黄连素在脊髓损伤中对线粒体的保护作用及机制
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摘要
目的探讨黄连素对脊髓损伤(SCI)后线粒体氧化损伤的作用和可能机制。方法将36只C57小鼠随机分为假手术组、SCI组(伤后立即腹腔注射10 mg/kg生理盐水)和黄连素组(SCI后立即腹腔注射10 mg/kg黄连素),每组12只。使用PSI-IH脊髓打击器建立小鼠SCI模型,于损伤后24 h处死小鼠,取脊髓组织。使用全自动酶标仪检测各组小鼠脊髓组织线粒体内丙二醛(MDA)、还原型谷胱甘肽(GSH)和超氧化物歧化酶(SOD)的变化;用蛋白质印迹法检测脊髓组织caspase-3、cleaved caspase-3的表达及细胞质内和线粒体内细胞色素C(Cyt C)的表达;用免疫荧光双标染色法检测脊髓组织中神经细胞凋亡情况。结果与假手术组相比,SCI组小鼠脊髓组织线粒体内MDA水平升高,GSH、SOD水平降低;细胞质内Cyt C和脊髓组织中caspase-3、cleaved caspase-3表达水平增高,线粒体内Cyt C表达水平降低;脊髓组织中神经元凋亡比例增高;差异均有统计学意义(P <0.05)。与SCI组相比,黄连素组小鼠脊髓组织线粒体内MDA水平降低,SOD和GSH水平增高;细胞质内Cyt C和脊髓组织中caspase-3、cleaved caspase-3表达水平降低,线粒体内Cyt C表达水平增高;脊髓组织中神经细胞凋亡比例减少;差异均有统计学意义(P <0.05)。结论黄连素可减轻SCI小鼠脊髓组织中神经细胞凋亡,这可能与其抑制线粒体氧化损伤、减少Cyt C释放、降低凋亡蛋白表达有关。
Objective To investigate the effects and possible mechanisms of berberine on the oxidative damage of mitochondria after spinal cord injury(SCI). Methods Thirty-six C57 mice were randomly divided into 3 groups(n=12):sham group,SCI group(immediately received 10 mg/kg saline injection intraperitoneally once post injury) and berberine group(immediately received 10 mg/kg berberine injection intraperitoneally once post injury). SCI model was established using the PSI-IH spinal cord impactor. The mice were sacrificed and the spinal cord tissue was taken out 24 h after injury. Malondialdehyde(MDA),reduced glutathione(GSH) and superoxide dismutase(SOD) in spinal cord mitochondria in each group were measured by automatic enzyme-linked immunosorbent assay. Cytochrome C(Cyt C) expression in cytoplasm and mitochondria and expressions of caspase-3 and cleaved caspase-3 in the spinal cord were analyzed by Western blotting. The neural apoptosis in the spinal cord was detected by immunofluorescent double labeling. Results Compared with the sham group,the level of MDA increased,while the levels of SOD and GSH decreased in the SCI group;the expression level of Cyt C in mitochondria decreased,while the expression of Cyt C in the cytoplasm,and the expressions of caspase-3 and cleaved caspase-3 in the spinal cord increased in the SCI group;the apoptosis rate of spinal neurons increased in the SCI group;the differences were all statistically significant(P < 0.05).Compared with the SCI group,the level of MDA decreased,and the level of SOD and GSH increased in the berberine group;the level of Cyt C in mitochondria increased,and the level of Cyt C in the cytoplasm,and the levels of caspase-3 and cleaved caspase-3 in the spinal cord decreased in the berberine group;the apoptosis rate of the spinal neurons decreased;the differences were all statistically significant(P < 0.05). Conclusion Berberine can inhibit the neuron apoptosis in SCI mice,which may be due to inhibition of mitochondrial oxidative damage,decrease of Cyt C releaseand activation of apoptotic molecules.
Objective To investigate the effects and possible mechanisms of berberine on the oxidative damage of mitochondria after spinal cord injury(SCI). Methods Thirty-six C57 mice were randomly divided into 3 groups(n=12):sham group,SCI group(immediately received 10 mg/kg saline injection intraperitoneally once post injury) and berberine group(immediately received 10 mg/kg berberine injection intraperitoneally once post injury). SCI model was established using the PSI-IH spinal cord impactor. The mice were sacrificed and the spinal cord tissue was taken out 24 h after injury. Malondialdehyde(MDA),reduced glutathione(GSH) and superoxide dismutase(SOD) in spinal cord mitochondria in each group were measured by automatic enzyme-linked immunosorbent assay. Cytochrome C(Cyt C) expression in cytoplasm and mitochondria and expressions of caspase-3 and cleaved caspase-3 in the spinal cord were analyzed by Western blotting. The neural apoptosis in the spinal cord was detected by immunofluorescent double labeling. Results Compared with the sham group,the level of MDA increased,while the levels of SOD and GSH decreased in the SCI group;the expression level of Cyt C in mitochondria decreased,while the expression of Cyt C in the cytoplasm,and the expressions of caspase-3 and cleaved caspase-3 in the spinal cord increased in the SCI group;the apoptosis rate of spinal neurons increased in the SCI group;the differences were all statistically significant(P < 0.05).Compared with the SCI group,the level of MDA decreased,and the level of SOD and GSH increased in the berberine group;the level of Cyt C in mitochondria increased,and the level of Cyt C in the cytoplasm,and the levels of caspase-3 and cleaved caspase-3 in the spinal cord decreased in the berberine group;the apoptosis rate of the spinal neurons decreased;the differences were all statistically significant(P < 0.05). Conclusion Berberine can inhibit the neuron apoptosis in SCI mice,which may be due to inhibition of mitochondrial oxidative damage,decrease of Cyt C releaseand activation of apoptotic molecules.
引文
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[12]Gomes AP,Duarte FV,Nunes P,et al.Berberine protects against high fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent mitochondrial biogenesis[J].Biochim Biophys Acta,2012,1822(2):185-195.
[13]Khuyagbaatar B,Kim K,Kim YH.Conversion equation between the drop height in the New York University impactor and the impact force in the Infinite Horizon impactor in the contusion spinal cord injury model[J].J Neurotrauma,2015,32(24):1987-1993.
[14]Chen CC,Hung TH,Lee CY,et al.Berberine protects against neuronal damage via suppression of glia-mediated inflammation in traumatic brain injury[J].PLoS One,2014,9(12):e115694.
[15]Hall ED,Wang JA,Bosken JM,et al.Lipid peroxidation in brain or spinal cord mitochondria after injury[J].J Bioenerg Biomembr,2016,48(2):169-174.
[16]Li H,Jia Z,Li G,et al.Neuroprotective effects of exendin-4 in rat model of spinal cord injury via inhibiting mitochondrial apoptotic pathway[J].Int J Clin Exp Pathol,2015,8(5):4837-4843.
[17]Yu W,Sheng M,Xu R,et al.Berberine protects human renal proximal tubular cells from hypoxia/reoxygenation injury via inhibiting endoplasmic reticulum and mitochondrial stress pathways[J].J Transl Med,2013,11:24.
[18]Lu J,Cao Y,Cheng K,et al.Berberine regulates neurite outgrowth through AMPK-dependent pathways by lowering energy status[J].Exp Cell Res,2015,334(2):194-206.
[2]李永华,王冬莲,梁一民,等.氢饱和生理盐水对兔脊髓缺血再灌注损伤的神经保护作用[J].脊柱外科杂志,2014,12(3):184-188.
[3]Spinal cord injury(SCI)2016 facts and figures at a glance[J].J Spinal Cord Med,2016,39(4):493-494.
[4]Cardozo MJ,Mysiak KS,Becker T,et al.Reduce,reuse,recycle-developmental signals in spinal cord regeneration[J].Dev Biol,2017,432(1):53-62.
[5]Sun X,Jones ZB,Chen XM,et al.Multiple organ dysfunction and systemic inflammation after spinal cord injury:a complex relationship[J].J Neuroinflammation,2016,13(1):260.
[6]Yan XJ,Xuan Y,Wang XP,et al.Mitochondria play an important role in the cell proliferation suppressing activity of berberine[J].Sci Rep,2017,7:41712.
[7]Imenshahidi M,Hosseinzadeh H.Berberis vulgaris and berberine:an update review[J].Phytother Res,2016,30(11):1745-1764.
[8]Hsu YY,Chen CS,Wu SN,et al.Berberine activates Nrf2 nuclear translocation and protects against oxidative damage via a phosphatidylinositol 3-kinase/Aktdependent mechanism in NSC34 motor neuron-like cells[J].Eur J Pharm Sci,2012,46(5):415-425.
[9]Jiang W,Li S,Li X.Therapeutic potential of berberine against neurodegenerative diseases[J].Sci China Life Sci,2015,58(6):564-569.
[10]Chen W,Wei S,Yu Y,et al.Pretreatment of rats with increased bioavailable berberine attenuates cerebral ischemia-reperfusion injury via down regulation of adenosine-5’monophosphate kinase activity[J].Eur JPharmacol,2016,779:80-90.
[11]Zhang W,Su X,Gao Y,et al.Berberine protects mesenchymal stem cells against hypoxia-induced apoptosis in vitro[J].Biol Pharm Bull,2009,32(8):1335-1342.
[12]Gomes AP,Duarte FV,Nunes P,et al.Berberine protects against high fat diet-induced dysfunction in muscle mitochondria by inducing SIRT1-dependent mitochondrial biogenesis[J].Biochim Biophys Acta,2012,1822(2):185-195.
[13]Khuyagbaatar B,Kim K,Kim YH.Conversion equation between the drop height in the New York University impactor and the impact force in the Infinite Horizon impactor in the contusion spinal cord injury model[J].J Neurotrauma,2015,32(24):1987-1993.
[14]Chen CC,Hung TH,Lee CY,et al.Berberine protects against neuronal damage via suppression of glia-mediated inflammation in traumatic brain injury[J].PLoS One,2014,9(12):e115694.
[15]Hall ED,Wang JA,Bosken JM,et al.Lipid peroxidation in brain or spinal cord mitochondria after injury[J].J Bioenerg Biomembr,2016,48(2):169-174.
[16]Li H,Jia Z,Li G,et al.Neuroprotective effects of exendin-4 in rat model of spinal cord injury via inhibiting mitochondrial apoptotic pathway[J].Int J Clin Exp Pathol,2015,8(5):4837-4843.
[17]Yu W,Sheng M,Xu R,et al.Berberine protects human renal proximal tubular cells from hypoxia/reoxygenation injury via inhibiting endoplasmic reticulum and mitochondrial stress pathways[J].J Transl Med,2013,11:24.
[18]Lu J,Cao Y,Cheng K,et al.Berberine regulates neurite outgrowth through AMPK-dependent pathways by lowering energy status[J].Exp Cell Res,2015,334(2):194-206.