Objective To investigate the protective effects and mechanism of insulin(INS) on brain in septic rats,and explore the possible role of uncoupling protein 2 (UCP2) in these effects.Methods Fifty male specific pathogen free(SPF) Sprague-Dawley rats were randomly divided into normal control (CN) group(n=10),lipopolysaccharide(LPS) group(n=20) and INS group (n=20) according to random number table.The septic rat model was established through an intraperitoneal injection of 15 mg/kg LPS of gram-negative bacteria.The rats in the INS group received a 1 U/kg INS injection subcutaneously 30 minutes before the injection of LPS,and the rats in the CN group were given equivalent 9 g/L saline in the same way.Eight rats in each group were killed,and their cerebral cortex were collected after the injection of LPS for 24 h.Pathological change of cerebral cortex was detected by Hematoxylin-Eosin(HE) staining.The cerebral cortex mitochondia were extracted for detecting the levels of reactive oxygen species(ROS),malondialdehyde (MDA) and the activity of superoxide dismutase(SOD).Neuronal apoptosis was detected by terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling staining.UCP2 mRNA expression was detected by quantitative real-time(RT)-PCR.Apoptosis-associated protein B lymphocyte tumor-2(Bcl-2),Bcl-2 associated X protein(Bax),cleaved cysteinyl aspartate specific protease(cleaved Caspase-9) and UCP2 protein expression were determined by Western blot.Results (1)Compared with the CN group,obvious abnormal pathological change was revealed by HE staining in cerebral cortex of rats in the LPS group and the INS group,but the pathological change was attenuated in the INS group compared with the LPS group.(2)Compared with the CN group,the levels of mitochondrial ROS[(210.01±14.09) RFU vs.(49.06±7.28) RFU] and MDA[(2.19±0.18) nmol/mg pro vs.(1.25±0.11)nmol/mg pro]in the LPS group significantly increased,whereas SOD activity significantly decreased [(238.49±35.60) U/g pro vs.(446.66±24.90)U/g pro],and the differences were significant(all P<0.05).Compared with the LPS group,the levels of ROS [(152.69±15.83) RFU vs.(210.01±14.09) RFU] and MDA[(1.55±0.14) nmol/mg pro vs.(2.19±0.18) nmol/mg pro] in the INS group decreased,while SOD activity increased[(327.8±23.26) U/g pro vs.(238.49± 35.60) U/g pro],and the differences were significant(all P<0.05).(3)Compared with the CN group,the neuronal apoptosis index of cortex in the LPS group was elevated[(54.16±6.84)% vs.(5.45±1.43)%],while the expression of Bcl-2 decreased (627±0.018 vs.0.739±0.020),but the expressions of Bax(0.768±0.019 vs.0.520±0.010) and cleaved Caspase-9(0.739±0.016 vs.0.467±0.030) increased,and the differences were significant(all P<0.05).Compared with the LPS group,the neuronal apoptosis index of cortex in the INS group decreased [(33.30±3.07)% vs.(54.16±6.84)%],but the Bcl-2 expression increased (0.743±0.022 vs.0.627±0.018),and Bax (0.687±0.034 vs.0.768±0.019) and cleaved Caspase-9(0.551±0.013 vs.0.739±0.016) were reduced,and the differences were significant (all P<0.05).(4)Compared with the CN group,the mRNA (2.248±0.155 vs.1.000±0.100) and protein expression of UCP2 (0.659±0.016 vs.0.599±0.018) were elevated in the LPS group.Compared with the LPS group,the UCP2 mRNA (2.944±0.117 vs.2.248±0.155) and UCP2 protein (0.719±0.018 vs.0.659±0.016) increased,and the differences were significant(all P<0.05).Conclusions INS can protect the brain of septic rats through alleviating mitochondrial oxidative stress and inhibiting the mitochondrial-initiated apoptotic pathway to reduce neuronal apoptosis.INS upregulates UCP2 expression in the brain of septic rats,which may play a role in the protective effects mentioned above.%目的 探讨胰岛素(INS)对脓毒症大鼠脑组织的保护作用及机制,分析解偶联蛋白2(UCP2)在其中的可能作用.方法 50只无特定病原体(SPF)级SD雄性大鼠,按随机数字表法分为正常对照组(CN组,10只)、脂多糖(LPS)组(20只)、INS组(20只).通过腹腔注射革兰阴性菌LPS 15 mg/kg建立脓毒症大鼠模型.INS组在造模前30 min皮下注射长效INS 1 U/kg,LPS组皮下注射等量9 g/L盐水.LPS造模后24 h,每组各取8只处死,留取大脑皮质.苏木精-伊红(HE)染色观察大脑皮质病理改变,提取皮质线粒体测定线粒体活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)水平,末端脱氧核苷酸转移酶介导的dUTP 缺口末端标记法检测皮质神经细胞凋亡,实时荧光定量聚合酶链反应(RT-PCR)检测皮质UCP2 mRNA表达,Western blot检测凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关蛋白(Bax)、活化的半胱氨酸天冬氨酸蛋白酶-9(cleaved Caspase-9)和UCP2蛋白的表达.结果 1.与CN组相比,LPS组和INS组大鼠皮质HE染色可见明显异常病理改变;与LPS组相比,INS组大鼠皮质病理改变减轻.2.与CN组相比,LPS组皮质线粒体ROS[(210.01±14.09) RFU比(49.06±7.28) RFU]和MDA[(2.19±0.18) nmol/mg pro比(1.25±0.11) nmol/mg pro]水平明显升高,SOD活力明显下降[(238.49±35.60) U/g pro比(446.66±24.90) U/g pro],差异均有统计学意义(均P<0.05);与LPS组相比,INS组皮质线粒体ROS[(152.69±15.83) RFU比(210.01±14.09) RFU]和MDA[(1.55±0.14) nmol/mg pro比(2.19±0.18) nmol/mg pro]水平下降,SOD活力回升[(327.8±23.26) U/g pro比(238.49±35.60) U/g pro],差异均有统计学意义(均P<0.05).3.与CN组相比,LPS组皮质神经细胞凋亡指数明显升高[(54.16±6.84)%比(5.45±1.43)%],Bcl-2表达下降(0.627±0.018比0.739±0.020),Bax(0.768±0.019比0.520±0.010)和cleaved Caspase-9(0.739±0.016比0.467±0.030)表达升高,差异均有统计学意义(均P<0.05);与LPS组相比,INS组皮质神经细胞凋亡指数明显下降[(33.30±3.07)%比(54.16±6.84)%],Bcl-2表达升高(0.743±0.022比0.627±0.018),Bax(0.687±0.034比0.768±0.019)和cleaved Caspase-9(0.551±0.013比0.739±0.016)表达下降,差异均有统计学意义(均P<0.05).4.与CN组相比,LPS组UCP2 mRNA(2.248±0.155比1.000±0.100)和蛋白(0.659±0.016比0.599±0.018)表达均升高,与LPS组相比,INS组大鼠UCP2 mRNA(2.944±0.117比2.248±0.155)和蛋白(0.719±0.018比0.659±0.016)表达升高,差异均有统计学意义(均P<0.05).结论 INS可减轻脓毒症大鼠脑组织线粒体氧化应激,抑制线粒体凋亡通路的激活并减少神经细胞凋亡,对脓毒症大鼠脑组织起保护作用.INS上调UCP2的表达而减少ROS生成可能是其发挥保护作用的机制之一.
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