线粒体Omi/HtrA2信号通路在颅脑损伤后神经细胞凋亡中的作用
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摘要
目的探讨大鼠颅脑损伤后线粒体Omi/HtrA2信号通路在大鼠神经细胞凋亡中的作用。方法 90只大鼠按随机数字表法分成假手术组、颅脑损伤组和UCF-101干预组,每组30只,然后每组按照12、24、48 h三个时间点分成三个亚组,每个亚组10只。使用大鼠自由落体颅脑损伤动物模型,UCF-101干预组大鼠在模型形成前0.5 h按1.5μmol/kg的剂量腹腔注射UCF-101,其余两组大鼠腹腔注射1 mL的等渗Na Cl溶液。按照12、24、48 h三个时间点每组分批断头处死10只大鼠,分离海马组织。采用末端脱氧核苷酸转移酶介导的d UTP缺口末端标记(TUNEL)测定法检测各组大鼠海马组织神经细胞凋亡情况,Western-blotting检测Omi/HtrA2、X染色体连锁凋亡抑制蛋白(XIAP)、pro-caspase-3、pro-caspase-9和剪切聚腺苷二磷酸-核糖多聚酶(PARP)蛋白表达,四肽荧光底物法检测caspase-3和caspase-9蛋白活性。结果三组大鼠海马神经细胞凋亡比例,Omi/HtrA2、pro-caspase-3、pro-caspase-9、剪切PARP和XIAP蛋白表达以及caspase-3和caspase-9蛋白活性比较,差异均有统计学意义(F=217.742、107.139、145.748、133.970、103.029、65.112、142.772、96.187,P均<0.05)。12、24、48 h时颅脑损伤组大鼠凋亡的海马神经细胞比例,Omi/HtrA2、pro-caspase-3、pro-caspase-9和剪切PARP蛋白表达以及caspase-3和caspase-9蛋白活性均较假手术组显著升高(P均<0.05);而UCF-101干预组大鼠凋亡的海马神经细胞比例,Omi/HtrA2、pro-caspase-3、pro-caspase-9和剪切PARP蛋白表达以及caspase-3和caspase-9蛋白活性均较颅脑损伤组显著下降(P均<0.05)。各时间点颅脑损伤组大鼠海马组织XIAP蛋白表达均较假手术组显著下降(P均<0.05),而UCF-101干预组大鼠海马组织XIAP蛋白表达均较颅脑损伤组显著升高(P均<0.05)。结论 Omi/HtrA2介导的线粒体途径可能参与大鼠颅脑损伤后神经细胞凋亡的发生过程,而UCF-101可有效抑制神经细胞凋亡。
Objective To investigate the role of the mitochondrial Omi/HtrA2 signaling pathway in neuronal apoptosis after craniocerebral injury in rats. Methods A total of 90 rats were randomly divided into the sham operation group, craniocerebral injury group and UCF-101 intervention group, 30 rats in each group; at 12, 24 and 48 h, each group was further divided into three subgroups at random, 10 rats in each subgroup. The animal model of free-fall brain injury in rats was used. Rats in the UCF-101 intervention group were intraperitoneally injected with UCF-101 at a dose of 1.5 μmol/kg 0.5 h before the formation of the model, and rats in the other two groups were intraperitoneally injected with 1 mL sodium chloride solution. At 12 h, 24 h and 48 h, 10 rats in each group were sacrificed to isolate the hippocampal tissues. Terminal deoxynucleotidyl transferase-mediated d UTP Nick end labeling(TUNEL) assay was used to detect neuronal apoptosis in hippocampal tissues of rats in each group, the Western-blotting method was used to detect the expressions of Omi/HtrA2, X-linked inhibitor of apoptosis protein(XIAP), procaspase-3, pro-caspase-9 and shearing poly-ADP-ribose polymerase(PARP), and the tetrapeptide fluorescence substrate method was used to detect the activity of caspase-3 and caspase-9.Results The ratio of apoptotic hippocampal neurons, the expressions of Omi/HtrA2, pro-caspase-3, pro-caspase-9, shearing PARP and XIAP, and the activity of caspase-3 and caspase-9 were all statistically significantly different in the three groups(F = 217.742, 107.139, 145.748, 133.970,103.029, 65.112, 142.772, 96.187; all P < 0.05). At 12 h, 24 h and 48 h, the ratio of apoptotic hippocampal neurons, the expressions of Omi/HtrA2, pro-caspase-3, pro-caspase-9 and shearing PARP, and the activity of caspase-3 and caspase-9 were significantly higher in the craniocerebral injury group than in the sham operation group(all P < 0.05), while they were significantly lower in the UCF-101 intervention group than in the craniocerebral injury group(all P < 0.05). The expressions of XIAP in hippocampi in the craniocerebral injury group at different time points were significantly lower as compared to the sham operation group(all P < 0.05), while the expressions of XIAP in the UCF-101 intervention group were significantly higher as compared to the craniocerebral injury group(all P < 0.05). Conclusion The mitochondrial pathway mediated by Omi/HtrA2 may be involved in the process of neuronal apoptosis after craniocerebral injury in rats, and UCF-101 can effectively inhibit the apoptosis of nerve cells.
Objective To investigate the role of the mitochondrial Omi/HtrA2 signaling pathway in neuronal apoptosis after craniocerebral injury in rats. Methods A total of 90 rats were randomly divided into the sham operation group, craniocerebral injury group and UCF-101 intervention group, 30 rats in each group; at 12, 24 and 48 h, each group was further divided into three subgroups at random, 10 rats in each subgroup. The animal model of free-fall brain injury in rats was used. Rats in the UCF-101 intervention group were intraperitoneally injected with UCF-101 at a dose of 1.5 μmol/kg 0.5 h before the formation of the model, and rats in the other two groups were intraperitoneally injected with 1 mL sodium chloride solution. At 12 h, 24 h and 48 h, 10 rats in each group were sacrificed to isolate the hippocampal tissues. Terminal deoxynucleotidyl transferase-mediated d UTP Nick end labeling(TUNEL) assay was used to detect neuronal apoptosis in hippocampal tissues of rats in each group, the Western-blotting method was used to detect the expressions of Omi/HtrA2, X-linked inhibitor of apoptosis protein(XIAP), procaspase-3, pro-caspase-9 and shearing poly-ADP-ribose polymerase(PARP), and the tetrapeptide fluorescence substrate method was used to detect the activity of caspase-3 and caspase-9.Results The ratio of apoptotic hippocampal neurons, the expressions of Omi/HtrA2, pro-caspase-3, pro-caspase-9, shearing PARP and XIAP, and the activity of caspase-3 and caspase-9 were all statistically significantly different in the three groups(F = 217.742, 107.139, 145.748, 133.970,103.029, 65.112, 142.772, 96.187; all P < 0.05). At 12 h, 24 h and 48 h, the ratio of apoptotic hippocampal neurons, the expressions of Omi/HtrA2, pro-caspase-3, pro-caspase-9 and shearing PARP, and the activity of caspase-3 and caspase-9 were significantly higher in the craniocerebral injury group than in the sham operation group(all P < 0.05), while they were significantly lower in the UCF-101 intervention group than in the craniocerebral injury group(all P < 0.05). The expressions of XIAP in hippocampi in the craniocerebral injury group at different time points were significantly lower as compared to the sham operation group(all P < 0.05), while the expressions of XIAP in the UCF-101 intervention group were significantly higher as compared to the craniocerebral injury group(all P < 0.05). Conclusion The mitochondrial pathway mediated by Omi/HtrA2 may be involved in the process of neuronal apoptosis after craniocerebral injury in rats, and UCF-101 can effectively inhibit the apoptosis of nerve cells.
引文
1 田正丰,俞文华,董晓巧,等.血浆和肽素浓度对急性创伤性进展性出血性脑损伤的预测价值[J/CD].中华危重症医学杂志(电子版),2016,9(3):169-173.
2 王林国,俞文华,董晓巧,等.血浆和肽素浓度对重型颅脑损伤后急性创伤性凝血病的预测价值[J/CD].中华危重症医学杂志(电子版),2015,8(5):300-305.
3 Huang TQ,Song JN,Zheng FW,et al.Protection of FK506 against neuronal apoptosis and axonal injury following experimental diffuse axonal injury[J].Mol Med Rep,2017,15(5):3001-3010.
4 Xue Z,Song Z,Wan Y,et al.Calcium-sensing receptor antagonist NPS2390 attenuates neuronal apoptosis though intrinsic pathway following traumatic brain injury in rats[J].Biochem Biophys Res Commun,2017,486(2):589-594.
5 Miao Q,Ge M,Huang L.Up-regulation of GBP2 is associated with neuronal apoptosis in rat brain cortex following traumatic brain injury[J].Neurochem Res,2017,42(5):1515-1523.
6 Kuhn H,Nieuwenhuijsen H,Karthe B,et al.Stretchinduced apoptosis in rat alveolar epithelial cells is mediated by the intrinsic mitochondrial pathway[J].Exp Lung Res,2017,43(1):49-56.
7 Hu J,Yan Q,Shi C,et al.BMSC paracrine activity attenuates interleukin-1β-induced inflammation and apoptosis in rat AF cells via inhibiting relative NF-κB signaling and the mitochondrial pathway[J].Am J Transl Res,2017,9(1):79-89.
8 Lin M,Tang S,Zhang C,et al.Euphorbia factor L2induces apoptosis in A549 cells through the mitochondrial pathway[J].Acta Pharm Sin B,2017,7(1):59-64.
9 Goo HG,Rhim H,Kang S.Htr A2/Omi influences the stability of LON protease 1 and prohibitin,proteins involved in mitochondrial homeostasis[J].Exp Cell Res,2014,328(2):456-465.
10 Goo HG,Jung MK,Han SS,et al.Htr A2/Omi deficiency causes damage and mutation of mitochondrial DNA[J].Biochim Biophys Acta,2013,1833(8):1866-1875.
11 Kang S,Fernandes-Alnemri T,Alnemri ES.A novel role for the mitochondrial HTRA2/OMI protease in aging[J].Autophagy,2013,9(3):420-421.
12 Dan HC,Sun M,Kaneko S,et al.Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein(XIAP)[J].J Biol Chem,2016,291(43):22846.
13 Han J,Liu Z,Wang N,et al.Micro RNA-874 inhibits growth,induces apoptosis and reverses chemoresistance in colorectal cancer by targeting X-linked inhibitor of apoptosis protein[J].Oncol Rep,2016,36(1):542-550.
14 Xu Y,Yang W,Shi J,et al.Prohibitin 1 regulates tumor cell apoptosis via the interaction with X-linked inhibitor of apoptosis protein[J].J Mol Cell Biol,2016,8(3):282-285.
15 Hu Y,Huang M,Wang P,et al.Ucf-101 protects against cerebral oxidative injury and cognitive impairment in septic rat[J].Int Immunopharmacol,2013,16(1):108-113.
16 Su D,Ma J,Zhang Z,et al.Protective effects of UCF-101 on cerebral ischemia-reperfusion(CIR)is depended on the MAPK/p38/ERK signaling pathway[J].Cell Mol Neurobiol,2016,36(6):907-914.
17 Su D,Su Z,Wang J,et al.UCF-101,a novel Omi/Htr A2 inhibitor,protects against cerebral ischemia/reperfusion injury in rats[J].Anat Rec(Hoboken),2009,292(6):854-861.
18 Feeney DM,Boyeson MG,Linn RT,et al.Responses to cortical injury:I.Methodology and local effects of contusions in the rat[J].Brain Res,1981,211(1):67-77.
19 Simon DW,Mc Geachy MJ,Bayir H,et al.The farreaching scope of neuroinflammation after traumatic brain injury[J].Nat Rev Neurol,2017,13(3):171-191.
20 Russo MV,Mc Gavern DB.Inflammatory neuroprotection following traumatic brain injury[J].Science,2016,353(6301):783-785.
21 Pearn ML,Niesman IR,Egawa J,et al.Pathophysiology associated with traumatic brain injury:current treatments and potential novel therapeutics[J].Cell Mol Neurobiol,2017,37(4):571-585.
22 Joseph B,Haider A,Rhee P.Traumatic brain injury advancements[J].Curr Opin Crit Care,2015,21(6):506-511.
23 Gao Y,Zhuang Z,Gao S,et al.Tetrahydrocurcumin reduces oxidative stress-induced apoptosis via the mitochondrial apoptotic pathway by modulating autophagy in rats after traumatic brain injury[J].Am J Transl Res,2017,9(3):887-899.
24 Choong CJ,Mochizuki H.Gene therapy targeting mitochondrial pathway in Parkinson's disease[J].J Neural Transm(Vienna),2017,124(2):193-207.
25 Gibson CJ,Davids MS.BCL-2 antagonism to target the intrinsic mitochondrial pathway of apoptosis[J].Clin Cancer Res,2015,21(22):5021-5029.
26 Fu K,Wang Y,Guo D,et al.Familial Parkinson's disease-associated L166P mutant DJ-1 is cleaved by mitochondrial serine protease Omi/Htr A2[J].Neurosci Bull,2017,33(6):685-694.
27 Westerlund M,Behbahani H,Gellhaar S,et al.Altered enzymatic activity and allele frequency of OMI/HTRA2 in Alzheimer's disease[J].FASEB J,2011,25(4):1345-1352.
2 王林国,俞文华,董晓巧,等.血浆和肽素浓度对重型颅脑损伤后急性创伤性凝血病的预测价值[J/CD].中华危重症医学杂志(电子版),2015,8(5):300-305.
3 Huang TQ,Song JN,Zheng FW,et al.Protection of FK506 against neuronal apoptosis and axonal injury following experimental diffuse axonal injury[J].Mol Med Rep,2017,15(5):3001-3010.
4 Xue Z,Song Z,Wan Y,et al.Calcium-sensing receptor antagonist NPS2390 attenuates neuronal apoptosis though intrinsic pathway following traumatic brain injury in rats[J].Biochem Biophys Res Commun,2017,486(2):589-594.
5 Miao Q,Ge M,Huang L.Up-regulation of GBP2 is associated with neuronal apoptosis in rat brain cortex following traumatic brain injury[J].Neurochem Res,2017,42(5):1515-1523.
6 Kuhn H,Nieuwenhuijsen H,Karthe B,et al.Stretchinduced apoptosis in rat alveolar epithelial cells is mediated by the intrinsic mitochondrial pathway[J].Exp Lung Res,2017,43(1):49-56.
7 Hu J,Yan Q,Shi C,et al.BMSC paracrine activity attenuates interleukin-1β-induced inflammation and apoptosis in rat AF cells via inhibiting relative NF-κB signaling and the mitochondrial pathway[J].Am J Transl Res,2017,9(1):79-89.
8 Lin M,Tang S,Zhang C,et al.Euphorbia factor L2induces apoptosis in A549 cells through the mitochondrial pathway[J].Acta Pharm Sin B,2017,7(1):59-64.
9 Goo HG,Rhim H,Kang S.Htr A2/Omi influences the stability of LON protease 1 and prohibitin,proteins involved in mitochondrial homeostasis[J].Exp Cell Res,2014,328(2):456-465.
10 Goo HG,Jung MK,Han SS,et al.Htr A2/Omi deficiency causes damage and mutation of mitochondrial DNA[J].Biochim Biophys Acta,2013,1833(8):1866-1875.
11 Kang S,Fernandes-Alnemri T,Alnemri ES.A novel role for the mitochondrial HTRA2/OMI protease in aging[J].Autophagy,2013,9(3):420-421.
12 Dan HC,Sun M,Kaneko S,et al.Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein(XIAP)[J].J Biol Chem,2016,291(43):22846.
13 Han J,Liu Z,Wang N,et al.Micro RNA-874 inhibits growth,induces apoptosis and reverses chemoresistance in colorectal cancer by targeting X-linked inhibitor of apoptosis protein[J].Oncol Rep,2016,36(1):542-550.
14 Xu Y,Yang W,Shi J,et al.Prohibitin 1 regulates tumor cell apoptosis via the interaction with X-linked inhibitor of apoptosis protein[J].J Mol Cell Biol,2016,8(3):282-285.
15 Hu Y,Huang M,Wang P,et al.Ucf-101 protects against cerebral oxidative injury and cognitive impairment in septic rat[J].Int Immunopharmacol,2013,16(1):108-113.
16 Su D,Ma J,Zhang Z,et al.Protective effects of UCF-101 on cerebral ischemia-reperfusion(CIR)is depended on the MAPK/p38/ERK signaling pathway[J].Cell Mol Neurobiol,2016,36(6):907-914.
17 Su D,Su Z,Wang J,et al.UCF-101,a novel Omi/Htr A2 inhibitor,protects against cerebral ischemia/reperfusion injury in rats[J].Anat Rec(Hoboken),2009,292(6):854-861.
18 Feeney DM,Boyeson MG,Linn RT,et al.Responses to cortical injury:I.Methodology and local effects of contusions in the rat[J].Brain Res,1981,211(1):67-77.
19 Simon DW,Mc Geachy MJ,Bayir H,et al.The farreaching scope of neuroinflammation after traumatic brain injury[J].Nat Rev Neurol,2017,13(3):171-191.
20 Russo MV,Mc Gavern DB.Inflammatory neuroprotection following traumatic brain injury[J].Science,2016,353(6301):783-785.
21 Pearn ML,Niesman IR,Egawa J,et al.Pathophysiology associated with traumatic brain injury:current treatments and potential novel therapeutics[J].Cell Mol Neurobiol,2017,37(4):571-585.
22 Joseph B,Haider A,Rhee P.Traumatic brain injury advancements[J].Curr Opin Crit Care,2015,21(6):506-511.
23 Gao Y,Zhuang Z,Gao S,et al.Tetrahydrocurcumin reduces oxidative stress-induced apoptosis via the mitochondrial apoptotic pathway by modulating autophagy in rats after traumatic brain injury[J].Am J Transl Res,2017,9(3):887-899.
24 Choong CJ,Mochizuki H.Gene therapy targeting mitochondrial pathway in Parkinson's disease[J].J Neural Transm(Vienna),2017,124(2):193-207.
25 Gibson CJ,Davids MS.BCL-2 antagonism to target the intrinsic mitochondrial pathway of apoptosis[J].Clin Cancer Res,2015,21(22):5021-5029.
26 Fu K,Wang Y,Guo D,et al.Familial Parkinson's disease-associated L166P mutant DJ-1 is cleaved by mitochondrial serine protease Omi/Htr A2[J].Neurosci Bull,2017,33(6):685-694.
27 Westerlund M,Behbahani H,Gellhaar S,et al.Altered enzymatic activity and allele frequency of OMI/HTRA2 in Alzheimer's disease[J].FASEB J,2011,25(4):1345-1352.