线粒体大麻素受体1在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响

目的:探讨线粒体CB1受体(mitochondrial cannabinoid receptor1,mtCB1)在大鼠海马神经元缺氧复氧损伤中对线粒体分裂的影响。方法:原代培养新生的Wistar大鼠海马神经元,将培养至第8天的海马神经元采用随机数字表分为5组(n=60):正常组(N组):正常培养,不做任何处理;缺氧复氧组(H/R组):采用氧糖剥夺法构建海马神经元缺氧复氧损伤模型,缺氧6h,复氧20 h;缺氧复氧组+ACEA+AM251组(H/R+ACEA+AM251组):缺氧6 h结束后立即加入ACEA和AM251,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+ACEA+Hemopressin(H/R+ACEA+Hemo组):缺氧6h结束后立即加入ACEA和Hemopressin,终浓度分别为1μmol/L、10μmol/L,复氧20 h;缺氧复氧+赋形剂组(H/R+V组):同样于缺氧6h结束后立即加入二甲基亚砜(DMSO),终浓度0.1%,复氧20 h。使用激光共聚焦显微镜检测细胞内Ca~(2+)的浓度,流式细胞仪检测细胞凋亡率,Western blot检测凋亡诱导因子(AIF)、线粒体分裂相关蛋白Drp1、Fis1,细胞凋亡相关蛋白细胞色素C(Cytc)和Rho相关的卷曲蛋白激酶1(ROCK1)的表达。结果:与N组相比,H/R组、H/R+ACEA+AM251组、H/R+ACEA+Hemo组和H/R+V组的细胞内Ca~(2+)浓度、细胞凋亡率、以及AIF、Drp1、Fis1、Cytc、ROCK1蛋白的表达水平均明显增加(P0.05);与H/R组相比,H/R+ACEA+Hem组上述各检测指标明显降低(P0.05),H/R+ACEA+AM251组和H/R+V组各指标比较差异无统计学意义(P0.05)。结论:线粒体CB1受体(mtCB1受体)可能通过降低细胞内ROS的含量来减少细胞内Ca~(2+)浓度和ROCK1的表达,进而抑制线粒体分裂,并最终减轻海马神经元缺氧复氧损伤。

Effect of Mitochondrial CB1 Receptor on the Mitochondrial Fission in a Rat Hippocampal Neuron Model of Hypoxia/Re-oxygenation Injury

ABSTRACT Objective: To evaluate the effect of mitochondrial CB1 receptor (mtCB1) on mitochondrial fission in a rat hippocampal neuron model of hypoxia/re-oxygenation (H/R) injury. Methods: Primarily cultured hippocampal neurons obtained from Wistar rats were divided into 5 groups(n=60) using a random number table: normal group (N group):were cultured in normal medium, without any administration; H/R group: the hippocampal neurons were subjected to oxygen-glucose deprivation (OGD) for 6h followed by re-oxygenation for 20h; H/R+ACEA+AM251 group: ACEA and AM251 were add with respectively final concentration 1 umol/L, 10 umol/L during the 20h re-oxygenation; H/R+ACEA+ Hemopressin group: ACEA and Hemopressin were add into culture medium with respectively final concentration 1 umol/L, 10 umol/L during the re-oxygenation for 20h; H/R + Vehicle group (H/R+V group): DMSO was add into culture medium with final concentration <0.1 % during the 20h re-oxygenation. Laser scanning confocal microscope was used to measure Ca²⁺ concentration in cytoplasm. The apoptosis rate was tested by flow cytometry. Western blot was adopted to examine the expression of apoptosis-inducing factor (AIF), dynamin-related protein 1(Drp1), fission 1(Fis1), apoptosis-related protein cytochrome c (Cytc), and Rho-associated coiled-coil containing protein kinase (ROCK1). Results: Compared to the N group, the apoptosis rate, Ca²⁺ concentration, the expression of AIF, Drp1, Fis1, Cytc and ROCK1were significantly increased in the other four groups (P<0.05); Compared to the H/R group, those detection indexes mentioned above were significantly decreased in H/R+ ACEA+ Hemopressin group (P<0.05), and there were no significant differences were observed in H/R+ACEA+AM251 group and H/R+ Vehicle group (P>0.05); Conclusion: The reduction of ROS induced by mtCB1 can alleviate the expression of ROCK1 and Ca²⁺ concentration in cytoplasm, and inhibit the mitochondrial fission, and eventually attenuate the H/R injury of hippocampal neurons.