丙泊酚诱导miR-378抑制大鼠肝脏缺血再灌注细胞凋亡的分子机制研究  

Molecular mechanism of propofol reducing hepatic ischemia reperfusion cell apoptosis via miR-378

作　　者：钮峥嵘[1] 杨飞[1] 苏涛[1] 姬翔 李学斌[1] 徐桂萍[1] NIU Zhengrong;YANG Fei;SU Tao;JI Xiang;LI Xuebin;XU Guiping(Department of Anesthesiology,Xinjiang Uygur Autonomous Region People＇s Hospital,Xinfiang,Urumqi 830000,Chin)

机构地区：[1]新疆维吾尔自治区人民医院麻醉科,乌鲁木齐830000

出　　处：《疑难病杂志》2018年第6期614-620,共7页Chinese Journal of Difficult and Complicated Cases

基　　金：国家自然科学基金资助项目(81160016)

摘　　要：目的探讨丙泊酚诱导miR-378抑制细胞凋亡从而保护大鼠肝脏缺血再灌注损伤的作用机制。方法于2017年3-6月在新疆医科大学动物实验中心完成实验。将健康成年SD大鼠45只按随机数字表法分为3组各15只:假手术组(sham组)、肝脏缺血再灌注损伤模型组(IR组)和丙泊酚处理的IR组(IR+propofol组)。采用缺血60 min后再灌注120 min实施肝脏的缺血再灌注损伤。通过自动生化分析仪检测血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)和乳酸脱氢酶(LDH)的水平以评价缺血再灌注的损伤程度。TUNEL方法检测3组大鼠的肝组织的细胞凋亡情况,Western blot和QPCR检测miR-378及凋亡相关因子的表达水平,采用人工合成miR-378 mimics,定点突变技术及双荧光素酶报告系统鉴定miR-378下游与凋亡相关的靶基因。结果 IR组的ALT、AST和LDH水平较sham组明显升高(t=17.236、16.527、14.349,P<0.01),IR+propofol组ALT、AST和LDH水平与IR组比较明显下降(t=15.626、14.995、11.376,P<0.01);与sham组相比,IR组的miR-378、Bcl-2和Bcl-xl表达明显降低(t=6.921、11.381、11.467,P<0.01),Cytochrome c、Bax、Bak、Caspase-9和Caspase-3的表达显著提高(t=14.486、12.653、11.434、15.712、15.934,P<0.01);丙泊酚处理的IR+propofol组逆转了此表达模式,使其与sham组趋于一致。TUNEL检测结果发现IR组的凋亡率远高于sham组(t=22.546,P=0.000),丙泊酚处理后,调亡率降低(t=9.765,P=0.000)。在人类正常肝细胞中,miR-378 mimics导致Bcl-2和Bcl-xl表达明显增加(t=15.589、15.715,P<0.01),而Cytochrome c、Bax、Bak、Caspase-9和Caspase-3表达显著下降(t=12.574、15.607、14.894、18.828、11.367,P<0.01),定点突变与双荧光素酶报告系统鉴定Bak及Caspase-3是miR-378的下游靶基因。结论丙泊酚诱导miR-378表达,调节凋亡相关基因的表达,阻遏细胞凋亡信号的产生,从而发挥保护肝脏缺血再灌注损伤的作用,因此miR-378可作为肝脏缺血再灌注Objective To investigate the mechanism of propofol induced miR-378 reduce apoptosis and then prevent liver from ischemia reperfusion injury. Methods Forty five healthy adult SD rats were randomly divided into 3 groups： sham group, IR group and IR group treated with propofol and then surgically treated. The ischemia reperfusion injury of the liver was performed 60 minutes for ischemia and 120 minutes for reperfusion. Serum levels of alanine aminotransferase（ ALT）, aspartate aminotransferase（AST） and lactate dehydrogenase（ LDH） were measured by automated biochemical analyzer to assess the degree of ischemia reperfusion injury. TUNEL assay was used to detect the apoptosis of liver tissue in three groups of rats.Western blot and QPCR were used to detect the expression of miR-378 and apoptosis related genes. Synthetic miR-378 mimics, site directed mutagenesis and dual luciferase reporer system were used to identify the target genes associated with apoptosis downstream of miR 378. Results The levels of ALT, AST and LDH in IR group were significantly increased compared with sham group（t=17.236,t=16.527, t= 14. 349,P〈0. 01）, whereas levels of ALT, AST and LDH in IR ＋ propofol group were significantly lower than those in IR group（t= 15.626, t =14.995, t =11.376,P〈0.01）. Compared with the sham group, the expressions of miR 378,Bcl-2 and Bcl-xl decreased in IR group（t =6.921, t = 11.381, t = 11.467, P〈0.01）.whereas the expression of Cytochrome c, Bax, Bak, caspase 9 and caspase 3 increased（ t = 14.486, t = 12. 653, t = 11.434,t = 15.712, t =15.934,P=0.000, P= 0.01）. Propofol treated IR reversed this expression pattern of IR group, made it more consistent with the sham group. TUNEL assay showed that the apoptotic rate in IR group was higher than sham group（ t =22.546,P = 0.000）, and the apoptosis rate in propofol group decreased significantly（t =9.765, P=0.000）. In human normal hepatocytes QSG 7701 transfected with miR-378 mimics, the expression of Bcl-2 and Bcl-xl was significan

关 键 词：肝脏 缺血再灌注损伤 细胞凋亡 miR-378 丙泊酚 分子机制 大鼠 

分 类 号：R614[医药卫生—麻醉学]