栝楼桂枝颗粒抑制MCAO大鼠神经元凋亡的机制研究
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摘要
目的研究栝楼桂枝颗粒(Gualou Guizhi granule,GLGZG)是否通过调控PI3K/AKT通路抑制脑缺血再灌注损伤大鼠神经元凋亡而达到抗脑缺血再灌注损伤的作用。方法 SD大鼠36只,♂,分为假手术组、大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)组、GLGZG组,每组12只。其中MCAO组、GLGZG组采用线栓法致MCAO建立大鼠脑缺血再灌注损伤模型,GLGZG灌胃给药,连续给药7 d。采用改良的神经功能缺损评分法对大鼠神经功能损伤进行评分,MRI观察大鼠脑梗死体积,Real-time PCR检测脑组织中PI3K、Akt m RNA的表达,Western blot法检测大鼠缺血侧脑组织PI3K(p85)、Akt、p-Akt、PDK1、Bcl-2、Bcl-x L、cleaved-caspase-3、Bad、Bax蛋白表达。结果与MCAO组比较,GLGZG组神经行为学评分降低,第5、7天神经评分显著低于MCAO组(P<0.05);与MCAO组比较,MRI观察发现GLGZG组大鼠脑梗死体积显著减小(P<0.05);PI3K(p85)、p-Akt、PDK1、Bcl-2、Bcl-x L蛋白的表达上调(P<0.01),cleaved-caspase-3、Bad、Bax蛋白的表达下调(P<0.01),对Akt的表达没有影响。结论 GLGZG能够提高MCAO大鼠神经功能,抑制神经细胞凋亡,其作用机制可能是通过激活PI3K/AKT信号通路抑制MCAO大鼠神经元凋亡。
OBJECTIVE To investigate the inhibitive effect of Gualou Guizhi granule(GLGZG) on nerve cell apoptosis in cerebral ischemia-reperfusion injury rat through activatig PI3 K/n AKT pathway. METHODS Thirty-six male SD rats were divided into sham operation group, middle cerebral artery occlusion(MCAO) model group and GLGZG group, 12 for each group. MCAO model group and GLGZG group subjected to focal cerebral ischemia reperfusion with suture-occluded induced MCAO method. GLGZG was gave by intragastric administration, once daily for 7 d. Neurobehavioral deficit was performed with modified neurological severity score(m NSS) scale. MRI was used to observe the cerebral infarct volume in rats. PI3 K, Akt m RNA were detected by q RT-PCR in the infarcted cortex. The protein expression of PI3 K(p85), p-Akt, PDK1, Bcl-2, Bcl-x L, cleaved-caspase-3, Bad, Bax were detected by Western blot. RESULTS The m NSS score of GLGZG group was reduced compared with the MACO model group, especially on 5 th and 7 th day(P<0.05). Compared with MCAO group, the cerebral infarct volume in GLGZG group rats were decreased significantly(P<0.05) and upregulated the proteins expression of PI3 K(p85), p-Akt, PDK1, Bcl-2, Bcl-x L(P<0.01), downregulated cleaved-caspase-3, Bad, Bax(P<0.01), however, the protein expression of Akt was not change. CONCLUSION GLGZG can improve the nerve function deficit of MCAO rat and inhibit the apoptosis of neural cell, which may be associated with activating the PI3 K/AKT pathway.
OBJECTIVE To investigate the inhibitive effect of Gualou Guizhi granule(GLGZG) on nerve cell apoptosis in cerebral ischemia-reperfusion injury rat through activatig PI3 K/n AKT pathway. METHODS Thirty-six male SD rats were divided into sham operation group, middle cerebral artery occlusion(MCAO) model group and GLGZG group, 12 for each group. MCAO model group and GLGZG group subjected to focal cerebral ischemia reperfusion with suture-occluded induced MCAO method. GLGZG was gave by intragastric administration, once daily for 7 d. Neurobehavioral deficit was performed with modified neurological severity score(m NSS) scale. MRI was used to observe the cerebral infarct volume in rats. PI3 K, Akt m RNA were detected by q RT-PCR in the infarcted cortex. The protein expression of PI3 K(p85), p-Akt, PDK1, Bcl-2, Bcl-x L, cleaved-caspase-3, Bad, Bax were detected by Western blot. RESULTS The m NSS score of GLGZG group was reduced compared with the MACO model group, especially on 5 th and 7 th day(P<0.05). Compared with MCAO group, the cerebral infarct volume in GLGZG group rats were decreased significantly(P<0.05) and upregulated the proteins expression of PI3 K(p85), p-Akt, PDK1, Bcl-2, Bcl-x L(P<0.01), downregulated cleaved-caspase-3, Bad, Bax(P<0.01), however, the protein expression of Akt was not change. CONCLUSION GLGZG can improve the nerve function deficit of MCAO rat and inhibit the apoptosis of neural cell, which may be associated with activating the PI3 K/AKT pathway.
引文
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[22]XU W,HUANG M,LI H,et al.Chemical profiling and quantification of Gua-Lou-Gui-Zhi decoction by high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry and ultra-performance liquid chromatography/triple quadrupole mass spectrometry[J].J Chromatogr B Analyt Technol Biomed Life Sci,2015(986/987):69-84.
[23]XU W,WU X,HUANG M Q,et al Analysis on pharmacodynamic constituents in Gualou Guizhi Decoction by serum pharmacochemistry[J].Chin Tradit Herb Drugs(中草药),2017,48(10):2033-2043.
[24]ZHANG Y Q,LI H,XU W,et al.Effects of Gualou Guizhi granule on blood brain barrier permeabillity and neuroprotection in cerebral ischemia-reperfusion injury rat[J].Chin J Tradit Chin Med Pharm(中华中医药杂志),2015,30(5):1410-1417.
[25]陈阿虹,刘杰,许文,等.RRLC-MS/MS法测定栝楼桂枝颗粒中γ-氨基丁酸和瓜氨酸含量[J].福建中医药,2015,46(4):43-45.
[2]杨春梅,陈立典,陶静.古方今用瓜蒌桂枝汤[J].辽宁中医杂志,2012,39(8):1599-1600.
[3]CHEN Y L,CHEN L D,TAO J.Clinical research on treating limbs spasm from cerebral apoplexy with the Gualou Guizhi decoction[J].Clin J Chin Med(中医临床研究),2013,5(4):7-9.
[4]ZHU Y P.Clinical Effect analysis of Trichosanthes Kirilowii Cassia Twig Decoction for treatment of lower limb spasm after cerebral apoplexy patients[J].Guide Chin Med(中国医药指南),2015,13(29):27-28.
[5]战美玲.脑卒中后下肢痉挛应用栝楼桂枝汤治疗的效果分析[J].双足与保健,2017,26(11):160-161.
[6]CHEN X,LI H,HUANG M,et al.Effect of Gua Lou Gui Zhi decoction on focal cerebral ischemia-reperfusion injury through regulating the expression of excitatory amino acids and their receptors[J].Mol Med Rep,2014,10(1):248-254.
[7]HUANG J,TAO J,XUE X,et al.Gua Lou Gui Zhi decoction exerts neuroprotective effects on post-stroke spasticity via the modulation of glutamate levels and AMPA receptor expression[J].Int J Mol Med,2013,31(4):841-848.
[8]LI Z F,LI R H,MAO J J,et al,Protective effect of Gualou Guizhi decoction on BV-2 cells injuried by glutamate[J].J Fujian Univ Tradit Chin Med(福建中医药大学学报),2013,23(5):14-17.
[9]LI Z,HU H,LIN R,et al.Neuroprotective effects of Gua Lou Gui Zhi decoction against glutamate-induced apoptosis in BV-2 cells[J].Int J Mol Med.2014,33(3):597-604.
[10]HUANG J H,HUANG X H,CHEN Z Y,et al.Dose conversion among different animals and healthy volunteers in pharmacological study[J].Chin J Clin Pharm Therap(中国临床药理学与治疗学),2004,9(9):106-107.
[11]GAO Y,CHEN T,LEI X,et al.Neuroprotective effects of polydatin against mitochondrial-dependent apoptosis in the rat cerebral cortex following ischemia/reperfusion injury[J].Mol Med Rep,2016,14(6):5481-5488.
[12]JI Y,TENG L,ZHANG R,et al.NRG-1βexerts neuroprotective effects against ischemia reperfusion-induced injury in rats through the JNK signaling pathway[J].Neuroscience,2017(362):13-24.
[13]LEVESLEY J,STEELE L,BRüNING-RICHARDSON A,et al.Selective BCL-x L inhibition promotes apoptosis in combination with MLN8237 in medulloblastoma and pediatric glioblastoma cells[J].Neuro Oncol,2017,20(2):203-214.
[14]林羽,徐伟,张玉琴,等.栝楼桂枝颗粒抗缺血性脑卒中大鼠神经元及原代海马神经元凋亡研究[J].康复学报,2015,(1):38-43.
[15]王宏运,张玉琴,李煌,等.栝楼桂枝颗粒对NMDA诱导海马神经元兴奋性毒性损伤的保护作用[J].中华中医药杂志,2017,32(9):4115-4120.
[16]JIANG X,TANG X,ZHANG P,et al.Cyanidin-3-O-β-glucoside protects primary mouse hepatocytes against high glucose-induced apoptosis by modulating mitochondrial dysfunction and the PI3K/Akt pathway[J].Biochem Pharmacol,2014,90(2):135-144.
[17]马陈晨.新型PI3K抑制剂的设计合成及抗肿瘤活性研究[D].济南:山东大学,2017.
[18]FAIVRE S,DJELLOUL S,RAYMOND E.New paradigms in anticancer therapy:targeting multiple signaling pathways with kinase inhibitors[J].Semin Oncol,2006,33(4):407-420.
[19]XUE X,YOU Y,TAO J,et al.Electro-acupuncture at points of Zusanli and Quchi exerts anti-apoptotic effect through the modulation of PI3K/Akt signaling pathway[J].Neurosci Lett,2014,(558):14-19.
[20]XUE M,JI X,XUE C,et al.Caspase-dependent and caspase-independent induction of apoptosis in breast cancer by fucoidan via the PI3K/AKT/GSK3βpathway in vivo and in vitro[J].Biomed Pharmacother,2017(94):898-908.
[21]ZHU X,JIANG X,LI A,et al.S-allylmercaptocysteine suppresses the growth of human gastric cancer xenografts through induction of apoptosis and regulation of MAPK and PI3K/Akt signaling pathways[J].Biochem Biophys Res Commun,2017,491(3):821-826.
[22]XU W,HUANG M,LI H,et al.Chemical profiling and quantification of Gua-Lou-Gui-Zhi decoction by high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry and ultra-performance liquid chromatography/triple quadrupole mass spectrometry[J].J Chromatogr B Analyt Technol Biomed Life Sci,2015(986/987):69-84.
[23]XU W,WU X,HUANG M Q,et al Analysis on pharmacodynamic constituents in Gualou Guizhi Decoction by serum pharmacochemistry[J].Chin Tradit Herb Drugs(中草药),2017,48(10):2033-2043.
[24]ZHANG Y Q,LI H,XU W,et al.Effects of Gualou Guizhi granule on blood brain barrier permeabillity and neuroprotection in cerebral ischemia-reperfusion injury rat[J].Chin J Tradit Chin Med Pharm(中华中医药杂志),2015,30(5):1410-1417.
[25]陈阿虹,刘杰,许文,等.RRLC-MS/MS法测定栝楼桂枝颗粒中γ-氨基丁酸和瓜氨酸含量[J].福建中医药,2015,46(4):43-45.