葛根素调节AMPK-mTOR信号通路抑制自噬改善大鼠脑缺血再灌注损伤研究
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摘要
目的探讨葛根素通过腺苷一磷酸活化蛋白激酶(AMPK)-哺乳动物雷帕霉素靶蛋白(m TOR)-Unc-51样激酶1(Ulk1)通路抑制自噬改善大鼠脑缺血再灌注损伤的作用。方法 40只雄性SD大鼠随机分为4组,即假手术组、模型组及葛根素低、高剂量(50、100 mg/kg)组。各组大鼠连续给药7 d,末次给药30min后,采用线栓法制备大鼠大脑中动脉栓塞(middle cerebral artery occlusion,MCAO)模型,缺血1.5 h再灌注24 h后,进行神经功能评分,TTC染色观察脑梗死体积,电镜观察自噬小体的形成,Western blotting法检测海马组织中微管相关蛋白1轻链3(LC3)、p62、AMPK、p-AMPK、m TOR、p-mTOR、Ulk1、p S757-Ulk1蛋白表达水平。结果与假手术组比较,模型组大鼠神经功能评分显著增加,脑梗死体积明显增大,自噬小体增多,LC3-II/LC3-I显著增加,p62蛋白表达水平显著降低,p-AMPK表达水平显著升高,p-m TOR和p S757-Ulk1蛋白表达水平显著降低。与模型组比较,葛根素各组大鼠的神经功能损伤明显改善,脑梗死体积减小,自噬小体减少,LC3-II/LC3-I显著降低,p62蛋白表达水平显著升高,p-AMPK蛋白表达水平显著降低,p-m TOR和p S757-Ulk1蛋白表达水平显著升高。结论葛根素可能通过调控AMPK-m TOR-Ulk1信号通路抑制自噬的过度发生,从而减轻脑缺血再灌注损伤的发生。
Objective To investigate the effect of puerarin on the regulation of AMPK-mTOR signaling pathway to inhibit autophagy and alleviate focal cerebral ischemia reperfusion injury. Methods Forty male Sprague-Dawley rats were randomly divided into four groups: Sham group, model group, puerarin low-dose(50 mg/kg) group and puerarin high-dose(100 mg/kg) group. Pretreatment with puerarin for 7 d, then the middle cerebral artery occlusion(MCAO) model was established 0.5 h after the last administration according to Longa's method. After 1.5 h of ischemia and 24 h of reperfusion, the neurological deficit scores were assessed, the infarct volume was calculated by TTC staining. The formation of autophagosome was observed by electron microscopy. The expression levels of LC3,p62, AMPK, p-AMPK, m TOR, p-mTOR, Ulk1, and pS757-Ulk1 were detected by Western blotting. Results Compared with the Sham group, the neurological deficit scores and infarct volume in model group were significantly increased, the numbers of autophagosome increased, and the rate of LC3-II/LC3-I significantly increased, the expression level of p62 gradually decreased. The expression of p-AMPK was markedly up-regulated, while the expression of p-mTOR and p S757-Ulk1 was significantly down-regulated. Compared with the model group, the neurological deficit scores and infarct volume were significantly reduced,the number of autophagosome and the rate of LC3-II/LC3-I decreased, the expression of p62 was significantly up-regulated, the expression of p-AMPK was markedly down-regulated, the levels of p-m TOR and p S757-Ulk1 were significantly up-regulated.Conclusion Puerarin alleviates cerebral ischemia-reperfusion injury may through suppressing autophagy via the AMPK-mTOR-Ulk1 signaling pathway.
Objective To investigate the effect of puerarin on the regulation of AMPK-mTOR signaling pathway to inhibit autophagy and alleviate focal cerebral ischemia reperfusion injury. Methods Forty male Sprague-Dawley rats were randomly divided into four groups: Sham group, model group, puerarin low-dose(50 mg/kg) group and puerarin high-dose(100 mg/kg) group. Pretreatment with puerarin for 7 d, then the middle cerebral artery occlusion(MCAO) model was established 0.5 h after the last administration according to Longa's method. After 1.5 h of ischemia and 24 h of reperfusion, the neurological deficit scores were assessed, the infarct volume was calculated by TTC staining. The formation of autophagosome was observed by electron microscopy. The expression levels of LC3,p62, AMPK, p-AMPK, m TOR, p-mTOR, Ulk1, and pS757-Ulk1 were detected by Western blotting. Results Compared with the Sham group, the neurological deficit scores and infarct volume in model group were significantly increased, the numbers of autophagosome increased, and the rate of LC3-II/LC3-I significantly increased, the expression level of p62 gradually decreased. The expression of p-AMPK was markedly up-regulated, while the expression of p-mTOR and p S757-Ulk1 was significantly down-regulated. Compared with the model group, the neurological deficit scores and infarct volume were significantly reduced,the number of autophagosome and the rate of LC3-II/LC3-I decreased, the expression of p62 was significantly up-regulated, the expression of p-AMPK was markedly down-regulated, the levels of p-m TOR and p S757-Ulk1 were significantly up-regulated.Conclusion Puerarin alleviates cerebral ischemia-reperfusion injury may through suppressing autophagy via the AMPK-mTOR-Ulk1 signaling pathway.
引文
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[17]Pan R,Cai J,Zhan L,et al.Buyang Huanwu decoction facilitates neurorehabilitation through an improvement of synaptic plasticity in cerebral ischemic rats[J].BMCCompl Altern Med,2017,doi:10.1186/s12906-017-1680-9.
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[20]Wang N,Zhang Y,Wu L,et al.Puerarin protected the brain from cerebral ischemia injury via astrocyte apoptosis inhibition[J].Neuropharmacology,2014,doi:10.1016/j.neuropharm.2013.12.004.
[21]Zheng Q H,Li X L,Mei Z G,et al.Efficacy and safety of puerarin injection in curing acute ischemic stroke:Ameta-analysis of randomized controlled trials[J].Medicine(Baltimore),2017,96(1):e5803.
[22]Liu X,Mei Z,Qian J,et al.Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway[J].Neural Regen Res,2013,8(34):3203-3215.
[23]Kuma A,Matsui M,Mizushima N.LC3,an autophagosome marker,can be incorporated into protein aggregates independent of autophagy:Caution in the interpretation of LC3 localization[J].Autophagy,2007,3(4):323-328.
[24]Lin X,Li S,Zhao Y,et al.Interaction domains of p62:Abridge between p62 and selective autophagy[J].DNACell Biol,2013,32(5):220-227.
[25]Buckley K M,Hess D L,Sazonova I Y,et al.Rapamycin up-regulation of autophagy reduces infarct size and improves outcomes in both permanent MCAL,and embolic MCAO,murine models of stroke[J].Exp Transl Stroke Med,2014,doi:10.1186/2040-7378-6-8.
[26]Sun Y,Zhang T,Zhang Y,et al.Ischemic postconditioning alleviates cerebral ischemia-reperfusion injury through activating autophagy during early reperfusion in rats[J].Neurochem Res,2018,43(9):1826-1840.
[27]Jiang W W,Huang B S,Han Y,et al.Sodium hydrosulfide attenuates cerebral ischemia/reperfusion injury by suppressing overactivated autophagy in rats[J].FEBS Open Bio,2017,7(11):1686-1695.
[28]Dong F,Yao R,Yu H,et al.Neuroprotection of Ro25-6981 against ischemia/reperfusion-induced brain injury via inhibition of autophagy[J].Cell Mol Neurobiol,2017,37(4):743-752.
[29]Tian F,Deguchi K,Yamashita T,et al.In vivo imaging of autophagy in a mouse stroke model[J].Autophagy,2010,6(8):1107-1114.
[30]Yang H,Li L,Zhou K,et al.Shengmai Injection attenuates the cerebral ischemia/reperfusion induced autophagy via modulation of the AMPK,mTOR and JNKpathways[J].Pharm Biol,2016,54(10):2288-2297.
[31]Xue T F,Ding X,Ji J,et al.PD149163 induces hypothermia to protect against brain injury in acute cerebral ischemic rats[J].J Pharmacol Sci,2017,135(3):105-113.
[32]Wataya-Kaneda M.Mammalian target of rapamycin and tuberous sclerosis complex[J].J Dermatol Sci,2015,79(2):93-100.
[33]Gwinn D M,Shackelford D B,Egan D F,et al.AMPKphosphorylation of raptor mediates a metabolic checkpoint[J].Mol Cell,2008,30(2):214-226.
[34]Tao R,Gong J,Luo X,et al.AMPK exerts dual regulatory effects on the PI3K pathway[J].J Mol Signal,2010,doi:10.1186/1750-2187-5-1.
[35]Hwang J Y,Gertner M,Pontarelli F,et al.Global ischemia induces lysosomal-mediated degradation of mTOR and activation of autophagy in hippocampal neurons destined to die[J].Cell Death Differ,2017,24(2):317-329.
[36]Russell R C,Tian Y,Yuan H,et al.ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase[J].Nat Cell Biol,2013,15(7):741-750.
[37]Tian W,Li W,Chen Y,et al.Phosphorylation of ULK1 by AMPK regulates translocation of ULK1 to mitochondria and mitophagy[J].FEBS Lett,2015,589(15):1847-1854.
[2]Chen Z,Jiang B,Ru X,et al.Mortality of stroke and its subtypes in China:Results from a nationwide populationbased survey[J].Neuroepidemiology,2017,48(3/4):95-102.
[3]Chang L,Zhang J,Liu L,et al.Fatty acid binding protein4 is associated with stroke risk and severity in patients with acute ischemic stroke[J].J Neuroimmunol,2017,doi:10.1016/j.jneuroim.2017.07.011.
[4]中国卒中学会,中国卒中学会神经介入分会,中华预防医学会卒中预防与控制专业委员会介入学组.急性缺血性卒中血管内治疗中国指南2018[J].中国卒中杂志,2018,13(7):706-729.
[5]Xin Q,Ji B,Cheng B,et al.Endoplasmic reticulum stress in cerebral ischemia[J].Neurochem Int,2014,doi:10.1016/j.neuint.2014.02.001.
[6]Wu J,Chen Y,Yu S,et al.Neuroprotective effects of sulfiredoxin-1 during cerebral ischemia/reperfusion oxidative stress injury in rats[J].Brain Res Bull,2017,doi:10.1016/j.brainresbull.2017.05.012.
[7]He Q,Li Z,Wang Y,et al.Resveratrol alleviates cerebral ischemia/reperfusion injury in rats by inhibiting NLRP3inflammasome activation through Sirt1-dependent autophagy induction[J].Int Immunopharmacol,2017,doi:10.1016/j.intimp.2017.06.029.
[8]Zhou F,Wang L,Liu P,et al.Puerarin protects brain tissue against cerebral ischemia/reperfusion injury by inhibiting the inflammatory response[J].Neural Regen Res,2014,9(23):2074-2080.
[9]Xu X,Zhang S,Zhang L,et al.The Neuroprotection of puerarin against cerebral ischemia is associated with the prevention of apoptosis in rats[J].Planta Med,2005,71(7):585-591.
[10]马鹍鹏,韩硕,赵淑敏,等.葛根素预处理对脑缺血再灌注大脑皮质微血管和血脑屏障的预防性保护作用[J].解剖学杂志,2015,38(6):694-697.
[11]Hongyun H,Tao G,Pengyue Z,et al.Puerarin provides a neuroprotection against transient cerebral ischemia by attenuating autophagy at the ischemic penumbra in neurons but not in astrocytes[J].Neurosci Lett,2017,doi:10.1016/j.neulet.2017.02.009.
[12]Fu L,Huang L,Cao C,et al.Inhibition of AMP-activated protein kinase alleviates focal cerebral ischemia injury in mice:Interference with m TOR and autophagy[J].Brain Res,2016,doi:10.1016/j.brainres.2016.08.035.
[13]Shen P,Hou S,Zhu M,et al.Cortical spreading depression preconditioning mediates neuroprotection against ischemic stroke by inducing AMP-activated protein kinase-dependent autophagy in a rat cerebral ischemic/reperfusion injury model[J].J Neurochem,2017,140(5):799-813.
[14]Alers S,Loffler A S,Wesselborg S,et al.Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy:Cross talk,shortcuts,and feedbacks[J].Mol Cell Biol,2012,32(1):2-11.
[15]Kim J,Kundu M,Viollet B,et al.AMPK and m TORregulate autophagy through direct phosphorylation of Ulk1[J].Nat Cell Biol,2011,13(2):132-141.
[16]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.
[17]Pan R,Cai J,Zhan L,et al.Buyang Huanwu decoction facilitates neurorehabilitation through an improvement of synaptic plasticity in cerebral ischemic rats[J].BMCCompl Altern Med,2017,doi:10.1186/s12906-017-1680-9.
[18]谭凌菁,王金凤,梅志刚.沉默信息调节因子1信号通路在脑缺血-再灌注损伤中的保护作用[J].中国脑血管病杂志,2016,13(8):442-445.
[19]郝春华,孙双勇,徐向伟,等.葛根素预处理对大鼠血栓栓塞性脑缺血急性期的保护作用[J].中药药理与临床,2015,31(6):11-14.
[20]Wang N,Zhang Y,Wu L,et al.Puerarin protected the brain from cerebral ischemia injury via astrocyte apoptosis inhibition[J].Neuropharmacology,2014,doi:10.1016/j.neuropharm.2013.12.004.
[21]Zheng Q H,Li X L,Mei Z G,et al.Efficacy and safety of puerarin injection in curing acute ischemic stroke:Ameta-analysis of randomized controlled trials[J].Medicine(Baltimore),2017,96(1):e5803.
[22]Liu X,Mei Z,Qian J,et al.Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway[J].Neural Regen Res,2013,8(34):3203-3215.
[23]Kuma A,Matsui M,Mizushima N.LC3,an autophagosome marker,can be incorporated into protein aggregates independent of autophagy:Caution in the interpretation of LC3 localization[J].Autophagy,2007,3(4):323-328.
[24]Lin X,Li S,Zhao Y,et al.Interaction domains of p62:Abridge between p62 and selective autophagy[J].DNACell Biol,2013,32(5):220-227.
[25]Buckley K M,Hess D L,Sazonova I Y,et al.Rapamycin up-regulation of autophagy reduces infarct size and improves outcomes in both permanent MCAL,and embolic MCAO,murine models of stroke[J].Exp Transl Stroke Med,2014,doi:10.1186/2040-7378-6-8.
[26]Sun Y,Zhang T,Zhang Y,et al.Ischemic postconditioning alleviates cerebral ischemia-reperfusion injury through activating autophagy during early reperfusion in rats[J].Neurochem Res,2018,43(9):1826-1840.
[27]Jiang W W,Huang B S,Han Y,et al.Sodium hydrosulfide attenuates cerebral ischemia/reperfusion injury by suppressing overactivated autophagy in rats[J].FEBS Open Bio,2017,7(11):1686-1695.
[28]Dong F,Yao R,Yu H,et al.Neuroprotection of Ro25-6981 against ischemia/reperfusion-induced brain injury via inhibition of autophagy[J].Cell Mol Neurobiol,2017,37(4):743-752.
[29]Tian F,Deguchi K,Yamashita T,et al.In vivo imaging of autophagy in a mouse stroke model[J].Autophagy,2010,6(8):1107-1114.
[30]Yang H,Li L,Zhou K,et al.Shengmai Injection attenuates the cerebral ischemia/reperfusion induced autophagy via modulation of the AMPK,mTOR and JNKpathways[J].Pharm Biol,2016,54(10):2288-2297.
[31]Xue T F,Ding X,Ji J,et al.PD149163 induces hypothermia to protect against brain injury in acute cerebral ischemic rats[J].J Pharmacol Sci,2017,135(3):105-113.
[32]Wataya-Kaneda M.Mammalian target of rapamycin and tuberous sclerosis complex[J].J Dermatol Sci,2015,79(2):93-100.
[33]Gwinn D M,Shackelford D B,Egan D F,et al.AMPKphosphorylation of raptor mediates a metabolic checkpoint[J].Mol Cell,2008,30(2):214-226.
[34]Tao R,Gong J,Luo X,et al.AMPK exerts dual regulatory effects on the PI3K pathway[J].J Mol Signal,2010,doi:10.1186/1750-2187-5-1.
[35]Hwang J Y,Gertner M,Pontarelli F,et al.Global ischemia induces lysosomal-mediated degradation of mTOR and activation of autophagy in hippocampal neurons destined to die[J].Cell Death Differ,2017,24(2):317-329.
[36]Russell R C,Tian Y,Yuan H,et al.ULK1 induces autophagy by phosphorylating Beclin-1 and activating VPS34 lipid kinase[J].Nat Cell Biol,2013,15(7):741-750.
[37]Tian W,Li W,Chen Y,et al.Phosphorylation of ULK1 by AMPK regulates translocation of ULK1 to mitochondria and mitophagy[J].FEBS Lett,2015,589(15):1847-1854.