基于PI3K/Akt/eNOS信号通路探讨补阳还五汤对大鼠蛛网膜下腔出血后脑血管痉挛的影响
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摘要
目的:探讨补阳还五汤改善蛛网膜下腔出血后脑血管痉挛的相关分子机制。方法:将雄性SD大鼠80只随机分为假手术组、模型组、补阳还五汤低、高剂量组(13,26 g·kg~(-1)·d~(-1)),每组20只。按照10 mL·kg~(-1)每天灌胃给药2次,连续7 d。采用二次枕大池注血法制作蛛网膜下腔出血模型,评估各组大鼠1,3,5,7 d的神经功能评分,利用苏木素-伊红(HE)染色对各组大鼠基底动脉直径进行测量,蛋白免疫印迹法(Western blot)检测基底动脉脑组织磷酸化磷脂酰肌醇3-激酶(p-PI3K),磷酸化蛋白激酶B(p-Akt),内皮型一氧化氮合酶(eNOS),神经型一氧化氮合酶(n NOS)蛋白的表达;酶联免疫吸附测定(ELISA)检测大鼠脑脊液中一氧化氮(NO),内皮素-1(ET-1)的表达。结果:与假手术组比较,模型组大鼠神经功能评分明显下降(P <0. 05),基底动脉直径明显缩小(P <0. 05),基底动脉脑组织p-PI3K,p-Akt,eNOS,n NOS蛋白的表达明显降低(P <0. 05),脑脊液中NO明显减少(P <0. 05),ET-1明显升高(P <0. 05);与模型组比较,不同剂量补阳还五汤组(26,13 g·kg~(-1)·d~(-1))在治疗后3～5 d后大鼠神经功能评分均有不同程度升高,基底动脉直径明显增宽(P <0. 05),p-PI3K,p-Akt,eNOS蛋白的表达明显升高(P <0. 05),NO明显升高(P <0. 05),ET-1明显下降(P <0. 05);与低剂量组比较,经过7 d的补阳还五汤治疗后高剂量组神经功能评分明显升高(P <0. 05),基底动脉直径增宽更明显(P <0. 05),p-PI3K,p-Akt,eNOS蛋白的表达更高(P <0. 05),NO升高更明显(P <0. 05),ET-1下降更明显(P <0. 05)。结论:补阳还五汤对蛛网膜下腔出血后脑血管痉挛的保护作用可能与上调PI3K/Akt/eNOS信号通路中的p-PI3K,p-Akt,eNOS的表达,从而增加NO产生有关。
Objective: To investigate the molecular mechanism of Buyang Huanwu Tang in improving cerebral vasospasm after subarachnoid hemorrhage. Method: Eighty male Sprague-Dawley rats were randomly divided into sham operation group,model group,Buyang Huanwu Tang low and high dose( 13,26 g·kg~(-1)·d~(-1))group. According to 10 mL·kg~(-1),the drug was administered twice a day for 7 days. The subarachnoid hemorrhage model was made by double occipital pool injection method. The neurological function scores of rats in each group were evaluated at 1,3,5 and 7 days. The diameter of basilar artery was measured by hematoxylin-eosin( HE)staining. The expressions of phosphp-phosphoinositide 3-kinases( p-PI3 K),phosphp-protein kinase B( p-Akt),endothelial nitric oxide synthase( eNOS) and neuronal nitric oxide synthase( nNOS) protein in basilar artery brain tissue were detected by Western blot. The expression of nitric oxide( NO) and endothelin~(-1)( ET-1) in rat cerebrospinal fluid was detected by enzyme-linked immunosorbent assay( ELISA). Result: Compared with sham operation group,the neurological function scores of the model group were significantly decreased( P < 0. 05),the diameter of the basilar artery was significantly reduced( P < 0. 05),and The expression of p-PI3 K,p-Akt,eNOS,nNOS proteins of the basilar artery brain tissue were significantly decreased( P < 0. 05). The level of NO in the cerebrospinal fluid was significantly decreased( P < 0. 05),and the ET-1 was significantly increased( P < 0. 05).Compared with model group,the different doses in Buyang Huanwu Tang group( 26,13 g·kg~(-1)·d~(-1)) increased the neurological function scores 3 to 5 days after treatment,and the basilar artery diameter was significant increased( P < 0. 05). The expression of p-PI3 K,p-Akt and eNOS protein was significantly increased( P < 0. 05). The level of NO was significantly increased( P < 0. 05),and ET-1 was significantly decreased( P < 0. 05). Compared with low-dose group,the neurological scores of high-dose group were significantly increased after 7 days of treatment with Buyang Huanwu Tang( P < 0. 05),and the diameter of the basilar artery was significantly increased( P < 0. 05),p-PI3 K,The expression of p-Akt and eNOS protein was higher( P < 0. 05),the increase of NO was more obvious( P < 0. 05),the decrease of ET-1 was more significant( P < 0. 05). Conclusion: The protective effect of Buyang Huanwu Tang on cerebral vasospasm after subarachnoid hemorrhage may be related to up-regulation of p-PI3 K,p-Akt and eNOS expression in PI3 K/Akt/eNOS signaling pathway,thereby increasing NO production.
Objective: To investigate the molecular mechanism of Buyang Huanwu Tang in improving cerebral vasospasm after subarachnoid hemorrhage. Method: Eighty male Sprague-Dawley rats were randomly divided into sham operation group,model group,Buyang Huanwu Tang low and high dose( 13,26 g·kg~(-1)·d~(-1))group. According to 10 mL·kg~(-1),the drug was administered twice a day for 7 days. The subarachnoid hemorrhage model was made by double occipital pool injection method. The neurological function scores of rats in each group were evaluated at 1,3,5 and 7 days. The diameter of basilar artery was measured by hematoxylin-eosin( HE)staining. The expressions of phosphp-phosphoinositide 3-kinases( p-PI3 K),phosphp-protein kinase B( p-Akt),endothelial nitric oxide synthase( eNOS) and neuronal nitric oxide synthase( nNOS) protein in basilar artery brain tissue were detected by Western blot. The expression of nitric oxide( NO) and endothelin~(-1)( ET-1) in rat cerebrospinal fluid was detected by enzyme-linked immunosorbent assay( ELISA). Result: Compared with sham operation group,the neurological function scores of the model group were significantly decreased( P < 0. 05),the diameter of the basilar artery was significantly reduced( P < 0. 05),and The expression of p-PI3 K,p-Akt,eNOS,nNOS proteins of the basilar artery brain tissue were significantly decreased( P < 0. 05). The level of NO in the cerebrospinal fluid was significantly decreased( P < 0. 05),and the ET-1 was significantly increased( P < 0. 05).Compared with model group,the different doses in Buyang Huanwu Tang group( 26,13 g·kg~(-1)·d~(-1)) increased the neurological function scores 3 to 5 days after treatment,and the basilar artery diameter was significant increased( P < 0. 05). The expression of p-PI3 K,p-Akt and eNOS protein was significantly increased( P < 0. 05). The level of NO was significantly increased( P < 0. 05),and ET-1 was significantly decreased( P < 0. 05). Compared with low-dose group,the neurological scores of high-dose group were significantly increased after 7 days of treatment with Buyang Huanwu Tang( P < 0. 05),and the diameter of the basilar artery was significantly increased( P < 0. 05),p-PI3 K,The expression of p-Akt and eNOS protein was higher( P < 0. 05),the increase of NO was more obvious( P < 0. 05),the decrease of ET-1 was more significant( P < 0. 05). Conclusion: The protective effect of Buyang Huanwu Tang on cerebral vasospasm after subarachnoid hemorrhage may be related to up-regulation of p-PI3 K,p-Akt and eNOS expression in PI3 K/Akt/eNOS signaling pathway,thereby increasing NO production.
引文
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[3] Kolias A G,Sen J,Belli A. Pathogenesis of cerebral vasospasm following aneurysmal subarachnoid hemorrhage:putative mechanisms and novel approaches[J]. J Neur Res,2010,87(1):1-11.
[4]王玉妹,唐思魏,石广志,等.蛛网膜下腔出血后迟发性脑血管痉挛的发病机制和治疗进展[J].中国卒中杂志,2016,11(6):494-500.
[5] CHEN H,SONG H,LIU X,et al. Buyanghuanwu Decoction alleviated pressure overload induced cardiac remodeling by suppressing TGF-β/Smads and MAPKs signaling activated fibrosis[J]. Biomed Pharmacother,2017,95:461-468.
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[7] HAO C Z,WU F,SHEN J,et al. Clinical efficacy and safety of Buyang Huanwu Tang for acute ischemic stroke:a systematic review and Meta-analysis of 19randomized controlled trials[J]. Evid Based Complement Alternat Med,2012,2012(3/4):630124-630137.
[8]熊录,范吉平.蛛网膜下腔出血的病机研究——兼论其并发症脑血管痉挛[J].中国中医急症,2002,11(3):200-202.
[9]刘欣.中西医结合治疗蛛网膜下腔出血23例[J].现代中西医结合杂志,2001,10(24):2380-2381.
[10] Raslan F. A modified double injection model of cisterna magna for the study of delayed cerebral vasospasm following subarachnoid hemorrhage in rats[J]. Exp Transl Stroke Med,2012,4(1):23-23.
[11] Sugawara T,Ayer R,Jadhav V,et al. A new grading system evaluating bleeding scale in filament perforation subarachnoid hemorrhage rat model[J]. J Neurosci Methods,2007,167(2):327-334.
[12]施新猷.医用实验动物学[M].西安:陕西科学技术出版社,1989:417.
[13]仇志富,吴晓光,孟杰,等.补阳还五汤提取物灌胃对脑出血大鼠脑组织中PI3K、AKT、Caspase-3表达的影响[J].山东医药,2016,56(9):29-31.
[14] Garcia J,Wagner S,LIU K F,et al. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats[J]. Stroke,1995,26(4):627-635.
[15] Poulos T L,LI H. Nitric oxide synthase and structurebased inhibitor design[J]. Nitric Oxide,2017,63(4):68-77.
[16] Atochin D N,HUANG P L. Endothelial nitric oxide synthase transgenic models of endothelial dysfunction[J]. Pflugers Arch,2010,460(6):965-974.
[17] LI X, XIA X, LI X. Plasmid p LXSN-mediated adrenomedullin gene therapy for cerebral vasospasm following subarachnoid hemorrhage in rats[J]. Med Sci Monit,2017,23(7):3293-3302.
[18] Drake C T,Iadecola C. The role of neuronal signaling in controlling cerebral blood flow[J]. Brain Lang,2007,102(2):141-152.
[19] ZHU J,SONG W,LI L,et al. Endothelial nitric oxide synthase:a potential therapeutic target for cerebrovascular diseases[J]. Mol Brain,2016,9(1):30-37.
[20]蔡俊,张继平,姚晖,等.补阳还五汤对急性脑缺血再灌注大鼠脑组织AKT和p-AKT蛋白表达的影响[J].中国实验方剂学杂志,2015,21(6):122-126.
[21] CUI H J,YANG A L,ZHOU H J,et al. Buyang Huanwu Tang promotes angiogenesis via vascular endothelial growth factor receptor-2 activation through the PI3K/Akt pathway in a mouse model of intracerebral hemorrhage[J]. BMC Complementary Altern Med,2015,15(1):91-102.
[22] CHOU C H,Reed S D,Allsbrook J S,et al. Costs of vasospasm in patients with aneurysmal subarachnoid hemorrhage[J]. Neurosurgery,2010,67(2):345-351,353.
[23] Mayberg M R,Batjer H H,Dacey R,et al. Guidelines for the management of aneurysmal subarachnoid hemorrhage:a statement for healthcare professionals from a special writing group of the stroke council,American heart association[J]. Stroke,1994,25(11):2315-2328.
[2] Rodríguez-Rodríguez A,Egea-Guerrero J J,Ruiz de Azúa-Lo'pez Z, et al. Biomarkers of vasospasm development and outcome in aneurysmal subarachnoid hemorrhage[J]. J Neurol Sci,2014,341(1/2):119-127.
[3] Kolias A G,Sen J,Belli A. Pathogenesis of cerebral vasospasm following aneurysmal subarachnoid hemorrhage:putative mechanisms and novel approaches[J]. J Neur Res,2010,87(1):1-11.
[4]王玉妹,唐思魏,石广志,等.蛛网膜下腔出血后迟发性脑血管痉挛的发病机制和治疗进展[J].中国卒中杂志,2016,11(6):494-500.
[5] CHEN H,SONG H,LIU X,et al. Buyanghuanwu Decoction alleviated pressure overload induced cardiac remodeling by suppressing TGF-β/Smads and MAPKs signaling activated fibrosis[J]. Biomed Pharmacother,2017,95:461-468.
[6] WEI R L,TENG H J,YIN B,et al. A systematic review and Meta-analysis of Buyang Huanwu Tang in animal model of focal cerebral ischemia[J]. Evid Based Complement Alternat Med,2013,4(6):1-13.
[7] HAO C Z,WU F,SHEN J,et al. Clinical efficacy and safety of Buyang Huanwu Tang for acute ischemic stroke:a systematic review and Meta-analysis of 19randomized controlled trials[J]. Evid Based Complement Alternat Med,2012,2012(3/4):630124-630137.
[8]熊录,范吉平.蛛网膜下腔出血的病机研究——兼论其并发症脑血管痉挛[J].中国中医急症,2002,11(3):200-202.
[9]刘欣.中西医结合治疗蛛网膜下腔出血23例[J].现代中西医结合杂志,2001,10(24):2380-2381.
[10] Raslan F. A modified double injection model of cisterna magna for the study of delayed cerebral vasospasm following subarachnoid hemorrhage in rats[J]. Exp Transl Stroke Med,2012,4(1):23-23.
[11] Sugawara T,Ayer R,Jadhav V,et al. A new grading system evaluating bleeding scale in filament perforation subarachnoid hemorrhage rat model[J]. J Neurosci Methods,2007,167(2):327-334.
[12]施新猷.医用实验动物学[M].西安:陕西科学技术出版社,1989:417.
[13]仇志富,吴晓光,孟杰,等.补阳还五汤提取物灌胃对脑出血大鼠脑组织中PI3K、AKT、Caspase-3表达的影响[J].山东医药,2016,56(9):29-31.
[14] Garcia J,Wagner S,LIU K F,et al. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats[J]. Stroke,1995,26(4):627-635.
[15] Poulos T L,LI H. Nitric oxide synthase and structurebased inhibitor design[J]. Nitric Oxide,2017,63(4):68-77.
[16] Atochin D N,HUANG P L. Endothelial nitric oxide synthase transgenic models of endothelial dysfunction[J]. Pflugers Arch,2010,460(6):965-974.
[17] LI X, XIA X, LI X. Plasmid p LXSN-mediated adrenomedullin gene therapy for cerebral vasospasm following subarachnoid hemorrhage in rats[J]. Med Sci Monit,2017,23(7):3293-3302.
[18] Drake C T,Iadecola C. The role of neuronal signaling in controlling cerebral blood flow[J]. Brain Lang,2007,102(2):141-152.
[19] ZHU J,SONG W,LI L,et al. Endothelial nitric oxide synthase:a potential therapeutic target for cerebrovascular diseases[J]. Mol Brain,2016,9(1):30-37.
[20]蔡俊,张继平,姚晖,等.补阳还五汤对急性脑缺血再灌注大鼠脑组织AKT和p-AKT蛋白表达的影响[J].中国实验方剂学杂志,2015,21(6):122-126.
[21] CUI H J,YANG A L,ZHOU H J,et al. Buyang Huanwu Tang promotes angiogenesis via vascular endothelial growth factor receptor-2 activation through the PI3K/Akt pathway in a mouse model of intracerebral hemorrhage[J]. BMC Complementary Altern Med,2015,15(1):91-102.
[22] CHOU C H,Reed S D,Allsbrook J S,et al. Costs of vasospasm in patients with aneurysmal subarachnoid hemorrhage[J]. Neurosurgery,2010,67(2):345-351,353.
[23] Mayberg M R,Batjer H H,Dacey R,et al. Guidelines for the management of aneurysmal subarachnoid hemorrhage:a statement for healthcare professionals from a special writing group of the stroke council,American heart association[J]. Stroke,1994,25(11):2315-2328.