阿魏酸钠对大鼠脑缺血再灌注炎症损伤的保护作用及机制分析
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摘要
目的:观察阿魏酸钠对大鼠脑缺血再灌注炎症损伤的保护作用,并探讨其作用机制。方法:取SD雄性大鼠,建立大脑中动脉阻塞(MCAO)致脑缺血/再灌注损伤模型,造模成功的36只随机分成模型组,阿魏酸钠低、中、高剂量组(25,50,100 mg·kg~(-1)),并另取9只作为假手术组。采用神经功能评分法观察大鼠神经功能情况;苏木精-伊红(HE)染色观察大鼠脑组织病理学形态变化;酶联免疫吸附测定(ELISA)检测大鼠血清和脑组织中肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL~(-1)β),白细胞介素-6(IL-6)含量变化;蛋白免疫印迹法(Western blot)检测缺血周边区核转录因子-кB p65 (NF-κB p65)蛋白表达。结果:与假手术组比较,模型组大鼠神经行为学评分升高,神经元坏死,大量炎症细胞产生,血清和脑组织中TNF-a,IL~(-1)β,IL-6水平显著升高(P <0. 01),细胞核/细胞浆NF-κB p65蛋白表达比例显著升高(P <0. 01);与模型组比较,阿魏酸钠可明显降低脑损伤大鼠神经行为学评分(P <0. 05,P <0. 01),抑制神经元坏死,减少炎症细胞产生,并可明显降低血清和脑组织中TNF-a,IL~(-1)β,IL-6水平(P <0. 05,P <0. 01),抑制NF-κB p65由细胞质向细胞核转位(P <0. 05,P <0. 01),最终减轻缺血再灌注造成的神经细胞炎症损伤。结论:阿魏酸钠可通过抑制NF-κB p65核转位减轻缺血再灌注大鼠脑神经细胞炎症反应,维持脑部正常形态,这可能是其发挥脑保护作用的机制之一。
Objective: To investigate the protective effect of sodium ferulate on cerebral ischemiareperfusion injury in rats and to explore its possible mechanism. Method: The cerebral ischemia reperfusion injury model was established by middle cerebral artery occlusion( MCAO) in SD male rats. 36 modeled rats with neurologic damage were randomly divided into 4 groups: model group,low,medium,high-dose sodium ferulate groups( 25,50,100 mg·kg~(-1)). Another nine rats were selected as a sham operation group. Neurological function was assessed by neurological scoring system in rats. Hematoxylin-eosin( HE) staining was performed to observe the pathological changes of the rats' brain. The levels of tumor necrosis factor α( TNF-α),interleukin-1β( IL~(-1)β) and interleukin-6( IL-6) in serum and brain tissues were detected by enzyme-linked immunosorbent assay( ELISA). Western blot was used to detect the protein expression of nucleus and cytoplasm nuclear factor-kappa B p65( NF-κB p65) in brain tissues. Result: As compared with normal group,neurological deficit score was increased; the neuronal necrosis and inflammatory cell number were present; the serum and brain tissue levels of TNF-α,IL~(-1)β and IL-6 were increased; nucleus/cytoplasm NF-κB p65 protein expression ratio was increased significantly in model group( P < 0. 01). As compared with model group,sodium ferulate distinctly decreased the neurological deficit score( P < 0. 05,P < 0. 01),inhibited neuronal necrosis,reduce inflammatory cell number,significantly reduce the serum and brain tissue levels of TNF-α,IL~(-1)β and IL-6( P < 0. 05,P < 0. 01). Western blot showed that sodium ferulate could inhibit the nuclear translocation of NF-κB p65 protein( P < 0. 05,P <0. 01),and finally,it can alleviate the inflammatory injury caused by ischemia reperfusion. Conclusion: Sodium ferulate protects the brain against focal cerebral ischemia reperfusion injury,and the mechanism may be related to inhibiting nuclear translocation of NF-κB p65 protein to alleviate inflammatory response.
Objective: To investigate the protective effect of sodium ferulate on cerebral ischemiareperfusion injury in rats and to explore its possible mechanism. Method: The cerebral ischemia reperfusion injury model was established by middle cerebral artery occlusion( MCAO) in SD male rats. 36 modeled rats with neurologic damage were randomly divided into 4 groups: model group,low,medium,high-dose sodium ferulate groups( 25,50,100 mg·kg~(-1)). Another nine rats were selected as a sham operation group. Neurological function was assessed by neurological scoring system in rats. Hematoxylin-eosin( HE) staining was performed to observe the pathological changes of the rats' brain. The levels of tumor necrosis factor α( TNF-α),interleukin-1β( IL~(-1)β) and interleukin-6( IL-6) in serum and brain tissues were detected by enzyme-linked immunosorbent assay( ELISA). Western blot was used to detect the protein expression of nucleus and cytoplasm nuclear factor-kappa B p65( NF-κB p65) in brain tissues. Result: As compared with normal group,neurological deficit score was increased; the neuronal necrosis and inflammatory cell number were present; the serum and brain tissue levels of TNF-α,IL~(-1)β and IL-6 were increased; nucleus/cytoplasm NF-κB p65 protein expression ratio was increased significantly in model group( P < 0. 01). As compared with model group,sodium ferulate distinctly decreased the neurological deficit score( P < 0. 05,P < 0. 01),inhibited neuronal necrosis,reduce inflammatory cell number,significantly reduce the serum and brain tissue levels of TNF-α,IL~(-1)β and IL-6( P < 0. 05,P < 0. 01). Western blot showed that sodium ferulate could inhibit the nuclear translocation of NF-κB p65 protein( P < 0. 05,P <0. 01),and finally,it can alleviate the inflammatory injury caused by ischemia reperfusion. Conclusion: Sodium ferulate protects the brain against focal cerebral ischemia reperfusion injury,and the mechanism may be related to inhibiting nuclear translocation of NF-κB p65 protein to alleviate inflammatory response.
引文
[1]陈丹丹,彭成,万峰,等.氢溴酸樟柳碱对抗大鼠急性脑缺血/再灌注损伤的作用机制研究[J].中国药理学通报,2017,33(8):1096-1102.
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[3] CHEN Y K,ZHANG L,NI J S,et al. Lldt-8 protects against cerebral ischemia/reperfusion injury by suppressing post-stroke inflammation[J]. J Pharmacol Sci,2016,131(2):131-137.
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[6] Ou S, Kwok K C. Ferulic acid:pharmaceutical functions,preparation and applications in foods[J]. J Sci Food Agric,2004,84(11):1261-1269.
[7]管咏梅,陶玲,朱小芳,等.乳香没药挥发油对川芎中阿魏酸促透机制的研究[J].中国中药杂志,2017,42(17):3350-3355.
[8] Mancuso C,Santangelo R. Ferulic acid:pharmacological and toxicological aspects[J]. Food Chem Toxicol,2014,65(2):185-195.
[9] REN Z K,ZHANG R P,LI Y Y,et al. Ferulic acid exerts neuroprotective effects against cerebral ischemia/reperfusion-induced injury via antioxidant and antiapoptotic mechanisms in vitro and in vivo[J]. Int J Mol Med,2017,40(5):1444-1456.
[10] Koh P O. Ferulic acid attenuates the down-regulation of mek/erk/p90rsk signaling pathway in focal cerebral ischemic injury[J]. Neurosci Lett,2015,588:18-23.
[11] ZHANG Q,ZHAO Y H,XU Y H,et al. Sodium ferulate and n-butylidenephthalate combined with bone marrow stromal cells(BMSCs)improve the therapeutic effects of angiogenesis and neurogenesis after rat focal cerebral ischemia[J]. J Transl Med,2016,14(1):223-238.
[12] Sasaki M, Honmou O, Kocsis J D. A rat middle cerebral artery occlusion model and intravenous cellular delivery[J]. Methods Mol Biol,2009,549:187-195.
[13] Longa E Z,Weinstein P R,Carlson S,et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke,1989,20(1):84-91.
[14]李子广,吴华璞,郭梅,等.阿魏酸钠对MCAO大鼠缺血/再灌注损伤p38MAPK信号通路的影响[J].中西医结合心脑血管病杂志,2009,7(2):193-195.
[15] TANG Q Q,HAN R D,XIAO H,et al. Protective effect of tanshinone IIA on the brain and its therapeutic time window in rat models of cerebral ischemia-reperfusion[J]. Exp Ther Med,2014,8(5):1616-1622.
[16]刘抒雯,杨丽华,马春,等.中医药保护脑缺血再灌注损伤后神经血管单元的作用[J].中国实验方剂学杂志,2018,24(21):225-234.
[17] Danton G H,Dietrich W D. Inflammatory mechanisms after ischemia and stroke[J]. J Neuropathol Exp Neurol,2003,62(2):127-136.
[18] JIN R,YANG G,LI G. Inflammatory mechanisms in ischemic stroke:role of inflammatory cells[J]. J Leukoc Biol,2010,87(5):779-789.
[19]张予阳,刘岩,付守廷.脑缺血与炎症反应[J].中国药理学通报,2006,22(1):5-9.
[20] Berti R,Williams A J,Moffett J R,et al. Quantitative real-time RT-PCR analysis of inflammatory gene expression associated with ischemia-reperfusion brain injury[J]. J Cereb Blood Flow Metab,2002,22(9):1068-1079.
[21]刘亭,刘香香,陈亭亭,等.灯盏细辛和赤芍配伍组方对大鼠脑缺血再灌注损伤的保护作用及对NF-κB通路的影响[J].中国实验方剂学杂志,2018,24(9):111-115.
[22] Stephenson D, YIN T, Smalstig E B, et al.Transcription factor nuclear factor-kappa B is activated in neurons after focal cerebral ischemia[J]. J Cereb Blood Flow Metab,2000,20(3):592-603.
[23] TU X K,YANG W Z,CHEN J P,et al. Curcumin inhibits TLR2/4-NF-κB signaling pathway and attenuates brain damage in permanent focal cerebral ischemia in rats[J]. Inflammation,2014,37(5):1544-1551.
[24]吴建良,沈敏敏,杨水新,等.阿魏酸对小胶质细胞炎性反应的抑制作用[J].中国药理学通报,2015,31(1):97-102.
[25]孟锐,陈逸青,陈勤.阿魏酸对痴呆小鼠脑内胶质细胞活化与炎性细胞因子表达的影响[J].中国医院药学杂志,2018,38(1):50-53.
[26] LIU Y M,SHEN J D,XU L P,et al. Ferulic acid inhibits neuro-inflammation in mice exposed to chronic unpredictable mild stress[J]. Int Immunopharmacol,2017,45:128-134.
[2]丁素菊,吴雄枫.脑卒中诊断治疗指南的缺陷——点评《中国急性缺血性脑卒中诊治指南2014》[J].医学与哲学(B),2016,37(6):13-16.
[3] CHEN Y K,ZHANG L,NI J S,et al. Lldt-8 protects against cerebral ischemia/reperfusion injury by suppressing post-stroke inflammation[J]. J Pharmacol Sci,2016,131(2):131-137.
[4]于凌志,左艳丽,贾孟辉,等.核转录因子NF-κB对脑缺血再灌注干预机制的研究进展[J].中国民族医药杂志,2015,21(7):42-44.
[5]秦文熠,荣晓凤,陶涛,等.核转录因子-κB拮抗剂对大鼠海马CA1区局灶性脑缺血再灌注损伤的影响[J].中华神经科杂志,2016,49(1):45-53.
[6] Ou S, Kwok K C. Ferulic acid:pharmaceutical functions,preparation and applications in foods[J]. J Sci Food Agric,2004,84(11):1261-1269.
[7]管咏梅,陶玲,朱小芳,等.乳香没药挥发油对川芎中阿魏酸促透机制的研究[J].中国中药杂志,2017,42(17):3350-3355.
[8] Mancuso C,Santangelo R. Ferulic acid:pharmacological and toxicological aspects[J]. Food Chem Toxicol,2014,65(2):185-195.
[9] REN Z K,ZHANG R P,LI Y Y,et al. Ferulic acid exerts neuroprotective effects against cerebral ischemia/reperfusion-induced injury via antioxidant and antiapoptotic mechanisms in vitro and in vivo[J]. Int J Mol Med,2017,40(5):1444-1456.
[10] Koh P O. Ferulic acid attenuates the down-regulation of mek/erk/p90rsk signaling pathway in focal cerebral ischemic injury[J]. Neurosci Lett,2015,588:18-23.
[11] ZHANG Q,ZHAO Y H,XU Y H,et al. Sodium ferulate and n-butylidenephthalate combined with bone marrow stromal cells(BMSCs)improve the therapeutic effects of angiogenesis and neurogenesis after rat focal cerebral ischemia[J]. J Transl Med,2016,14(1):223-238.
[12] Sasaki M, Honmou O, Kocsis J D. A rat middle cerebral artery occlusion model and intravenous cellular delivery[J]. Methods Mol Biol,2009,549:187-195.
[13] Longa E Z,Weinstein P R,Carlson S,et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke,1989,20(1):84-91.
[14]李子广,吴华璞,郭梅,等.阿魏酸钠对MCAO大鼠缺血/再灌注损伤p38MAPK信号通路的影响[J].中西医结合心脑血管病杂志,2009,7(2):193-195.
[15] TANG Q Q,HAN R D,XIAO H,et al. Protective effect of tanshinone IIA on the brain and its therapeutic time window in rat models of cerebral ischemia-reperfusion[J]. Exp Ther Med,2014,8(5):1616-1622.
[16]刘抒雯,杨丽华,马春,等.中医药保护脑缺血再灌注损伤后神经血管单元的作用[J].中国实验方剂学杂志,2018,24(21):225-234.
[17] Danton G H,Dietrich W D. Inflammatory mechanisms after ischemia and stroke[J]. J Neuropathol Exp Neurol,2003,62(2):127-136.
[18] JIN R,YANG G,LI G. Inflammatory mechanisms in ischemic stroke:role of inflammatory cells[J]. J Leukoc Biol,2010,87(5):779-789.
[19]张予阳,刘岩,付守廷.脑缺血与炎症反应[J].中国药理学通报,2006,22(1):5-9.
[20] Berti R,Williams A J,Moffett J R,et al. Quantitative real-time RT-PCR analysis of inflammatory gene expression associated with ischemia-reperfusion brain injury[J]. J Cereb Blood Flow Metab,2002,22(9):1068-1079.
[21]刘亭,刘香香,陈亭亭,等.灯盏细辛和赤芍配伍组方对大鼠脑缺血再灌注损伤的保护作用及对NF-κB通路的影响[J].中国实验方剂学杂志,2018,24(9):111-115.
[22] Stephenson D, YIN T, Smalstig E B, et al.Transcription factor nuclear factor-kappa B is activated in neurons after focal cerebral ischemia[J]. J Cereb Blood Flow Metab,2000,20(3):592-603.
[23] TU X K,YANG W Z,CHEN J P,et al. Curcumin inhibits TLR2/4-NF-κB signaling pathway and attenuates brain damage in permanent focal cerebral ischemia in rats[J]. Inflammation,2014,37(5):1544-1551.
[24]吴建良,沈敏敏,杨水新,等.阿魏酸对小胶质细胞炎性反应的抑制作用[J].中国药理学通报,2015,31(1):97-102.
[25]孟锐,陈逸青,陈勤.阿魏酸对痴呆小鼠脑内胶质细胞活化与炎性细胞因子表达的影响[J].中国医院药学杂志,2018,38(1):50-53.
[26] LIU Y M,SHEN J D,XU L P,et al. Ferulic acid inhibits neuro-inflammation in mice exposed to chronic unpredictable mild stress[J]. Int Immunopharmacol,2017,45:128-134.