目的 探究鈣激活氯通道參與七氟醚對大鼠腦缺血后神經(jīng)血管單元的保護機制研究。方法 將TMEM16A局部腦內(nèi)基因敲除大鼠分為假手術組、模型組、沉默TMEM-16A(LV-shTMEM16A)組、七氟醚組、七氟醚+LV-shTMEM16A組。除假手術組外,其余各組大鼠建立中動脈腦缺血模型,七氟醚組與七氟醚+LV-shTMEM16A組大鼠中動脈缺血期間,持續(xù)吸入3%七氟醚治療。大鼠腦缺血2h后,拔除線栓,檢測神經(jīng)元中Ca²⁺與Cl^-含量。3 d后評估神經(jīng)功能。將大鼠處死,取大鼠腦組織分別開展TTC染色分析各組大鼠腦梗死區(qū)面積,TUNEL染色觀察大鼠腦組織中TUNEL染色陽性細胞率,CD31免疫組化法檢測新生血管生成標記物CD31,Western blotting檢測凋亡和神經(jīng)營養(yǎng)相關蛋白表達。結果 與模型組相比,七氟醚組大鼠神經(jīng)元中Ca²⁺與Cl^-濃度降低,神經(jīng)功能評分降低且腦梗死面積明顯減少。七氟醚組大鼠腦組織中CD31水平與VEGF蛋白表達進一步升高,TUNEL染色陽性細胞率減少,凋亡相關蛋白Bcl-2相關X蛋白(Bax)、活化的半胱氨酸蛋白酶-3(cleaved-Caspase-3)表達量降低,神經(jīng)營養(yǎng)相關蛋白轉化生長因子-β(TGF-β)、腦源性神經(jīng)營養(yǎng)因子(BDNF)與酪氨酸激酶受體B(TrkB)蛋白表達量升高。與七氟醚組相比,七氟醚+LV-shTMEM16A組大鼠神經(jīng)元中雖然Cl^-沒有變化,但Ca²⁺濃度增加,神經(jīng)功能評分升高且腦梗死面積增加,大鼠腦組織中CD31水平與VEGF蛋白表達減少,TUNEL染色陽性細胞率增加,Bax、cleaved-Caspase-3蛋白表達量增加,而TGF-β、BDNF與TrkB蛋白表達量減少。結論 七氟醚可能通過抑制TMEM16A蛋白表達,降低Ca²⁺與Cl^-電流,一方面抑制了神經(jīng)元凋亡,另一方面促進了神經(jīng)血管單元的新生,最終達到減輕神經(jīng)功能損傷的作用。

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

新疆維吾爾自治區(qū)自然科學基金項目(2021D01C429)