining. TUNEL staining was used to observe the TUNEL staining positive cell rate in the brain tissues of the rats, and CD31 immunohistochemical method was used to detect the neovascularization marker CD31. The expressions of apoptosis-related and neurotrophic proteins were detected by Western blotting. Results Compared with the model group, the concentrations of Ca²⁺ and Cl^- in the neurons of sevoflurane group were decreased, the neural function scores were decreased, and the cerebral infarction size was significantly reduced. In sevoflurane group, the expressions of CD31 and VEGF protein were further increased, the rate of TUNEL stained positive cells was decreased, the expressions of apoptosis-related proteins Bax and cleaved Caspase-3 were decreased, and the expressions of neurotrophic proteins TGF-β, BDNF and TrkB were increased. Compared with sevoflurane group, although there was no change in Cl^- in sevoflurane + LV-shTMEM16A group, there was an increase in Ca²⁺ concentration, an increase in neural function score, an increase in cerebral infarction area, a decrease in the expression of CD31 and VEGF protein, and an increase in the rate of TUNEL staining positive cells. Expression of apoptosis-associated proteins Bax and cleaved Caspase-3 increased, while expression of neurotrophic proteins TGF-β, BDNF, and TrkB decreased. Conclusion Sevoflurane may inhibit the expression of TMEM16A protein and reduce the Cl^- and Ca²⁺ concentration. It inhibits neuronal apoptosis and promotes the regeneration of neurovascular units, which finally achieves the effect of reducing neurological damage.

R965

新疆维吾尔自治区自然科学基金项目(2021D01C429)