七氟烷对大鼠离体海马神经元程序性坏死的影响:与兰尼碱受体的关系  

Effect of sevoflurane on necroptosis in isolated hippocampal neurons in rats:relationship with ryanodine receptor

作　　者：张琦 李亚南[2] 尹春平[2] 张亚辉[3] 朱炼[4] 侯志勇[4] 王秋筠[2] Zhang Qi;Li Yanan;Yin Chunping;Zhang Yahui;Zhu Lian;Hou Zhiyong;Wang Qiujun(Department of Anesthesiology,Children′s Hospital of Hebei Province,Shijiazhuang 050031,China;Department of Anesthesiology,Third Hospital of Hebei Medical University,Shijiazhuang 050051,China;Department of Nursing,Third Hospital of Hebei Medical University,Shijiazhuang 050051,China;Center of Emergency and Trauma,Third Hospital of Hebei Medical University,Shijiazhuang 050051,China)

机构地区：[1]河北省儿童医院麻醉科,石家庄050031 [2]河北医科大学第三医院麻醉科,石家庄050051 [3]河北医科大学第三医院护理部,石家庄050051 [4]河北医科大学第三医院创伤与急救中心,石家庄050051

出　　处：《中华麻醉学杂志》2021年第1期52-55,共4页Chinese Journal of Anesthesiology

基　　金：国家自然科学基金面上项目(81771134);河北省自然科学基金面上项目(H2018206305)。

摘　　要：目的评价七氟烷对大鼠离体海马神经元程序性坏死的影响及其与兰尼碱受体的关系。方法原代培养SD大鼠胎鼠的海马神经元,培养7 d时,以5×105个/ml细胞密度接种于培养孔(100μl/孔)或培养瓶(3 ml/瓶)中,采用随机数字表法分为3组(n=24):对照组(C组)、七氟烷组(S组)和兰尼碱受体拮抗剂组(R组)。C组常规培养,R组加入兰尼碱受体拮抗剂丹曲林,终浓度为3μmol/L,30 min后将S组和R组细胞放置于含2%七氟烷的培养箱中,37℃培养5 h。收集细胞,倒置显微镜下观察神经元形态,采用流式细胞术检测细胞胞浆游离钙离子浓度([Ca^(2+)]i),采用Western blot法检测兰尼碱受体和磷酸化人混合系列蛋白激酶样结构域(p-MLKL)的表达,采用免疫荧光法检测受体相互作用蛋白激酶3的表达,计算程序性坏死率。结果与C组比较,S组和R组神经元[Ca^(2+)]i升高,兰尼碱受体和p-MLKL表达上调,程序性坏死率升高(P<0.05);与S组比较,R组神经元[Ca^(2+)]i降低,兰尼碱受体和p-MLKL表达下调,程序性坏死率降低(P<0.05)。C组神经元形态未见明显异常,S组神经元胞体皱缩,突起断裂及突起间网络稀疏;R组神经元胞体圆润,形态接近正常。结论七氟烷可导致大鼠离体海马神经元程序性坏死,其机制与其上调兰尼碱受体表达,导致钙超载有关。Objective To evaluate the effect of sevoflurane on necroptosis in isolated hippocampal neurons and the relationship with ryanodine receptor.Methods Primarily cultured hippocampal neurons of fetal rats of Sprague-Dawley rats were inoculated in culture wells(100μl/well)or culture flasks(3 ml/bottle)at a density of 5×105 cells/ml at 7 days of culture and divided into 3 groups(n=24 each)using a random number table method:control group(group C),sevoflurane group(group S)and ryanodine receptor antagonist group(group R).Group C received routine culture.Ryanodine receptor antagonist Dantrolene at a final concentration of 3μmol/L was added in group R.Thirty minutes later,the cells were placed in the incubator containing 2%sevoflurane and cultured for 5 h at 37℃in S and R groups.Then cells were collected,the morphology of neurons was observed with an inverted microscope,the concentrations of free calcium ion([Ca^(2+)]i)in cytoplasm were determined by flow cytometry,the expression of ryanodine receptor and phosphorylated MLKL protein(p-MLKL)was detected by Western blot,the expression of RIP3 was measured by immunofluorescence,and necroptosis rate was calculated.Results Compared with group C,the[Ca^(2+)]i were significantly increased,the expression of ryanodine receptor and p-MLKL was up-regulated,and the necroptosis rate was increased in S and R groups(P<0.05).Compared with group S,the expression of ryanodine receptor and p-MLKL was significantly down-regulated,and the[Ca^(2+)]i and necroptosis rate were decreased in group R(P<0.05).There was no obvious abnormality in the morphology of neurons in group C.The cell body of neurons were shrunk,the processes were broken,and the network between processes was sparse in group S.The cell body was round,and the morphology was close to normal in group R.Conclusion Sevoflurane can cause necroptosis in isolated hippocampal neurons of rats,and the mechanism is related to up-regulating the expression of ryanodine receptors and leading to calcium overload.

关 键 词：麻醉药 吸入 海马 神经元 坏死 兰尼碱受体钙释放通道 

分 类 号：R965[医药卫生—药理学]