PARP-1依赖性程序性细胞死亡（Parthanatos）在布比卡因致SH-SY5Y细胞损伤中作用的研究
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摘要
局麻药(local anesthetics, LA)是神经阻滞及术后镇痛的常用药物,但随着局麻药的广泛应用,关于其毒性反应的报道也逐渐增多。因此局麻药的细胞毒性成为近年来的研究热点,研究者们试图以此阐明其机制从而指导局麻药所致周围神经损伤的治疗。
研究表明,布比卡因是最具毒性的局麻药,可能通过氧化磷酸化解偶联(uncoupling of oxidative phosphorylation)、活性氧(reactive oxygen, ROS)生成增加及三磷酸腺苷(adenosine triphosphate, ATP)生成减少等引起细胞的凋亡及坏死。凋亡是细胞程序性死亡的一种,主要通过激活半胱天冬酶-3、7(caspase-3、7)而裂解细胞活性相关蛋白,并引起凋亡特有的形态学改变。经典的细胞凋亡信号转导通路可以分为两种：分别是外源性通路(extrinsic pathway)和内源性通路(intrinsic pathway)。前者即死亡受体途径,由细胞表面的死亡受体如肿瘤坏死因子受体(tumour necrosis factor receptor, TNF-R)等介导,以激活caspase-8、10为主；后者也称为线粒体途径(mitochondrial pathway),主要是通过线粒体释放细胞色素-c(cytochrome-c)至胞浆并激活caspase-9。两条通路最终都能激活caspase-3和caspase-7。 Unami等研究证实,在布比卡因所致的细胞毒性中,caspase-8、9及caspase-3均被激活,且与凋亡小体、DNA碎片的形成正相关,这意味着caspase依赖性内、外源性凋亡通路均与布比卡因的细胞毒性有关。但Perez等研究发现,布比卡因处理的细胞虽然有caspase的激活,却存在caspase的激活滞后的现象,他们用1mM的布比卡因处理SH-SY5Y细胞10分钟,即有接近50%的细胞死亡,而3小时后才出现caspase的激活。Perez等把这种先于caspase激活的细胞死亡归结为坏死,但随着对细胞死亡研究的深入,Perez所依据的这种基于形态学的“凋亡-坏死”的细胞死亡分类显得过于简单。目前学者们将细胞死亡分为两大类,一类为非程序性死亡,另一类则为程序性死亡。前者即坏死,其特点是不能为细胞信号转导抑制剂所阻断,为被动死亡；后者能为细胞信号转导抑制剂所阻断,为主动死亡。程序性细胞死亡又可以分为两大类：caspase依赖性和非caspase依赖性,前者即典型的凋亡,后者包括自吞噬性程序性细胞死亡、副凋亡(paraptosis)、胀亡等,其最大特点是细胞的主动死亡不依赖caspase的激活。那么,Perez的实验结果是否提示在布比卡因所致的细胞死亡中,存在非caspase依赖性的细胞程序性死亡途径?
Parthanatos是新近发现的非caspase依赖性细胞程序性死亡。Parthanatos的特点为多聚腺嘌呤二核苷酸核糖聚合酶(Poly(ADP-ribose) polymerase-1, PARP-1)依赖性,其主要表现为PARP-1激活及随后的细胞内烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide, NAD+)水平的降低。
在Parthanatos中,PARP-1的激活起到关键作用。PARP-1是PARP家族成员中被广泛研究的细胞内的酶类,大约占细胞内PARP活性的90%以上,生理条件下,PARP-1能为DNA碎片等激活,与受损的DNA结合,主要起到DNA修复酶的作用；在病理条件下,如兴奋性毒素损伤、氧化应激、缺血再灌注损伤中,PARP-1出现过度激活,并水解NAD+,同时生成PAR多聚体。由于NAD+在能量代谢中扮演重要角色,因此细胞内NAD+水平的过度消耗将导致能量代谢衰竭；同时NAD+消耗也将导致继发的细胞损伤如ROS增加、线粒体膜电位降低、细胞核及DNA的损伤等,这些变化将最终导致细胞凋亡及死亡。因此,NAD+水平的降低是Parthanatos中细胞死亡的主要原因,而维持细胞内的NAD+水平,有可能明显降低细胞死亡率。研究表明,外源性NAD+能够通过细胞膜上的P2X7受体门控通道进入细胞,使细胞内NAD+水平显著提升,并以此维持细胞能量代谢而减少细胞死亡率。
既然氧化应激能激活PARP-1,而布比卡因能导致细胞ROS增加,且在布比卡因所致的细胞死亡中存在caspase延迟现象,我们有理由推测：在布比卡因所致的细胞死亡中,存在Parthanatos,即因PARP-1的激活而导致的细胞内NAD+水平下降及由此导致细胞死亡；外源性的NAD能维持细胞内NAD+水平并由此减少布比卡因所致的细胞ROS生成增加、线粒体膜电位降低,进而减少细胞凋亡的发生。
本研究拟应用SH-SY5Y细胞培养技术,通过检测细胞内PARP-1的表达、PAR含量变化及细胞内NAD+水平的变化,初步明确布比卡因所致的神经毒性中是否存在Parthanatos;同时通过外源性NAD对布比卡因所致的细胞内ROS含量增加、线粒体膜电位降低、细胞核损伤及细胞凋亡的影响,确定外源性NAD是否有助于减轻布比卡因的神经毒性。其目的在于从新的角度探讨在布比卡因神经毒性的机制,为临床上防治局麻药的神经毒性提供实验依据。
第1章布比卡因神经毒性是否与多聚腺嘌呤二核苷酸核糖聚合酶-1(Poly(ADP-ribose) polymerase-1, PARP-1)激活有关
目的明确布比卡因是否导致SH-SY5Y细胞PARP-1激活,其神经毒性是否与激活PARP-1有关。
方法以1-10mM布比卡因培养液处理SH-SY5Y细胞30分钟-6小时,以Western-blot法检测细胞内PARP-1及其代谢产物-多聚腺苷二磷酸核糖(Poly-(ADP) ribose, PAR)的表达情况；以100nM、200nM的PARP-1抑制剂-PJ34培养液与5mM布比卡因培养液共同处理细胞30分钟,或单独以5mM布比卡因处理细胞30分钟,光镜下观察其对布比卡因所致细胞形态改变的影响,并以CCK-8法其对细胞存活率的影响。
计量资料以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。不同浓度布比卡因处理30分钟后细胞内PARP-1及PAR的表达、1mM布比卡因处理0-6小时后细胞内PAPR-1及PAR的表达采用完全随机单因素方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)；PJ34对布比卡因处理后细胞存活率的影响采用两因素析因设计资料方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)。P<0.05为差异有统计学意义。
结果以1～10mM布比卡因处理SH-SY5Y细胞30分钟后,各组细胞PARP-1蛋白表达水平不同,其中5mM及10mM处理组细胞内PARP-1蛋白的表达明显高于对照组,差异有统计学意义(P=0.011,P=0.006)；各组细胞PAR含量不同,2mM、5mM及10mM处理组均明显高于对照组(P=0.029,P=0.032,P=0.008)。
以1mM布比卡因处理细胞0-6小时后,各组细胞PARP-1蛋白表达水平不同,其中布比卡因处理1小时、2小时及4小时后,PARP-1蛋白表达明显高于对照组(P=0.013、P=0.001、P=0.002)；各组PAR含量不同,在布比卡因处理2小时、4小时及6小时后高于对照组(P=0.003,P=0.004,P=0.033)。
以5mM布比卡因处理30分钟之后,光镜下可见细胞出现胞体变圆、皱缩、突触减少甚至消失及细胞贴壁性差等形态学变化,提示细胞受到明显的损伤,PARP-1抑制剂PJ34干预后,较少出现上述变化；CCK-8细胞存活率检测显示：PJ34处理和布比卡因处理之间存在交互效应(F=7.608,P=0.001),PJ34100nM+5mM布比卡因组及PJ34200nM+5mM布比卡因组细胞存活率分别达到60.01±16.71%、74.00±22.35%,明显高于5mM布比卡因处理组的39.14±12.46%(P=0.007,P=0.000)。
结论布比卡因能导致SH-SY5Y细胞内PARP-1的激活,PARP-1激活与布比卡因的神经毒性有关。
第2章布比卡因的神经细胞毒性是否与细胞内NAD+水平降低相关
目的观察布比卡因是否降低细胞内NAD+水平,细胞内NAD+水平下降是否由PARP-1激活引起,布比卡因神经细胞毒性是否与细胞内NAD+水平降低有关。
方法以1～10mM布比卡因培养液处理SH-SY5Y细胞30分钟～7小时,以酶循环法检测SH-SY5Y细胞内NAD+含量,并以CCK-8法检测相同处理条件下SH-SY5Y细胞的存活率；以1～10mM利多卡因培养液处理SH-SY5Y细胞30分钟,以酶循环法检测SH-SY5Y细胞内NAD+含量,以CCK-8法检测细胞死亡率；以100nM、200nM的PARP-1抑制剂-PJ34培养液与5mM布比卡因共孵育30分钟,或单独以5mM布比卡因处理细胞30分钟,以酶循环法检测对照组、5mM布比卡因组、100nM PJ34组及200nM PJ34组细胞内NAD+水平的变化；以0.1～10mM NAD培养液预处理SH-SY5Y细胞30分钟,再以5mM布比卡因细胞30分钟,以酶循环法检测各组细胞内NAD+水平的变化；并以100μMP2X7受体抑制剂-Ox-ATP培养液预处理SH-SY5Y细胞30分钟后,再以0-10mM NAD培养液处理细胞30分钟,或直接以0～10mM NAD培养液处理细胞30分钟,以酶循环法检测细胞内NAD+水平；以1～10mM NAD培养液对SH-SY5Y细胞进行预处理或后处理,分别再以2mM、5mM及10mM布比卡因培养液处理30分钟,以CCK-8法检测各组细胞死亡率。
计量资料以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。1mM布比卡因处理30分钟～7小时后细胞内NAD+水平的变化及细胞存活率、不同浓度布比卡因、利多卡因处理后细胞内NAD+水平变化及存活率、PJ34干预对布比卡因所致细胞内NAD+水平降低的影响、外源性NAD对细胞内NAD+水平及细胞存活率的影响,均采用完全随机单因素方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)；Ox-ATP对细胞内NAD水平的影响、外源性NAD预处理或后处理对布比卡因处理后细胞毒性的影响采用两因素析因设计资料方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)。P<0.05为差异有统计学意义。
结果1mM布比卡因处理后3小时,细胞内NAD+水平开始明显下降,由处理前的100.38±11.87%下降至57.40±3.91%(P=0.000),此后保持逐渐下降的趋势,至处理后7小时,细胞内NAD+水平降至处理前的27.8±8.47%(P=0.000)；不同浓度布比卡因处理30分钟后,细胞内NAD+呈浓度依赖性下降(F=16.827,P=0.004),以2mM、5mM、10mM布比卡因处理细胞30分钟后,细胞内NAD+水平分别降至对照组的21.50±3.15%、25.73±7.22%及16.07±13.93%,明显低于对照组(P=0.043、0.031、0.015)。
相同处理条件下,细胞存活率的变化较为缓和：1mM布比卡因处理各时点细胞存活率无明显差异；1～10mM布比卡因处理后,细胞存活率随着布比卡因浓度的增高而降低(F=130.140,P=0.000),其中2mM、5mM、10mM布比卡因组,细胞存活率分别降至对照组的49.44%±8.55%、35.75±15.83%及25.58±4.45%,明显低于对照组(P=0.000、0.000、0.000)。在利多卡因处理组,以10mM利多卡因处理细胞30分钟后,细胞内NAD+水平降至对照组的41.57±1.73%,明显低于对照组(P=0.004)；以5mM、10mM利多卡因处理细胞30分钟后,细胞存活率分别降至对照组的86.56%±11.28%、71.86±12.29%,明显低于对照组(P=0.048、0.000)。
以PJ34与5mM布比卡因共同处理细胞后,NAD+水平较5mM布比卡因组明显升高,其中以100nM PJ34及5mM布比卡因处理细胞30分钟后,细胞内NAD+水平虽上升至对照组的69.25±25.25%,但未明显高于单独布比卡因组(P=0.072)；以200nM PJ34及5mM布比卡因处理细胞30分钟后,细胞内NAD+水平升高至对照组的80.81±21.66%,明显高于单独布比卡因处理组(P=0.026),且与对照组相比,差异无统计学意义(P=0.318)。
以NAD预处理30分钟之后再以5mM布比卡因处理,细胞内NAD+水平下降较5mM布比卡因处理组缓和,且与对照组相比,差异无统计学意义。其中以2.5mM、5mM、10mM NAD预处理的细胞,其细胞内NAD+水平分别达到对照组的85.87+11.82%、89.21±11.55%及105.05±58.82%,明显高于单独以布比卡因处理的细胞(P=0.043、0.033、0.008)。
未行Ox-ATP预处理的细胞,以5mM、10mM NAD处理细胞后,细胞内NAD+水平分别达到对照组的192.87+27.68%及279.37±20.00%,明显高于对照组(P=0.003,P=0.000),且明显高于相同NAD处理条件下的Ox-ATP预处理组细胞(t=-3.248, P=0.031、t=-14.923, P=0.000)。
以不同浓度NAD预处理或后处理干预后,细胞存活率明显高于单独以2mM、5mM、10mM布比卡因处理的细胞,且预处理效果好于后处理。
结论布比卡因能通过激活PARP-1引起细胞内NAD+水平降低；外源性NAD能通过P2X7受体门控通道进入细胞,并提高细胞内NAD+水平；布比卡因所致的NAD+下降早于细胞死亡的发生,外源性NAD能提高布比卡因处理后的细胞存活率。上述结果提示NAD+水平降低由PARP-1激活引起,且与布比卡因的神经细胞毒性有关。
第3章外源性NAD是否能减轻布比卡因所致的细胞损伤
目的观察不同处理条件下,NAD+对布比卡因所致的细胞内ROS增加、线粒体膜电位衰减及核损伤的影响,并明确外源性NAD是否能由此减少布比卡因所致的细胞凋亡。
方法以1～10mM NAD预处理SH-SY5Y细胞30分钟,然后以1mM布比卡因处理3小时,以流式细胞仪(FCM)检测细胞内DCFH-DA探针含量,由此确定细胞内ROS含量；以FCM检测JC-1多聚体／单体比值,以观察线粒体膜电位的变化；以FCM检测Annexin V-FITC及PI染色后各组细胞的凋亡率,以Hochest33258核染色观察核固缩率。
结果以1mM布比卡因处理3小时后,细胞内ROS含量为对照组的1.67±0.15倍,明显高于对照组(P=0.000)；以1～10mM NAD预处理30分钟后,再以相同条件的布比卡因处理SH-SY5Y细胞,细胞内ROS含量分别为对照组的1.43±0.09倍、1.43±0.05倍、1.46±0.13倍及1.20±0.07倍,明显低于单独布比卡因处理组(P=0.017、P＝0.017、P=0.033、P=0.000)。
布比卡因处理组JC-1多聚体/单体比值为9.49±1.85,明显低于对照组的36.98±6.32(P=0.000)；以5mM、10mM NAD预处理组细胞JC-1多聚体/单体比值升则至33.19±12.95、46.09±10.24,与对照组相比差异无统计学意义(P=0.579,P=0.192),且明显高于单独布比卡因处理组(P=0.003,P=0.000)。
布比卡因处理组核固缩发生率高达22.49±2.23%,表现为多处、散在的高亮蓝色荧光,明显高于对照组的6.29±2.12%(P=0.000)；而以2.5mM、5mM及10mM NAD预处理后,核固缩率明显降低,分别降至8.09±2.87%、7.00±1.39%及6.53±0.24%,与对照组相比无明显差异(P=0.319、P=0.688、P=0.894),且明显低于单独布比卡因处理组(P=0.000、P=0.000、P=0.000)
1mM布比卡因处理组细胞早期凋亡率为18.73±1.66%,明显高于对照组的5.40±0.69%,1mM、2.5mM及5mM NAD预处理组细胞早期凋亡率分别为10.87±0.49%、7.80±0.61%及7.10±0.17%,已明显低于布比卡因处理组(P=0.000, P=0.000,P=0.000),10mM NAD预处理组细胞早期凋亡率降至6.10±0.52%,与对照组相比,差异无统计学意义(P=0.323),且明显低于布比卡因处理组(P=0.000);1mM布比卡因处理组细胞晚期凋亡率为5.70±0.70%,明显高于对照组的1.00±0.30%,1mM NAD预处理组细胞晚期凋亡率明显低于单独布比卡因处理组(P=0.000),2.5mM、5mM及10mM NAD预处理组细胞晚期凋亡率分别降至1.67±0.67%、1.50±0.30%及1.27±0.39%,与对照组比较,差异无统计学意义(P=0.247,P=0.379,P=0.3247),且明显低于单独布比卡因处理组(P=0.000,P=0.000,P=0.000)。
结论NAD预处理、后处理均能减少布比卡因所致的ROS生产增加、线粒体膜电位降低及核固缩等细胞损伤,并最终减少了布比卡因所致的细胞凋亡,且预处理组效果优于后处理组。
Local anesthetics(LA) is commonly used in nerve block and postoperative analgesia, however, the reports of the toxic effects induced by LA are also gradually increasing with its extensive application. The cytotoxic of LA therefore become a research hotspot in recent years, and researchers have sought to clarify its mechanism so as to guide the treatment of peripheral nerve injury caused by LA.
Previous study has shown that bupivacaine is the most toxic local anesthetic, which can cause apoptosis and necrosis by uncoupling of oxidative phosphorylation, increasing intracellular reactive oxygen(ROS) production and decreasing adenosine triphosphate(ATP) generation. Apotosis is a form of programmed cell death, characterized by caspase-3/7activation and subsequent cytoactive relevant protein and special morphological changes of apoptosis. There are two signal transduction pathways in classic apoptosis:extrinsic pathway and intrinsic pathway. The former, namely the former death receptor pathway, is induced by the cell surface death receptors, such as tumor necrosis factor receptor (TNF-R) and actives caspase-8/9mainly. The latter is also known as mitochondria pathway, actives caspase-9by release mitochondrial cytochrome-c to the cytoplasm. Both pathways will eventually activate caspase-3and caspase-7.Unami A etc have proven that both caspase-8and caspase-9are activated and relative to formation of apoptosis body and DNA fragments, which means that caspase dependent internal and extrinsic apoptotic apoptotic pathways are associated with the cyotoxicity induced by bupivacaine. However, Perez etc demonstrated a delayed caspase activation in SH-SY5Y cell with bupivacaine treatment. They found nearly50%cell death immediately after10-min bupivacaine treatment (1mM), and the caspase was activated3hours later. Perez ascribed this kind of cell death to necrosis. However, this classification of cell death basing on the morphology is found too simple with the development of research on the cell death. Cell death can be divided into two categories at present, one is unprogrammed cell death, and the other is programmed cell death. The former is necrosis namely, characterized by invalid signal transduction inhibitors interrupt, and is a passived death. The latter is initiative cell death, which can be blocked by intracellular signal transduction inhibitors. The programmed cell death can also be divided into two categories:caspase-dependent and caspase-independent programmed cell death. The former is apoptosis namely, and the latter is characterized by an initiative death independent of caspase activation, including autophagy programmed cell death, paraptosis and oncosis. Then, whether the study of Perez suggests that there is caspase-independent programmed cell death in bupivacaine-induced cell death?
Parthanatos is a newly discovered caspase-independent programmed cell death. Parthanatos has the characteristic of Poly (ADP-ribose) polymerase-1(PARP-1) dependent, and mainly manifests as PARP-1activation and subsequently intracellular nicotinamide adenine dinucleotide (NAD+) decrease.
PARP-1activation plays a pivotal role in Parthanatos.PARP-1is a widely studied intracellular enzyme belonging to PARP family, and accounts for more than90%enzyme activity of PARP family. Under physiology condition, PARP-1is activated by DNA fragment, and mainly exerts role in DNA repairase by combining with injured DNA. However, under pathological condition such as excitotoxicity, oxidative stress, and ischemia-reperfusion, PARP-1is overactivated and produces poly(ADP) ribose(PAR) by hydrolyzing NAD+. As NAD+plays an important role in energy metabolism, the intracellular NAD+decrease thus will lead to energy failure. Meanwhile, the NAD+depletion will also induce subsequent cellular damages like ROS production increase, mitochondrial depolarization, nucleus and DNA injury, and so on, which eventually result in cell apoptosis and death. Therefore, intracellular NAD+level decline is the primary cause leading to cell death in Parthanatos, and the maintenance of intracellular NAD+content will decrease cell mortality obviously. Previous studies have shown exogenous NAD increases intracellular NAD+level markedly by entering cells through P2Xy-gated channel, and for this reason, exogenous NAD will maintain cellular energy metabolism and reduce cell mortality. Since PARP-1can be activated by oxidative stress, and there are intracellular ROS increase and delayed-caspase activation in cell death induced by bupivacaine, we speculate that Parthanatos is present in cell death induced by bupivacaine, in other words, intracellular NAD+decline resulting from PARP-1activation leads to cell death induced by bupivacaine. Exogenous NAD will maintain intracellular NAD+level, and thence reduces ROS production, mitochondrial depolarization, and then reduce the occurrence of apoptosis.
In this study, we determines whether Parthanatos presents in bupivacaine-induced neurotoxicity by examination of intracellular PARP-1expression, and detection of PAR and NAD+contents by application of SH-SY5Ycell culture technology. We also determine the effect of exogenous NAD on intracellular ROS increase, mitochondrial depolarization, nucleus injury and apoptosis resulting from bupivacaine, in order to confirm whether exogenous NAD contributes to alleviate bupivacaine-induced neurotoxicity. The aim of this study is to seek the mechanism of bupivacaine-induced neurotoxicity with a new point of view, and provide the experimental basis for the prevention and treatment of clinical LA neurotoxicity.
Chapter1Whether the Poly(ADP-ribose) polymerase-1(PARP-1) activation involves in the neurotoxicity of bupivacaine?
Objective To determine whether bupivacaine induces PARP-1activation in SH-SY5Ycell, and whether PARP-1activation involves in its neurotoxicity.
Methods SH-SY5Y cells were treated with bupivacaine at various concentration (1to10mM) for indicated times (30minutes to6hours), and the expression level of intracellular PARP-1and its metabolite-PAR were detected by western-blot analysis. SH-SY5Ycells were treated with100nM、200nM PJ34(PARP-1inhibitor) for30minutes, with or without5mM bupivacaine treatment, the effect of PJ34on bupivacaine-induced morphological changes was detected by light microscopy, and cell viability was detected by CCK-8.
Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment. The intracellular PARP-1protein and PAR expression levels were analyzed by one-way ANOVA, and mutiple comparisons test were perform by LSD(homogenous) or Dunnett's T3(heterogeneity of variance).The effect of PJ34on cell viability after bupivacaine treatment was analyzed by factorial design ANOVA, multiple comparisons tests were performed by LSD (homogeneous variance) or Dunnett's T3(heterogeneity of variance). A probability value of P<0.05was considered to be statistically significant.
Results Differences in the expression level of PARP-1among different experimental groups receiving30-min treatment of bupivacaine at various concentrations ranging from1to10mM were found to be statistically significant, and the PARP-1expression levels in5mM and10mM group were significantly higher than in control group (P=0.011, P=0.006) Differences in the PAR content among different experimental groups were also found to be statistically significant, the PAR contents detected in2mM,5mM and10mM group were significantly higher than in control group (P=0.029, P=0.032, P=0.008)
Differences in the expression level of PARP-1among different experimental groups with1mM bupivacaine treatment at indicated times from0to6hours were also found to be statistically significant, and the PARP-1expression levels detected after1h,2h, and4h bupivacaine treatment were significantly higher than untreated control (P=0.013、P=0.001、P=0.002), Differences in the PAR content among different experimental groups were also found to be statistically significant, the PAR contents detected after2h,4h, and6h bupivacaine treatment were significantly higher than in control group(P=0.003, P=0.004, P=0.033)
The cells exhibited representative morphological changes including round and shrunken shapes, diminishment and disappearance of neurites, and diminished wall-adhesion capacity were detected by light microscopy after30treatment with5mM bupivacaine, which suggest an obvious cellular damage. These morphological changes were much fewer in cells co-incubated with PJ34and bupivacaine than in bupivacaine-treated group. The treatment of PJ34and bupivacaine had interactive effect to the cell viability detected by CCK-8(F=7.608, P=0.001).The cell viabilities in100nM and200nM PJ34-treated group were60.01±16.71%and74.00±22.35%, which were higher than in bupivacaine group(39.14±12.46%)(P=0.007, P=0.000)
Conclusion Bupivacaine induces PARP-1activation in SH-SY5Y cell, and PARP-1activation involves in bupivacaine-induced neurotoxicity.
Chapter2Whether the intracellular NAD+decrease involves in bupivacaine-induced neurotoxicity.
Objective To investigate whether bupivacaine induces decline of intracellular NAD+level, and whether NAD+decline results from PARP-1activation, whether the NAD+level decrease involves in bupivacaine-induced neurotoxicity.
Methods SH-SY5Y cell were treated with bupivacaine at various concentration (1to10mM) for indicated times(30minutes to7hours). The intracellular NAD+content was detected by enzymatic cycling technology, and cell viability was detected by CCK-8. SH-SY5Y cell were treated with lidocaine at various concentration (1to10mM) for30minutes, and the intracellular NAD+content was detected by enzymatic cycling technology, and cell viability was detected by CCK-8. SH-SY5Y cell were incubated with100nM,200nM PJ34(PARP-1inhibitor), with or without5mM bupivacaine treatment, and the intracellular NAD levels in5mM bupivacaine group,100nM and200nM PJ34group were measured by enzymatic cycling technology. SH-SY5Y cells were treated with exogenous NAD for30minutes at different concentrations in the range of0.1-10mM, and next, received30-min5mM bupivacaine treatment. Then the intracellular NAD+levels in all groups were examined by enzymatic cycling technology. SH-SY5Y cells were treated with exogenous NAD for30minutes at different concentrations in the range of1-10mM in the present or absent of100μM Ox-ATP(P2X7receptor inhibitor), and then, the intracellular NAD+levels in all groups were examined by enzymatic cycling technology. SH-SY5Y cells were treated with30-min exogenous NAD pre-incubation or post-incubation, and next, underwent30-min bupivacaine treatment at concentrations of2mM,5mM, and10mM, the cell viabilities in all groups were detected then by CCK-8.
Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment. The intracellular NAD+level and cell viability after1mM bupivacaine treatment for indicated times(30minutes to7hours), and after30-min treatment of bupivacaine or lidocaine at various concentrations, the effect of PJ34on intracellular NAD+level decline induced by bupivacaine, the effect of exogenous NAD on intracellular NAD+level and cell viability, were analyzed by one-way ANOVA, and mutiple comparisons test were perform by LSD(homogenous) or Dunnett's T3(heterogeneity of variance).The effect of Ox-ATP on intracellular NAD+level, the effect of exogenous NAD pre-treatment or post-treatment on bupivacaine-induced cytotoxicity were analyzed by factorial design ANOVA, multiple comparisons tests were performed by LSD (homogeneous variance) or Dunnett's T3(heterogeneity of variance). A probability value of P<0.05was considered to be statistically significant.
Result The intracellular NAD+level significantly reduced to57.40q3.91%from100.38±11.87%(bupivacaine untreated)(P=0.000),and followed by gradual decline, and the intracellular NAD+level reduced to27.8±8.47%after7-hr bupivacaine treatment (P=0.000).The intracellular NAD+level decreased in a concentration-dependent manner after SH-SY5Y cells treated with bupivacaine at different concentrations (F=16.827, P=0.004),and the NAD+content significantly decreased to21.50±3.15%,25.73±7.22%, and16.07±13.93%, after SH-SY5Y cells received2mM,5mM,10mM bupivacaine treatment for30minutes, respectively, compared with untreated control (P=0.043、0.031、0.015)
Upon same conditions, bupivacaine caused a modest decline of cell viability. There were no significant differences among different experimental groups with1mM bupivacaine treatment at indicated times from0to7hours. And bupivacaine caused a concentration-dependent decrease of viability at concentrations in range of1to10mM. The cell viability significantly declined to49.44%±8.55%,35.75±15.83%, and25.58±4.45%, respectively, at the concentration of2mM,5mM, and10mM, compared with untreated control (P=0.000.0.000、0.000).The intracellular NAD±level significantly declined to41.57+1.73%after30-min lidocaine treatment, compared with control group (P=0.004), and the cell viability significantly declined to86.56%±11.28%、71.86±12.29%when cells received5mM and10mM lidocaine treatment for30minutes, compared with control group(P=0.048、0.000)
The intracellular NAD±level was significantly higher in SH-SY5Y cells receiving PJ34and bupivacaine co-incubation than in bupivacaine-treated group. The intracellular NAD±level increased to69.25±25.25%in cells treated with bupivacaine in the present of100nM PJ34, however, which is not significantly higher than in cells treated with bupivacaine alone(JP=0.07). The NAD+level increased to80.81±21.66%in cells treated with bupivacaine in the present of200nM PJ34, which was significantly higher than in cells treated with bupivacaine alone (P=0.072), and showed no significantly difference when compare with untreated control(P=0.318)
A milder intracellular NAD+level decline was observed in cells treated with5mM bupivacaine in present of NAD pre-treatment compared to cells treated with5mM bupivacaine alone, and the NAD level in cells upon NAD pre-treatment was not significantly different from that in untreated control. The cellular NAD+levels reached85.87±11.82%,89.21±11.55%, and105.05±58.82%in2.5mM,5mM,10mM NAD pre-treatment groups respectively, compared with control group, and were significantly higher than in bupivacaine group (P=0.043、0.033、0.008)
The intracellular NAD±levels reached192.87±27.68%and279.37±20.00% respectively, in cells treated with5mM and10mM NAD in the absence of Ox-ATP, which were significantly higher than in control group (P=0.003, P=0.000), and in Ox-ATP pre-treatment group upon same NAD treatment (t=-3.248, P=0.031、 t=-14.923, P=0.000)
The cell viability was higher in cells receiving NAD pre-treatment or post-treatment than in cell treated with2mM,5mM,10mM bupivacaine alone, and the pre-treatment group achieved a better result.
Conclusion Bupivacaine causes intracellular NAD+level decline by activating PARP-1. Exogenous NAD enters cell through P2X7-gated channel, and increases cellular NAD+level. Cellular NAD+level decline resulting from bupivacaine treatment precedes cell death, and exogenous NAD improves declined cell viability induced by bupivacaine treatment. Taken together, these results suggest that intracellular NAD+level decline resulting from PARP-1activation involves in bupivacaine-induced neurotoxicity.
Chapter3Whether exogenous NAD alleviates cellular damage induced by bupivacaine.
Objective To investigate the effect of NAD+upon different treatment on intracellular ROS increase, mitochondrial depolarization, and nucleus injury induced by bupivacaine, and determine whether exogenous NAD thence reduces apoptosis resulting from bupivacaine treatment.
Methods SH-SY5Y cells were treated with1mM bupivacaine for3hours with30-min NAD pre-treatment at different concentrations ranging from1to10mM. Intracellular ROS content was assayed with DCFH-DA probe by FCM, and mitochondrial membrane potential was shown as ratio of JC-1ploymer over monomer detected by FCM, and cell apoptosis was detected with Annexin V-FITC and PI staining by FCM, and nuclear condensation was determined by Hochest33258staining.
Results The intracellular ROS content significantly increased to1.67±0.15fold after1mM bupivacaine treatment for3hours, compared with untreated control (P=0.000).Cellular ROS were1.43±0.09,1.43±0.05,1.46±0.13,and1.20±0.07fold in cells received same bupivacaine treatment upon NAD pretreatment at different concentrations(1to10mM), respectively, which were significantly lower than in bupivacaine-treated group (P=0.017、P=0.017、P=0.033、P=0.000)
The ratio of polymer over monomer declined to9.49±1.85in bupivacaine-treated group, significantly lower than in control group(36.98±6.32)(P=0.000).The ratio raised to33.19±12.95and46.09±10.24in5mM and10mM NAD-pretreated group, which were not significantly different from control group (P=0.579、P=0.192) and significantly higher than in bupivacaine treated group (P=0.003, P=0.000)
The percentage of damaged nuclei was22.49±2.23%in bupivacaine-treated group, exhibiting as mutiple and scattered brighter blue fluorescence, which was higher than in control group(6.29±2.12%)(P=0.000),the percentage significantly decreased in2.5mM,5mM, and10mM NAD pretreated group, which were not significantly different from control group (P=0.319、P=0.688、P=0.894) and significantly higher than in bupivacaine-treated group (P=0.000、P=0.000、 P=0.000)
The early apoptosis rates was18.73±1.66%in1mM bupivacaine-treated group, significantly higher than in control group(5.40±0.69%). The early apoptosis rates was10.87±0.49%,7.80±0.61%, and7.10±0.17%in1mM,2.5mM, and5mM NAD-pretreated group, respectively, significantly lower than in bupivacaine-treated group (P=0.000, P=0.000, P=0.000).The rate decreased to6.10±0.52%in10mM NAD-pretreated group, which was not significantly different from control group (P=0.323) and significantly lower than in bupivacaine treated group (P=0.000)
The late apoptosis rates was5.70±0.70%in1mM bupivacaine-treated group, significantly higher than in control group(1.00±0.30%). The late apoptosis rates was significantly lower than in bupivacaine-treated group (P=0.000). The rate decreased to 1.67±0.67%、1.50±0.30%及1.27±0.39%in2.5mM,5mM, and10mM NAD-pretreated group, respectively, which was not significantly different from control group (P=0.247, P=0.379, P=0.3247) and significantly lower than in bupivacaine-treated group (P=0.000, P=0.000, P=0.000)
Conclusion NAD pretreatment and posttreatment alleviate intracellular cellular damage including ROS increase, mitochondrial depolarization, and nucleus condensation induced by bupivacaine, and reduces apoptosis resulting from bupivacaine treatment eventually. NAD pre-treatment group achieved a better result than post-treatment.
研究表明,布比卡因是最具毒性的局麻药,可能通过氧化磷酸化解偶联(uncoupling of oxidative phosphorylation)、活性氧(reactive oxygen, ROS)生成增加及三磷酸腺苷(adenosine triphosphate, ATP)生成减少等引起细胞的凋亡及坏死。凋亡是细胞程序性死亡的一种,主要通过激活半胱天冬酶-3、7(caspase-3、7)而裂解细胞活性相关蛋白,并引起凋亡特有的形态学改变。经典的细胞凋亡信号转导通路可以分为两种：分别是外源性通路(extrinsic pathway)和内源性通路(intrinsic pathway)。前者即死亡受体途径,由细胞表面的死亡受体如肿瘤坏死因子受体(tumour necrosis factor receptor, TNF-R)等介导,以激活caspase-8、10为主；后者也称为线粒体途径(mitochondrial pathway),主要是通过线粒体释放细胞色素-c(cytochrome-c)至胞浆并激活caspase-9。两条通路最终都能激活caspase-3和caspase-7。 Unami等研究证实,在布比卡因所致的细胞毒性中,caspase-8、9及caspase-3均被激活,且与凋亡小体、DNA碎片的形成正相关,这意味着caspase依赖性内、外源性凋亡通路均与布比卡因的细胞毒性有关。但Perez等研究发现,布比卡因处理的细胞虽然有caspase的激活,却存在caspase的激活滞后的现象,他们用1mM的布比卡因处理SH-SY5Y细胞10分钟,即有接近50%的细胞死亡,而3小时后才出现caspase的激活。Perez等把这种先于caspase激活的细胞死亡归结为坏死,但随着对细胞死亡研究的深入,Perez所依据的这种基于形态学的“凋亡-坏死”的细胞死亡分类显得过于简单。目前学者们将细胞死亡分为两大类,一类为非程序性死亡,另一类则为程序性死亡。前者即坏死,其特点是不能为细胞信号转导抑制剂所阻断,为被动死亡；后者能为细胞信号转导抑制剂所阻断,为主动死亡。程序性细胞死亡又可以分为两大类：caspase依赖性和非caspase依赖性,前者即典型的凋亡,后者包括自吞噬性程序性细胞死亡、副凋亡(paraptosis)、胀亡等,其最大特点是细胞的主动死亡不依赖caspase的激活。那么,Perez的实验结果是否提示在布比卡因所致的细胞死亡中,存在非caspase依赖性的细胞程序性死亡途径?
Parthanatos是新近发现的非caspase依赖性细胞程序性死亡。Parthanatos的特点为多聚腺嘌呤二核苷酸核糖聚合酶(Poly(ADP-ribose) polymerase-1, PARP-1)依赖性,其主要表现为PARP-1激活及随后的细胞内烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide, NAD+)水平的降低。
在Parthanatos中,PARP-1的激活起到关键作用。PARP-1是PARP家族成员中被广泛研究的细胞内的酶类,大约占细胞内PARP活性的90%以上,生理条件下,PARP-1能为DNA碎片等激活,与受损的DNA结合,主要起到DNA修复酶的作用；在病理条件下,如兴奋性毒素损伤、氧化应激、缺血再灌注损伤中,PARP-1出现过度激活,并水解NAD+,同时生成PAR多聚体。由于NAD+在能量代谢中扮演重要角色,因此细胞内NAD+水平的过度消耗将导致能量代谢衰竭；同时NAD+消耗也将导致继发的细胞损伤如ROS增加、线粒体膜电位降低、细胞核及DNA的损伤等,这些变化将最终导致细胞凋亡及死亡。因此,NAD+水平的降低是Parthanatos中细胞死亡的主要原因,而维持细胞内的NAD+水平,有可能明显降低细胞死亡率。研究表明,外源性NAD+能够通过细胞膜上的P2X7受体门控通道进入细胞,使细胞内NAD+水平显著提升,并以此维持细胞能量代谢而减少细胞死亡率。
既然氧化应激能激活PARP-1,而布比卡因能导致细胞ROS增加,且在布比卡因所致的细胞死亡中存在caspase延迟现象,我们有理由推测：在布比卡因所致的细胞死亡中,存在Parthanatos,即因PARP-1的激活而导致的细胞内NAD+水平下降及由此导致细胞死亡；外源性的NAD能维持细胞内NAD+水平并由此减少布比卡因所致的细胞ROS生成增加、线粒体膜电位降低,进而减少细胞凋亡的发生。
本研究拟应用SH-SY5Y细胞培养技术,通过检测细胞内PARP-1的表达、PAR含量变化及细胞内NAD+水平的变化,初步明确布比卡因所致的神经毒性中是否存在Parthanatos;同时通过外源性NAD对布比卡因所致的细胞内ROS含量增加、线粒体膜电位降低、细胞核损伤及细胞凋亡的影响,确定外源性NAD是否有助于减轻布比卡因的神经毒性。其目的在于从新的角度探讨在布比卡因神经毒性的机制,为临床上防治局麻药的神经毒性提供实验依据。
第1章布比卡因神经毒性是否与多聚腺嘌呤二核苷酸核糖聚合酶-1(Poly(ADP-ribose) polymerase-1, PARP-1)激活有关
目的明确布比卡因是否导致SH-SY5Y细胞PARP-1激活,其神经毒性是否与激活PARP-1有关。
方法以1-10mM布比卡因培养液处理SH-SY5Y细胞30分钟-6小时,以Western-blot法检测细胞内PARP-1及其代谢产物-多聚腺苷二磷酸核糖(Poly-(ADP) ribose, PAR)的表达情况；以100nM、200nM的PARP-1抑制剂-PJ34培养液与5mM布比卡因培养液共同处理细胞30分钟,或单独以5mM布比卡因处理细胞30分钟,光镜下观察其对布比卡因所致细胞形态改变的影响,并以CCK-8法其对细胞存活率的影响。
计量资料以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。不同浓度布比卡因处理30分钟后细胞内PARP-1及PAR的表达、1mM布比卡因处理0-6小时后细胞内PAPR-1及PAR的表达采用完全随机单因素方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)；PJ34对布比卡因处理后细胞存活率的影响采用两因素析因设计资料方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)。P<0.05为差异有统计学意义。
结果以1～10mM布比卡因处理SH-SY5Y细胞30分钟后,各组细胞PARP-1蛋白表达水平不同,其中5mM及10mM处理组细胞内PARP-1蛋白的表达明显高于对照组,差异有统计学意义(P=0.011,P=0.006)；各组细胞PAR含量不同,2mM、5mM及10mM处理组均明显高于对照组(P=0.029,P=0.032,P=0.008)。
以1mM布比卡因处理细胞0-6小时后,各组细胞PARP-1蛋白表达水平不同,其中布比卡因处理1小时、2小时及4小时后,PARP-1蛋白表达明显高于对照组(P=0.013、P=0.001、P=0.002)；各组PAR含量不同,在布比卡因处理2小时、4小时及6小时后高于对照组(P=0.003,P=0.004,P=0.033)。
以5mM布比卡因处理30分钟之后,光镜下可见细胞出现胞体变圆、皱缩、突触减少甚至消失及细胞贴壁性差等形态学变化,提示细胞受到明显的损伤,PARP-1抑制剂PJ34干预后,较少出现上述变化；CCK-8细胞存活率检测显示：PJ34处理和布比卡因处理之间存在交互效应(F=7.608,P=0.001),PJ34100nM+5mM布比卡因组及PJ34200nM+5mM布比卡因组细胞存活率分别达到60.01±16.71%、74.00±22.35%,明显高于5mM布比卡因处理组的39.14±12.46%(P=0.007,P=0.000)。
结论布比卡因能导致SH-SY5Y细胞内PARP-1的激活,PARP-1激活与布比卡因的神经毒性有关。
第2章布比卡因的神经细胞毒性是否与细胞内NAD+水平降低相关
目的观察布比卡因是否降低细胞内NAD+水平,细胞内NAD+水平下降是否由PARP-1激活引起,布比卡因神经细胞毒性是否与细胞内NAD+水平降低有关。
方法以1～10mM布比卡因培养液处理SH-SY5Y细胞30分钟～7小时,以酶循环法检测SH-SY5Y细胞内NAD+含量,并以CCK-8法检测相同处理条件下SH-SY5Y细胞的存活率；以1～10mM利多卡因培养液处理SH-SY5Y细胞30分钟,以酶循环法检测SH-SY5Y细胞内NAD+含量,以CCK-8法检测细胞死亡率；以100nM、200nM的PARP-1抑制剂-PJ34培养液与5mM布比卡因共孵育30分钟,或单独以5mM布比卡因处理细胞30分钟,以酶循环法检测对照组、5mM布比卡因组、100nM PJ34组及200nM PJ34组细胞内NAD+水平的变化；以0.1～10mM NAD培养液预处理SH-SY5Y细胞30分钟,再以5mM布比卡因细胞30分钟,以酶循环法检测各组细胞内NAD+水平的变化；并以100μMP2X7受体抑制剂-Ox-ATP培养液预处理SH-SY5Y细胞30分钟后,再以0-10mM NAD培养液处理细胞30分钟,或直接以0～10mM NAD培养液处理细胞30分钟,以酶循环法检测细胞内NAD+水平；以1～10mM NAD培养液对SH-SY5Y细胞进行预处理或后处理,分别再以2mM、5mM及10mM布比卡因培养液处理30分钟,以CCK-8法检测各组细胞死亡率。
计量资料以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。1mM布比卡因处理30分钟～7小时后细胞内NAD+水平的变化及细胞存活率、不同浓度布比卡因、利多卡因处理后细胞内NAD+水平变化及存活率、PJ34干预对布比卡因所致细胞内NAD+水平降低的影响、外源性NAD对细胞内NAD+水平及细胞存活率的影响,均采用完全随机单因素方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)；Ox-ATP对细胞内NAD水平的影响、外源性NAD预处理或后处理对布比卡因处理后细胞毒性的影响采用两因素析因设计资料方差分析,组间比较采用LSD法(方差齐)或Dunnett's T3法(方差不齐)。P<0.05为差异有统计学意义。
结果1mM布比卡因处理后3小时,细胞内NAD+水平开始明显下降,由处理前的100.38±11.87%下降至57.40±3.91%(P=0.000),此后保持逐渐下降的趋势,至处理后7小时,细胞内NAD+水平降至处理前的27.8±8.47%(P=0.000)；不同浓度布比卡因处理30分钟后,细胞内NAD+呈浓度依赖性下降(F=16.827,P=0.004),以2mM、5mM、10mM布比卡因处理细胞30分钟后,细胞内NAD+水平分别降至对照组的21.50±3.15%、25.73±7.22%及16.07±13.93%,明显低于对照组(P=0.043、0.031、0.015)。
相同处理条件下,细胞存活率的变化较为缓和：1mM布比卡因处理各时点细胞存活率无明显差异；1～10mM布比卡因处理后,细胞存活率随着布比卡因浓度的增高而降低(F=130.140,P=0.000),其中2mM、5mM、10mM布比卡因组,细胞存活率分别降至对照组的49.44%±8.55%、35.75±15.83%及25.58±4.45%,明显低于对照组(P=0.000、0.000、0.000)。在利多卡因处理组,以10mM利多卡因处理细胞30分钟后,细胞内NAD+水平降至对照组的41.57±1.73%,明显低于对照组(P=0.004)；以5mM、10mM利多卡因处理细胞30分钟后,细胞存活率分别降至对照组的86.56%±11.28%、71.86±12.29%,明显低于对照组(P=0.048、0.000)。
以PJ34与5mM布比卡因共同处理细胞后,NAD+水平较5mM布比卡因组明显升高,其中以100nM PJ34及5mM布比卡因处理细胞30分钟后,细胞内NAD+水平虽上升至对照组的69.25±25.25%,但未明显高于单独布比卡因组(P=0.072)；以200nM PJ34及5mM布比卡因处理细胞30分钟后,细胞内NAD+水平升高至对照组的80.81±21.66%,明显高于单独布比卡因处理组(P=0.026),且与对照组相比,差异无统计学意义(P=0.318)。
以NAD预处理30分钟之后再以5mM布比卡因处理,细胞内NAD+水平下降较5mM布比卡因处理组缓和,且与对照组相比,差异无统计学意义。其中以2.5mM、5mM、10mM NAD预处理的细胞,其细胞内NAD+水平分别达到对照组的85.87+11.82%、89.21±11.55%及105.05±58.82%,明显高于单独以布比卡因处理的细胞(P=0.043、0.033、0.008)。
未行Ox-ATP预处理的细胞,以5mM、10mM NAD处理细胞后,细胞内NAD+水平分别达到对照组的192.87+27.68%及279.37±20.00%,明显高于对照组(P=0.003,P=0.000),且明显高于相同NAD处理条件下的Ox-ATP预处理组细胞(t=-3.248, P=0.031、t=-14.923, P=0.000)。
以不同浓度NAD预处理或后处理干预后,细胞存活率明显高于单独以2mM、5mM、10mM布比卡因处理的细胞,且预处理效果好于后处理。
结论布比卡因能通过激活PARP-1引起细胞内NAD+水平降低；外源性NAD能通过P2X7受体门控通道进入细胞,并提高细胞内NAD+水平；布比卡因所致的NAD+下降早于细胞死亡的发生,外源性NAD能提高布比卡因处理后的细胞存活率。上述结果提示NAD+水平降低由PARP-1激活引起,且与布比卡因的神经细胞毒性有关。
第3章外源性NAD是否能减轻布比卡因所致的细胞损伤
目的观察不同处理条件下,NAD+对布比卡因所致的细胞内ROS增加、线粒体膜电位衰减及核损伤的影响,并明确外源性NAD是否能由此减少布比卡因所致的细胞凋亡。
方法以1～10mM NAD预处理SH-SY5Y细胞30分钟,然后以1mM布比卡因处理3小时,以流式细胞仪(FCM)检测细胞内DCFH-DA探针含量,由此确定细胞内ROS含量；以FCM检测JC-1多聚体／单体比值,以观察线粒体膜电位的变化；以FCM检测Annexin V-FITC及PI染色后各组细胞的凋亡率,以Hochest33258核染色观察核固缩率。
结果以1mM布比卡因处理3小时后,细胞内ROS含量为对照组的1.67±0.15倍,明显高于对照组(P=0.000)；以1～10mM NAD预处理30分钟后,再以相同条件的布比卡因处理SH-SY5Y细胞,细胞内ROS含量分别为对照组的1.43±0.09倍、1.43±0.05倍、1.46±0.13倍及1.20±0.07倍,明显低于单独布比卡因处理组(P=0.017、P＝0.017、P=0.033、P=0.000)。
布比卡因处理组JC-1多聚体/单体比值为9.49±1.85,明显低于对照组的36.98±6.32(P=0.000)；以5mM、10mM NAD预处理组细胞JC-1多聚体/单体比值升则至33.19±12.95、46.09±10.24,与对照组相比差异无统计学意义(P=0.579,P=0.192),且明显高于单独布比卡因处理组(P=0.003,P=0.000)。
布比卡因处理组核固缩发生率高达22.49±2.23%,表现为多处、散在的高亮蓝色荧光,明显高于对照组的6.29±2.12%(P=0.000)；而以2.5mM、5mM及10mM NAD预处理后,核固缩率明显降低,分别降至8.09±2.87%、7.00±1.39%及6.53±0.24%,与对照组相比无明显差异(P=0.319、P=0.688、P=0.894),且明显低于单独布比卡因处理组(P=0.000、P=0.000、P=0.000)
1mM布比卡因处理组细胞早期凋亡率为18.73±1.66%,明显高于对照组的5.40±0.69%,1mM、2.5mM及5mM NAD预处理组细胞早期凋亡率分别为10.87±0.49%、7.80±0.61%及7.10±0.17%,已明显低于布比卡因处理组(P=0.000, P=0.000,P=0.000),10mM NAD预处理组细胞早期凋亡率降至6.10±0.52%,与对照组相比,差异无统计学意义(P=0.323),且明显低于布比卡因处理组(P=0.000);1mM布比卡因处理组细胞晚期凋亡率为5.70±0.70%,明显高于对照组的1.00±0.30%,1mM NAD预处理组细胞晚期凋亡率明显低于单独布比卡因处理组(P=0.000),2.5mM、5mM及10mM NAD预处理组细胞晚期凋亡率分别降至1.67±0.67%、1.50±0.30%及1.27±0.39%,与对照组比较,差异无统计学意义(P=0.247,P=0.379,P=0.3247),且明显低于单独布比卡因处理组(P=0.000,P=0.000,P=0.000)。
结论NAD预处理、后处理均能减少布比卡因所致的ROS生产增加、线粒体膜电位降低及核固缩等细胞损伤,并最终减少了布比卡因所致的细胞凋亡,且预处理组效果优于后处理组。
Local anesthetics(LA) is commonly used in nerve block and postoperative analgesia, however, the reports of the toxic effects induced by LA are also gradually increasing with its extensive application. The cytotoxic of LA therefore become a research hotspot in recent years, and researchers have sought to clarify its mechanism so as to guide the treatment of peripheral nerve injury caused by LA.
Previous study has shown that bupivacaine is the most toxic local anesthetic, which can cause apoptosis and necrosis by uncoupling of oxidative phosphorylation, increasing intracellular reactive oxygen(ROS) production and decreasing adenosine triphosphate(ATP) generation. Apotosis is a form of programmed cell death, characterized by caspase-3/7activation and subsequent cytoactive relevant protein and special morphological changes of apoptosis. There are two signal transduction pathways in classic apoptosis:extrinsic pathway and intrinsic pathway. The former, namely the former death receptor pathway, is induced by the cell surface death receptors, such as tumor necrosis factor receptor (TNF-R) and actives caspase-8/9mainly. The latter is also known as mitochondria pathway, actives caspase-9by release mitochondrial cytochrome-c to the cytoplasm. Both pathways will eventually activate caspase-3and caspase-7.Unami A etc have proven that both caspase-8and caspase-9are activated and relative to formation of apoptosis body and DNA fragments, which means that caspase dependent internal and extrinsic apoptotic apoptotic pathways are associated with the cyotoxicity induced by bupivacaine. However, Perez etc demonstrated a delayed caspase activation in SH-SY5Y cell with bupivacaine treatment. They found nearly50%cell death immediately after10-min bupivacaine treatment (1mM), and the caspase was activated3hours later. Perez ascribed this kind of cell death to necrosis. However, this classification of cell death basing on the morphology is found too simple with the development of research on the cell death. Cell death can be divided into two categories at present, one is unprogrammed cell death, and the other is programmed cell death. The former is necrosis namely, characterized by invalid signal transduction inhibitors interrupt, and is a passived death. The latter is initiative cell death, which can be blocked by intracellular signal transduction inhibitors. The programmed cell death can also be divided into two categories:caspase-dependent and caspase-independent programmed cell death. The former is apoptosis namely, and the latter is characterized by an initiative death independent of caspase activation, including autophagy programmed cell death, paraptosis and oncosis. Then, whether the study of Perez suggests that there is caspase-independent programmed cell death in bupivacaine-induced cell death?
Parthanatos is a newly discovered caspase-independent programmed cell death. Parthanatos has the characteristic of Poly (ADP-ribose) polymerase-1(PARP-1) dependent, and mainly manifests as PARP-1activation and subsequently intracellular nicotinamide adenine dinucleotide (NAD+) decrease.
PARP-1activation plays a pivotal role in Parthanatos.PARP-1is a widely studied intracellular enzyme belonging to PARP family, and accounts for more than90%enzyme activity of PARP family. Under physiology condition, PARP-1is activated by DNA fragment, and mainly exerts role in DNA repairase by combining with injured DNA. However, under pathological condition such as excitotoxicity, oxidative stress, and ischemia-reperfusion, PARP-1is overactivated and produces poly(ADP) ribose(PAR) by hydrolyzing NAD+. As NAD+plays an important role in energy metabolism, the intracellular NAD+decrease thus will lead to energy failure. Meanwhile, the NAD+depletion will also induce subsequent cellular damages like ROS production increase, mitochondrial depolarization, nucleus and DNA injury, and so on, which eventually result in cell apoptosis and death. Therefore, intracellular NAD+level decline is the primary cause leading to cell death in Parthanatos, and the maintenance of intracellular NAD+content will decrease cell mortality obviously. Previous studies have shown exogenous NAD increases intracellular NAD+level markedly by entering cells through P2Xy-gated channel, and for this reason, exogenous NAD will maintain cellular energy metabolism and reduce cell mortality. Since PARP-1can be activated by oxidative stress, and there are intracellular ROS increase and delayed-caspase activation in cell death induced by bupivacaine, we speculate that Parthanatos is present in cell death induced by bupivacaine, in other words, intracellular NAD+decline resulting from PARP-1activation leads to cell death induced by bupivacaine. Exogenous NAD will maintain intracellular NAD+level, and thence reduces ROS production, mitochondrial depolarization, and then reduce the occurrence of apoptosis.
In this study, we determines whether Parthanatos presents in bupivacaine-induced neurotoxicity by examination of intracellular PARP-1expression, and detection of PAR and NAD+contents by application of SH-SY5Ycell culture technology. We also determine the effect of exogenous NAD on intracellular ROS increase, mitochondrial depolarization, nucleus injury and apoptosis resulting from bupivacaine, in order to confirm whether exogenous NAD contributes to alleviate bupivacaine-induced neurotoxicity. The aim of this study is to seek the mechanism of bupivacaine-induced neurotoxicity with a new point of view, and provide the experimental basis for the prevention and treatment of clinical LA neurotoxicity.
Chapter1Whether the Poly(ADP-ribose) polymerase-1(PARP-1) activation involves in the neurotoxicity of bupivacaine?
Objective To determine whether bupivacaine induces PARP-1activation in SH-SY5Ycell, and whether PARP-1activation involves in its neurotoxicity.
Methods SH-SY5Y cells were treated with bupivacaine at various concentration (1to10mM) for indicated times (30minutes to6hours), and the expression level of intracellular PARP-1and its metabolite-PAR were detected by western-blot analysis. SH-SY5Ycells were treated with100nM、200nM PJ34(PARP-1inhibitor) for30minutes, with or without5mM bupivacaine treatment, the effect of PJ34on bupivacaine-induced morphological changes was detected by light microscopy, and cell viability was detected by CCK-8.
Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment. The intracellular PARP-1protein and PAR expression levels were analyzed by one-way ANOVA, and mutiple comparisons test were perform by LSD(homogenous) or Dunnett's T3(heterogeneity of variance).The effect of PJ34on cell viability after bupivacaine treatment was analyzed by factorial design ANOVA, multiple comparisons tests were performed by LSD (homogeneous variance) or Dunnett's T3(heterogeneity of variance). A probability value of P<0.05was considered to be statistically significant.
Results Differences in the expression level of PARP-1among different experimental groups receiving30-min treatment of bupivacaine at various concentrations ranging from1to10mM were found to be statistically significant, and the PARP-1expression levels in5mM and10mM group were significantly higher than in control group (P=0.011, P=0.006) Differences in the PAR content among different experimental groups were also found to be statistically significant, the PAR contents detected in2mM,5mM and10mM group were significantly higher than in control group (P=0.029, P=0.032, P=0.008)
Differences in the expression level of PARP-1among different experimental groups with1mM bupivacaine treatment at indicated times from0to6hours were also found to be statistically significant, and the PARP-1expression levels detected after1h,2h, and4h bupivacaine treatment were significantly higher than untreated control (P=0.013、P=0.001、P=0.002), Differences in the PAR content among different experimental groups were also found to be statistically significant, the PAR contents detected after2h,4h, and6h bupivacaine treatment were significantly higher than in control group(P=0.003, P=0.004, P=0.033)
The cells exhibited representative morphological changes including round and shrunken shapes, diminishment and disappearance of neurites, and diminished wall-adhesion capacity were detected by light microscopy after30treatment with5mM bupivacaine, which suggest an obvious cellular damage. These morphological changes were much fewer in cells co-incubated with PJ34and bupivacaine than in bupivacaine-treated group. The treatment of PJ34and bupivacaine had interactive effect to the cell viability detected by CCK-8(F=7.608, P=0.001).The cell viabilities in100nM and200nM PJ34-treated group were60.01±16.71%and74.00±22.35%, which were higher than in bupivacaine group(39.14±12.46%)(P=0.007, P=0.000)
Conclusion Bupivacaine induces PARP-1activation in SH-SY5Y cell, and PARP-1activation involves in bupivacaine-induced neurotoxicity.
Chapter2Whether the intracellular NAD+decrease involves in bupivacaine-induced neurotoxicity.
Objective To investigate whether bupivacaine induces decline of intracellular NAD+level, and whether NAD+decline results from PARP-1activation, whether the NAD+level decrease involves in bupivacaine-induced neurotoxicity.
Methods SH-SY5Y cell were treated with bupivacaine at various concentration (1to10mM) for indicated times(30minutes to7hours). The intracellular NAD+content was detected by enzymatic cycling technology, and cell viability was detected by CCK-8. SH-SY5Y cell were treated with lidocaine at various concentration (1to10mM) for30minutes, and the intracellular NAD+content was detected by enzymatic cycling technology, and cell viability was detected by CCK-8. SH-SY5Y cell were incubated with100nM,200nM PJ34(PARP-1inhibitor), with or without5mM bupivacaine treatment, and the intracellular NAD levels in5mM bupivacaine group,100nM and200nM PJ34group were measured by enzymatic cycling technology. SH-SY5Y cells were treated with exogenous NAD for30minutes at different concentrations in the range of0.1-10mM, and next, received30-min5mM bupivacaine treatment. Then the intracellular NAD+levels in all groups were examined by enzymatic cycling technology. SH-SY5Y cells were treated with exogenous NAD for30minutes at different concentrations in the range of1-10mM in the present or absent of100μM Ox-ATP(P2X7receptor inhibitor), and then, the intracellular NAD+levels in all groups were examined by enzymatic cycling technology. SH-SY5Y cells were treated with30-min exogenous NAD pre-incubation or post-incubation, and next, underwent30-min bupivacaine treatment at concentrations of2mM,5mM, and10mM, the cell viabilities in all groups were detected then by CCK-8.
Measurement data were presented as mean±standard deviation, and were analysed by SPSS17.0software for statistical treatment. The intracellular NAD+level and cell viability after1mM bupivacaine treatment for indicated times(30minutes to7hours), and after30-min treatment of bupivacaine or lidocaine at various concentrations, the effect of PJ34on intracellular NAD+level decline induced by bupivacaine, the effect of exogenous NAD on intracellular NAD+level and cell viability, were analyzed by one-way ANOVA, and mutiple comparisons test were perform by LSD(homogenous) or Dunnett's T3(heterogeneity of variance).The effect of Ox-ATP on intracellular NAD+level, the effect of exogenous NAD pre-treatment or post-treatment on bupivacaine-induced cytotoxicity were analyzed by factorial design ANOVA, multiple comparisons tests were performed by LSD (homogeneous variance) or Dunnett's T3(heterogeneity of variance). A probability value of P<0.05was considered to be statistically significant.
Result The intracellular NAD+level significantly reduced to57.40q3.91%from100.38±11.87%(bupivacaine untreated)(P=0.000),and followed by gradual decline, and the intracellular NAD+level reduced to27.8±8.47%after7-hr bupivacaine treatment (P=0.000).The intracellular NAD+level decreased in a concentration-dependent manner after SH-SY5Y cells treated with bupivacaine at different concentrations (F=16.827, P=0.004),and the NAD+content significantly decreased to21.50±3.15%,25.73±7.22%, and16.07±13.93%, after SH-SY5Y cells received2mM,5mM,10mM bupivacaine treatment for30minutes, respectively, compared with untreated control (P=0.043、0.031、0.015)
Upon same conditions, bupivacaine caused a modest decline of cell viability. There were no significant differences among different experimental groups with1mM bupivacaine treatment at indicated times from0to7hours. And bupivacaine caused a concentration-dependent decrease of viability at concentrations in range of1to10mM. The cell viability significantly declined to49.44%±8.55%,35.75±15.83%, and25.58±4.45%, respectively, at the concentration of2mM,5mM, and10mM, compared with untreated control (P=0.000.0.000、0.000).The intracellular NAD±level significantly declined to41.57+1.73%after30-min lidocaine treatment, compared with control group (P=0.004), and the cell viability significantly declined to86.56%±11.28%、71.86±12.29%when cells received5mM and10mM lidocaine treatment for30minutes, compared with control group(P=0.048、0.000)
The intracellular NAD±level was significantly higher in SH-SY5Y cells receiving PJ34and bupivacaine co-incubation than in bupivacaine-treated group. The intracellular NAD±level increased to69.25±25.25%in cells treated with bupivacaine in the present of100nM PJ34, however, which is not significantly higher than in cells treated with bupivacaine alone(JP=0.07). The NAD+level increased to80.81±21.66%in cells treated with bupivacaine in the present of200nM PJ34, which was significantly higher than in cells treated with bupivacaine alone (P=0.072), and showed no significantly difference when compare with untreated control(P=0.318)
A milder intracellular NAD+level decline was observed in cells treated with5mM bupivacaine in present of NAD pre-treatment compared to cells treated with5mM bupivacaine alone, and the NAD level in cells upon NAD pre-treatment was not significantly different from that in untreated control. The cellular NAD+levels reached85.87±11.82%,89.21±11.55%, and105.05±58.82%in2.5mM,5mM,10mM NAD pre-treatment groups respectively, compared with control group, and were significantly higher than in bupivacaine group (P=0.043、0.033、0.008)
The intracellular NAD±levels reached192.87±27.68%and279.37±20.00% respectively, in cells treated with5mM and10mM NAD in the absence of Ox-ATP, which were significantly higher than in control group (P=0.003, P=0.000), and in Ox-ATP pre-treatment group upon same NAD treatment (t=-3.248, P=0.031、 t=-14.923, P=0.000)
The cell viability was higher in cells receiving NAD pre-treatment or post-treatment than in cell treated with2mM,5mM,10mM bupivacaine alone, and the pre-treatment group achieved a better result.
Conclusion Bupivacaine causes intracellular NAD+level decline by activating PARP-1. Exogenous NAD enters cell through P2X7-gated channel, and increases cellular NAD+level. Cellular NAD+level decline resulting from bupivacaine treatment precedes cell death, and exogenous NAD improves declined cell viability induced by bupivacaine treatment. Taken together, these results suggest that intracellular NAD+level decline resulting from PARP-1activation involves in bupivacaine-induced neurotoxicity.
Chapter3Whether exogenous NAD alleviates cellular damage induced by bupivacaine.
Objective To investigate the effect of NAD+upon different treatment on intracellular ROS increase, mitochondrial depolarization, and nucleus injury induced by bupivacaine, and determine whether exogenous NAD thence reduces apoptosis resulting from bupivacaine treatment.
Methods SH-SY5Y cells were treated with1mM bupivacaine for3hours with30-min NAD pre-treatment at different concentrations ranging from1to10mM. Intracellular ROS content was assayed with DCFH-DA probe by FCM, and mitochondrial membrane potential was shown as ratio of JC-1ploymer over monomer detected by FCM, and cell apoptosis was detected with Annexin V-FITC and PI staining by FCM, and nuclear condensation was determined by Hochest33258staining.
Results The intracellular ROS content significantly increased to1.67±0.15fold after1mM bupivacaine treatment for3hours, compared with untreated control (P=0.000).Cellular ROS were1.43±0.09,1.43±0.05,1.46±0.13,and1.20±0.07fold in cells received same bupivacaine treatment upon NAD pretreatment at different concentrations(1to10mM), respectively, which were significantly lower than in bupivacaine-treated group (P=0.017、P=0.017、P=0.033、P=0.000)
The ratio of polymer over monomer declined to9.49±1.85in bupivacaine-treated group, significantly lower than in control group(36.98±6.32)(P=0.000).The ratio raised to33.19±12.95and46.09±10.24in5mM and10mM NAD-pretreated group, which were not significantly different from control group (P=0.579、P=0.192) and significantly higher than in bupivacaine treated group (P=0.003, P=0.000)
The percentage of damaged nuclei was22.49±2.23%in bupivacaine-treated group, exhibiting as mutiple and scattered brighter blue fluorescence, which was higher than in control group(6.29±2.12%)(P=0.000),the percentage significantly decreased in2.5mM,5mM, and10mM NAD pretreated group, which were not significantly different from control group (P=0.319、P=0.688、P=0.894) and significantly higher than in bupivacaine-treated group (P=0.000、P=0.000、 P=0.000)
The early apoptosis rates was18.73±1.66%in1mM bupivacaine-treated group, significantly higher than in control group(5.40±0.69%). The early apoptosis rates was10.87±0.49%,7.80±0.61%, and7.10±0.17%in1mM,2.5mM, and5mM NAD-pretreated group, respectively, significantly lower than in bupivacaine-treated group (P=0.000, P=0.000, P=0.000).The rate decreased to6.10±0.52%in10mM NAD-pretreated group, which was not significantly different from control group (P=0.323) and significantly lower than in bupivacaine treated group (P=0.000)
The late apoptosis rates was5.70±0.70%in1mM bupivacaine-treated group, significantly higher than in control group(1.00±0.30%). The late apoptosis rates was significantly lower than in bupivacaine-treated group (P=0.000). The rate decreased to 1.67±0.67%、1.50±0.30%及1.27±0.39%in2.5mM,5mM, and10mM NAD-pretreated group, respectively, which was not significantly different from control group (P=0.247, P=0.379, P=0.3247) and significantly lower than in bupivacaine-treated group (P=0.000, P=0.000, P=0.000)
Conclusion NAD pretreatment and posttreatment alleviate intracellular cellular damage including ROS increase, mitochondrial depolarization, and nucleus condensation induced by bupivacaine, and reduces apoptosis resulting from bupivacaine treatment eventually. NAD pre-treatment group achieved a better result than post-treatment.
引文
[1]KANAI Y, KATSUKI H, TAKASAKI M. Graded, irreversible changes in crayfish giant axon as manifestations of lidocaine neurotoxicity in vitro [J]. Anesth Analg,1998,86(3):569-73.
[2]KASABA T, ONIZUKA S, TAKASAKI M. Procaine and mepivacaine have less toxicity in vitro than other clinically used local anesthetics [J]. Anesth Analg, 2003,97(1):85-90, table of contents.
[3]RAD WAN I A, SAITO S, GOTO F. The neurotoxicity of local anesthetics on growing neurons:a comparative study of lidocaine, bupivacaine, mepivacaine, and ropivacaine [J]. Anesth Analg,2002,94(2):319-24, table of contents.
[4]FRIEDERICH P, SCHMITZ T P. Lidocaine-induced cell death in a human model of neuronal apoptosis [J]. Eur J Anaesthesiol,2002,19(8):564-70.
[5]PEREZ-CASTRO R, PATEL S, GARAVITO-AGUILAR Z V, et al. Cytotoxicity of Local Anesthetics in Human Neuronal Cells [J]. Anesthesia & Analgesia, 2009,108(3):997-1007.
[6]PARK C J, PARK S A, YOON T G, et al. Bupivacaine Induces Apoptosis via ROS in the Schwann Cell Line [J]. Journal of Dental Research,2005,84(9): 852-7.
[7]WANG S, XING Z, VOSLER P S, et al. Cellular NAD replenishment confers marked neuroprotection against ischemic cell death:role of enhanced DNA repair [J]. Stroke,2008,39(9):2587-95.
[8]UNAMI A, SHINOHARA Y, ICHIKAWA T, et al. Biochemical and microarray analyses of bupivacaine-induced apoptosis [J]. J Toxicol Sci,2003,28(2):77-94.
[9]LEIST M, JAATTELA M. Four deaths and a funeral:from caspases to alternative mechanisms [J]. Nat Rev Mol Cell Biol,2001,2(8):589-98.
[10]FADEEL B, ORRENIUS S, ZHIVOTOVSKY B. Apoptosis in human disease:a new skin for the old ceremony? [J]. Biochem Biophys Res Commun,1999, 266(3):699-717.
[11]GIANSANTI V, DONA F, TILLHON M, et al. PARP inhibitors:new tools to protect from inflammation [J]. Biochem Pharmacol,2010,80(12):1869-77.
[12]HARRAZ M M, DAWSON T M, DAWSON V L. Advances in neuronal cell death 2007 [J]. Stroke,2008,39(2):286-8.
[13]YU S W, WANG H, POITRAS M F, et al. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor [J]. Science, 2002,297(5579):259-63.
[14]YU S W, ANDRABI S A, WANG H, et al. Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death [J]. Proceedings of the National Academy of Sciences,2006,103(48):18314-9.
[15]WANG Y, KIM N S, HAINCE J F, et al. Poly(ADP-Ribose) (PAR) Binding to Apoptosis-inducing Factor Is Critical for PAR Polymerase-1-Dependent Cell Death (Parthanatos) [J]. Science Signaling,2011,4(167):ra20-ra.
[16]ALANO C C, GARNIER P, YING W, et al. NAD+ Depletion Is Necessary and Sufficient forPoly(ADP-Ribose) Polymerase-1-Mediated Neuronal Death [J]. Journal of Neuroscience,2010,30(8):2967-78.
[17]KIM S H, LU H F, ALANO C C. Neuronal Sirt3 protects against excitotoxic injury in mouse cortical neuron culture [J]. PLoS One,2011,6(3):e14731.
[18]ALANO C C, TRAN A, TAO R, et al. Differences among cell types in NAD(+) compartmentalization:a comparison of neurons, astrocytes, and cardiac myocytes [J]. J Neurosci Res,2007,85(15):3378-85.
[19]MORONI F. Poly(ADP-ribose)polymerase 1 (PARP-1) and postischemic brain damage [J]. Curr Opin Pharmacol,2008,8(1):96-103.
[20]PACHER P, SZABO C. Role of the peroxynitrite-poly(ADP-ribose) polymerase pathway in human disease [J]. Am J Pathol,2008,173(1):2-13.
[21]ALANO C C. Poly(ADP-ribose) Polymerase-1-mediated Cell Death in Astrocytes Requires NAD+Depletion and Mitochondrial Permeability Transition [J]. Journal of Biological Chemistry,2004,279(18):18895-902.
[22]WANG J. A local mechanism mediates NAD-dependent protection of axon degeneration [J]. The Journal of Cell Biology,2005,170(3):349-55.
[23]ARAKI T. Increased Nuclear NAD Biosynthesis and SIRT1 Activation Prevent Axonal Degeneration [J]. Science,2004,305(5686):1010-3.
[24]LORENZO H K, SUSIN S A, PENNINGER J, et al. Apoptosis inducing factor (AIF):a phylogenetically old, caspase-independent effector of cell death [J]. Cell Death Differ,1999,6(6):516-24.
[25]MATE M J, ORTIZ-LOMBARDIA M, BOITEL B, et al. The crystal structure of the mouse apoptosis-inducing factor AIF [J]. Nat Struct Biol,2002,9(6):442-6.
[26]ISHITSUKA K, HIDESHIMA T, HAMASAKI M, et al. Novel inosine monophosphate dehydrogenase inhibitor VX-944 induces apoptosis in multiple myeloma cells primarily via caspase-independent AIF/Endo G pathway [J]. Oncogene,2005,24(38):5888-96.
[27]CHIARUGI A, MOSKOWITZ M A. Cell biology. PARP-1--a perpetrator of apoptotic cell death? [J]. Science,2002,297(5579):200-1.
[28]LU J, XU S Y, ZHANG Q G, et al. Bupivacaine induces apoptosis via mitochondria and p38 MAPK dependent pathways [J]. European Journal of Pharmacology,2011,657(1-3):51-8.
[29]MA R, WANG X, LU C, et al. Dexamethasone attenuated bupivacaine-induced neuron injury in vitro through a threonine-serine protein kinase B-dependent mechanism [J]. Neuroscience,2010,167(2):329-42.
[30]YING W, SEVIGNY M B, CHEN Y, et al. Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death [J]. Proc Natl Acad Sci U S A, 2001,98(21):12227-32.
[31]FINKEL T. Oxidant signals and oxidative stress [J]. Curr Opin Cell Biol,2003, 15(2):247-54.
[32]OLEK R A, ZIOLKOWSKI W, KACZOR J J, et al. Antioxidant activity of NADH and its analogue--an in vitro study [J]. J Biochem Mol Biol,2004,37(4): 416-21.
[33]QUINTANA-CABRERA R, BOLANOS J P. Glutathione and gamma-glutamylcysteine in the antioxidant and survival functions of mitochondria [J]. Biochem Soc Trans,2013,41(1):106-10.
[34]SHI F, LI Y, WANG X. Molecular properties, functions, and potential applications of NAD kinases [J]. Acta Biochim Biophys Sin (Shanghai),2009, 41(5):352-61.
[35]ZHANG H, FORMAN H J. Glutathione synthesis and its role in redox signaling [J]. Semin Cell Dev Biol,2012,23(7):722-8.
[36]HIPKISS A R. NAD+Availability and Proteotoxicity [J]. NeuroMolecular Medicine,2009,11(2):97-100.
[37]DESAI K M, WU L. Free radical generation by methylglyoxal in tissues [J]. Drug Metabol Drug Interact,2008,23(1-2):151-73.
[38]SZTARK F, NOUETTE-GAULAIN K, MALGAT M, et al. Absence of stereospecific effects of bupivacaine isomers on heart mitochondrial bioenergetics [J]. Anesthesiology,2000,93(2):456-62.
[39]WANG J, HE Z. NAD and axon degeneration:from the Wlds gene to neurochemistry [J]. Cell Adh Migr,2009,3(1):77-87.
[40]JIANG X, WANG X. Cytochrome C-mediated apoptosis [J]. Annu Rev Biochem, 2004,73(87-106.
[41]SPERLAGH B, VIZI E S, WIRKNER K, et al. P2X7 receptors in the nervous system [J]. Prog Neurobiol,2006,78(6):327-46.
[42]CICCARONE V, SPENGLER B A, MEYERS M B, et al. Phenotypic diversification in human neuroblastoma cells:expression of distinct neural crest lineages [J]. Cancer Res,1989,49(1):219-25.
[43]XIE H R, HU L S, LI G Y. SH-SY5Y human neuroblastoma cell line:in vitro cell model of dopaminergic neurons in Parkinson's disease [J]. Chin Med J (Engl),2010,123(8):1086-92.
[44]BEILIN Y, HALPERN S. Focused review:ropivacaine versus bupivacaine for epidural labor analgesia [J]. Anesth Analg,2010,111(2):482-7.
[45]CHAN V W, PENG P, CHINYANGA H, et al. Determining minimum effective anesthetic concentration of hyperbaric bupivacaine for spinal anesthesia [J]. Anesth Analg,2000,90(5):1135-40.
[46]WANG Y, KIM N S, HAINCE J F, et al. Poly(ADP-ribose) (PAR) binding to apoptosis-inducing factor is critical for PAR polymerase-1-dependent cell death (parthanatos) [J]. Sci Signal,2011,4(167):ra20.
[47]LIU D, GHARAVI R, PITTA M, et al. Nicotinamide prevents NAD+depletion and protects neurons against excitotoxicity and cerebral ischemia:NAD+ consumption by SIRT1 may endanger energetically compromised neurons [J]. Neuromolecular Med,2009,11(1):28-42.
[48]MATTSON M P, LIU D. Energetics and oxidative stress in synaptic plasticity and neurodegenerative disorders [J]. Neuromolecular Med,2002,2(2):215-31.
[49]MASSUDI H, GRANT R, GUILLEMIN G J, et al. NAD+ metabolism and oxidative stress: the golden nucleotide on a crown of thorns [J]. Redox Report,2012,17(1):28-46.
[50]ANDRABI S A, KIM N S, YU S W, et al. Poly(ADP-ribose) (PAR) polymer is a death signal [J]. Proceedings of the National Academy of Sciences,2006, 103(48):18308-13.
[51]DAVID K K, ANDRABI S A, DAWSON T M, et al. Parthanatos, a messenger of death [J]. Front Biosci (Landmark Ed),2009,14(1116-28.
[52]EDINGER A L, THOMPSON C B. Death by design:apoptosis, necrosis and autophagy [J]. Curr Opin Cell Biol,2004,16(6):663-9.
[53]KROEMER G, GALLUZZI L, VANDENABEELE P, et al. Classification of cell death:recommendations of the Nomenclature Committee on Cell Death 2009 [J]. Cell Death Differ,2009,16(1):3-11.
[54]GALLUZZI L, KROEMER G. Necroptosis:a specialized pathway of programmed necrosis [J]. Cell,2008,135(7):1161-3.
[55]ROSENBAUM D M, DEGTEREV A, DAVID J, et al. Necroptosis, a novel form of caspase-independent cell death, contributes to neuronal damage in a retinal ischemia-reperfusion injury model [J]. J Neurosci Res,2010,88(7):1569-76.
[56]HONG S J, DAWSON T M, DAWSON V L. Nuclear and mitochondrial conversations in cell death:PARP-1 and AIF signaling [J]. Trends in Pharmacological Sciences,2004,25(5):259-64.
[57]SIEGEL C, MCCULLOUGH L D. NAD+depletion or PAR polymer formation: which plays the role of executioner in ischaemic cell death? [J]. Acta Physiologica,2011,203(1):225-34.
[58]SAIRANEN T, SZEPESI R, KARJALAINEN-LINDSBERG M-L, et al. Neuronal caspase-3 and PARP-1 correlate differentially with apoptosis and necrosis in ischemic human stroke [J]. Acta Neuropathologica,2009,118(4): 541-52.
[59]MAYSINGER D, AIKIN R, ROSENBERG L, et al. Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans [J]. Journal of Molecular Medicine,2004,82(6):389-97.
[60]SHIH M F, CHERNG J Y. Protective Effects of Chlorella-Derived Peptide Against UVC-Induced Cytotoxicity through Inhibition of Caspase-3 Activity and Reduction of the Expression of Phosphorylated FADD and Cleaved PARP-1 in Skin Fibroblasts [J]. Molecules,2012,17(12):9116-28.
[61]KIM M Y. Poly(ADP-ribosyl)ation by PARP-1:PAR-laying' NAD+ into a nuclear signal [J]. Genes & Development,2005,19(17):1951-67.
[62]LUO X, KRAUS W L. On PAR with PARP:cellular stress signaling through poly(ADP-ribose) and PARP-1 [J]. Genes & Development,2012,26(5):417-32.
[63]YING W, WEI G, WANG D, et al. Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia [J]. Front Biosci,2007,12(2728-34.
[64]YING W. NAD+/NADH and NADP+/NADPH in Cellular Functions and Cell Death:Regulation and Biological Consequences [J]. Antioxidants & Redox Signaling,2008,10(2):179-206.
[65]HIPKISS A R. NAD+availability and proteotoxicity [J]. Neuromolecular Med, 2009,11(2):97-100.
[66]ALANO C C, TRAN A, TAO R, et al. Differences among cell types in NAD+ compartmentalization:A comparison of neurons, astrocytes, and cardiac myocytes [J]. Journal of Neuroscience Research,2007,85(15):3378-85.
[67]HUANG F, VEMURI M C, SCHNEIDER J S. Modulation of ATP levels alters the mode of hydrogen peroxide-induced cell death in primary cortical cultures: effects of putative neuroprotective agents [J]. Brain Res,2004,997(1):79-88.
[68]LIU D, GHARAVIR, PITTA M, et al. Nicotinamide Prevents NAD+Depletion and Protects Neurons Against Excitotoxicity and Cerebral Ischemia:NAD+ Consumption by SIRT1 may Endanger Energetically Compromised Neurons [J]. NeuroMolecular Medicine,2009,11(1):28-42.
[69]LU J, XU S Y, ZHANG Q G, et al. Bupivacaine induces reactive oxygen species production via activation of the AMP-activated protein kinase-dependent pathway [J]. Pharmacology,2011,87(3-4):12.1-9.
[70]LU S C. Regulation of glutathione synthesis [J]. Mol Aspects Med,2009, 30(1-2):42-59.
[2]KASABA T, ONIZUKA S, TAKASAKI M. Procaine and mepivacaine have less toxicity in vitro than other clinically used local anesthetics [J]. Anesth Analg, 2003,97(1):85-90, table of contents.
[3]RAD WAN I A, SAITO S, GOTO F. The neurotoxicity of local anesthetics on growing neurons:a comparative study of lidocaine, bupivacaine, mepivacaine, and ropivacaine [J]. Anesth Analg,2002,94(2):319-24, table of contents.
[4]FRIEDERICH P, SCHMITZ T P. Lidocaine-induced cell death in a human model of neuronal apoptosis [J]. Eur J Anaesthesiol,2002,19(8):564-70.
[5]PEREZ-CASTRO R, PATEL S, GARAVITO-AGUILAR Z V, et al. Cytotoxicity of Local Anesthetics in Human Neuronal Cells [J]. Anesthesia & Analgesia, 2009,108(3):997-1007.
[6]PARK C J, PARK S A, YOON T G, et al. Bupivacaine Induces Apoptosis via ROS in the Schwann Cell Line [J]. Journal of Dental Research,2005,84(9): 852-7.
[7]WANG S, XING Z, VOSLER P S, et al. Cellular NAD replenishment confers marked neuroprotection against ischemic cell death:role of enhanced DNA repair [J]. Stroke,2008,39(9):2587-95.
[8]UNAMI A, SHINOHARA Y, ICHIKAWA T, et al. Biochemical and microarray analyses of bupivacaine-induced apoptosis [J]. J Toxicol Sci,2003,28(2):77-94.
[9]LEIST M, JAATTELA M. Four deaths and a funeral:from caspases to alternative mechanisms [J]. Nat Rev Mol Cell Biol,2001,2(8):589-98.
[10]FADEEL B, ORRENIUS S, ZHIVOTOVSKY B. Apoptosis in human disease:a new skin for the old ceremony? [J]. Biochem Biophys Res Commun,1999, 266(3):699-717.
[11]GIANSANTI V, DONA F, TILLHON M, et al. PARP inhibitors:new tools to protect from inflammation [J]. Biochem Pharmacol,2010,80(12):1869-77.
[12]HARRAZ M M, DAWSON T M, DAWSON V L. Advances in neuronal cell death 2007 [J]. Stroke,2008,39(2):286-8.
[13]YU S W, WANG H, POITRAS M F, et al. Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor [J]. Science, 2002,297(5579):259-63.
[14]YU S W, ANDRABI S A, WANG H, et al. Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death [J]. Proceedings of the National Academy of Sciences,2006,103(48):18314-9.
[15]WANG Y, KIM N S, HAINCE J F, et al. Poly(ADP-Ribose) (PAR) Binding to Apoptosis-inducing Factor Is Critical for PAR Polymerase-1-Dependent Cell Death (Parthanatos) [J]. Science Signaling,2011,4(167):ra20-ra.
[16]ALANO C C, GARNIER P, YING W, et al. NAD+ Depletion Is Necessary and Sufficient forPoly(ADP-Ribose) Polymerase-1-Mediated Neuronal Death [J]. Journal of Neuroscience,2010,30(8):2967-78.
[17]KIM S H, LU H F, ALANO C C. Neuronal Sirt3 protects against excitotoxic injury in mouse cortical neuron culture [J]. PLoS One,2011,6(3):e14731.
[18]ALANO C C, TRAN A, TAO R, et al. Differences among cell types in NAD(+) compartmentalization:a comparison of neurons, astrocytes, and cardiac myocytes [J]. J Neurosci Res,2007,85(15):3378-85.
[19]MORONI F. Poly(ADP-ribose)polymerase 1 (PARP-1) and postischemic brain damage [J]. Curr Opin Pharmacol,2008,8(1):96-103.
[20]PACHER P, SZABO C. Role of the peroxynitrite-poly(ADP-ribose) polymerase pathway in human disease [J]. Am J Pathol,2008,173(1):2-13.
[21]ALANO C C. Poly(ADP-ribose) Polymerase-1-mediated Cell Death in Astrocytes Requires NAD+Depletion and Mitochondrial Permeability Transition [J]. Journal of Biological Chemistry,2004,279(18):18895-902.
[22]WANG J. A local mechanism mediates NAD-dependent protection of axon degeneration [J]. The Journal of Cell Biology,2005,170(3):349-55.
[23]ARAKI T. Increased Nuclear NAD Biosynthesis and SIRT1 Activation Prevent Axonal Degeneration [J]. Science,2004,305(5686):1010-3.
[24]LORENZO H K, SUSIN S A, PENNINGER J, et al. Apoptosis inducing factor (AIF):a phylogenetically old, caspase-independent effector of cell death [J]. Cell Death Differ,1999,6(6):516-24.
[25]MATE M J, ORTIZ-LOMBARDIA M, BOITEL B, et al. The crystal structure of the mouse apoptosis-inducing factor AIF [J]. Nat Struct Biol,2002,9(6):442-6.
[26]ISHITSUKA K, HIDESHIMA T, HAMASAKI M, et al. Novel inosine monophosphate dehydrogenase inhibitor VX-944 induces apoptosis in multiple myeloma cells primarily via caspase-independent AIF/Endo G pathway [J]. Oncogene,2005,24(38):5888-96.
[27]CHIARUGI A, MOSKOWITZ M A. Cell biology. PARP-1--a perpetrator of apoptotic cell death? [J]. Science,2002,297(5579):200-1.
[28]LU J, XU S Y, ZHANG Q G, et al. Bupivacaine induces apoptosis via mitochondria and p38 MAPK dependent pathways [J]. European Journal of Pharmacology,2011,657(1-3):51-8.
[29]MA R, WANG X, LU C, et al. Dexamethasone attenuated bupivacaine-induced neuron injury in vitro through a threonine-serine protein kinase B-dependent mechanism [J]. Neuroscience,2010,167(2):329-42.
[30]YING W, SEVIGNY M B, CHEN Y, et al. Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death [J]. Proc Natl Acad Sci U S A, 2001,98(21):12227-32.
[31]FINKEL T. Oxidant signals and oxidative stress [J]. Curr Opin Cell Biol,2003, 15(2):247-54.
[32]OLEK R A, ZIOLKOWSKI W, KACZOR J J, et al. Antioxidant activity of NADH and its analogue--an in vitro study [J]. J Biochem Mol Biol,2004,37(4): 416-21.
[33]QUINTANA-CABRERA R, BOLANOS J P. Glutathione and gamma-glutamylcysteine in the antioxidant and survival functions of mitochondria [J]. Biochem Soc Trans,2013,41(1):106-10.
[34]SHI F, LI Y, WANG X. Molecular properties, functions, and potential applications of NAD kinases [J]. Acta Biochim Biophys Sin (Shanghai),2009, 41(5):352-61.
[35]ZHANG H, FORMAN H J. Glutathione synthesis and its role in redox signaling [J]. Semin Cell Dev Biol,2012,23(7):722-8.
[36]HIPKISS A R. NAD+Availability and Proteotoxicity [J]. NeuroMolecular Medicine,2009,11(2):97-100.
[37]DESAI K M, WU L. Free radical generation by methylglyoxal in tissues [J]. Drug Metabol Drug Interact,2008,23(1-2):151-73.
[38]SZTARK F, NOUETTE-GAULAIN K, MALGAT M, et al. Absence of stereospecific effects of bupivacaine isomers on heart mitochondrial bioenergetics [J]. Anesthesiology,2000,93(2):456-62.
[39]WANG J, HE Z. NAD and axon degeneration:from the Wlds gene to neurochemistry [J]. Cell Adh Migr,2009,3(1):77-87.
[40]JIANG X, WANG X. Cytochrome C-mediated apoptosis [J]. Annu Rev Biochem, 2004,73(87-106.
[41]SPERLAGH B, VIZI E S, WIRKNER K, et al. P2X7 receptors in the nervous system [J]. Prog Neurobiol,2006,78(6):327-46.
[42]CICCARONE V, SPENGLER B A, MEYERS M B, et al. Phenotypic diversification in human neuroblastoma cells:expression of distinct neural crest lineages [J]. Cancer Res,1989,49(1):219-25.
[43]XIE H R, HU L S, LI G Y. SH-SY5Y human neuroblastoma cell line:in vitro cell model of dopaminergic neurons in Parkinson's disease [J]. Chin Med J (Engl),2010,123(8):1086-92.
[44]BEILIN Y, HALPERN S. Focused review:ropivacaine versus bupivacaine for epidural labor analgesia [J]. Anesth Analg,2010,111(2):482-7.
[45]CHAN V W, PENG P, CHINYANGA H, et al. Determining minimum effective anesthetic concentration of hyperbaric bupivacaine for spinal anesthesia [J]. Anesth Analg,2000,90(5):1135-40.
[46]WANG Y, KIM N S, HAINCE J F, et al. Poly(ADP-ribose) (PAR) binding to apoptosis-inducing factor is critical for PAR polymerase-1-dependent cell death (parthanatos) [J]. Sci Signal,2011,4(167):ra20.
[47]LIU D, GHARAVI R, PITTA M, et al. Nicotinamide prevents NAD+depletion and protects neurons against excitotoxicity and cerebral ischemia:NAD+ consumption by SIRT1 may endanger energetically compromised neurons [J]. Neuromolecular Med,2009,11(1):28-42.
[48]MATTSON M P, LIU D. Energetics and oxidative stress in synaptic plasticity and neurodegenerative disorders [J]. Neuromolecular Med,2002,2(2):215-31.
[49]MASSUDI H, GRANT R, GUILLEMIN G J, et al. NAD+ metabolism and oxidative stress: the golden nucleotide on a crown of thorns [J]. Redox Report,2012,17(1):28-46.
[50]ANDRABI S A, KIM N S, YU S W, et al. Poly(ADP-ribose) (PAR) polymer is a death signal [J]. Proceedings of the National Academy of Sciences,2006, 103(48):18308-13.
[51]DAVID K K, ANDRABI S A, DAWSON T M, et al. Parthanatos, a messenger of death [J]. Front Biosci (Landmark Ed),2009,14(1116-28.
[52]EDINGER A L, THOMPSON C B. Death by design:apoptosis, necrosis and autophagy [J]. Curr Opin Cell Biol,2004,16(6):663-9.
[53]KROEMER G, GALLUZZI L, VANDENABEELE P, et al. Classification of cell death:recommendations of the Nomenclature Committee on Cell Death 2009 [J]. Cell Death Differ,2009,16(1):3-11.
[54]GALLUZZI L, KROEMER G. Necroptosis:a specialized pathway of programmed necrosis [J]. Cell,2008,135(7):1161-3.
[55]ROSENBAUM D M, DEGTEREV A, DAVID J, et al. Necroptosis, a novel form of caspase-independent cell death, contributes to neuronal damage in a retinal ischemia-reperfusion injury model [J]. J Neurosci Res,2010,88(7):1569-76.
[56]HONG S J, DAWSON T M, DAWSON V L. Nuclear and mitochondrial conversations in cell death:PARP-1 and AIF signaling [J]. Trends in Pharmacological Sciences,2004,25(5):259-64.
[57]SIEGEL C, MCCULLOUGH L D. NAD+depletion or PAR polymer formation: which plays the role of executioner in ischaemic cell death? [J]. Acta Physiologica,2011,203(1):225-34.
[58]SAIRANEN T, SZEPESI R, KARJALAINEN-LINDSBERG M-L, et al. Neuronal caspase-3 and PARP-1 correlate differentially with apoptosis and necrosis in ischemic human stroke [J]. Acta Neuropathologica,2009,118(4): 541-52.
[59]MAYSINGER D, AIKIN R, ROSENBERG L, et al. Inhibition of caspase-mediated PARP-1 cleavage results in increased necrosis in isolated islets of Langerhans [J]. Journal of Molecular Medicine,2004,82(6):389-97.
[60]SHIH M F, CHERNG J Y. Protective Effects of Chlorella-Derived Peptide Against UVC-Induced Cytotoxicity through Inhibition of Caspase-3 Activity and Reduction of the Expression of Phosphorylated FADD and Cleaved PARP-1 in Skin Fibroblasts [J]. Molecules,2012,17(12):9116-28.
[61]KIM M Y. Poly(ADP-ribosyl)ation by PARP-1:PAR-laying' NAD+ into a nuclear signal [J]. Genes & Development,2005,19(17):1951-67.
[62]LUO X, KRAUS W L. On PAR with PARP:cellular stress signaling through poly(ADP-ribose) and PARP-1 [J]. Genes & Development,2012,26(5):417-32.
[63]YING W, WEI G, WANG D, et al. Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia [J]. Front Biosci,2007,12(2728-34.
[64]YING W. NAD+/NADH and NADP+/NADPH in Cellular Functions and Cell Death:Regulation and Biological Consequences [J]. Antioxidants & Redox Signaling,2008,10(2):179-206.
[65]HIPKISS A R. NAD+availability and proteotoxicity [J]. Neuromolecular Med, 2009,11(2):97-100.
[66]ALANO C C, TRAN A, TAO R, et al. Differences among cell types in NAD+ compartmentalization:A comparison of neurons, astrocytes, and cardiac myocytes [J]. Journal of Neuroscience Research,2007,85(15):3378-85.
[67]HUANG F, VEMURI M C, SCHNEIDER J S. Modulation of ATP levels alters the mode of hydrogen peroxide-induced cell death in primary cortical cultures: effects of putative neuroprotective agents [J]. Brain Res,2004,997(1):79-88.
[68]LIU D, GHARAVIR, PITTA M, et al. Nicotinamide Prevents NAD+Depletion and Protects Neurons Against Excitotoxicity and Cerebral Ischemia:NAD+ Consumption by SIRT1 may Endanger Energetically Compromised Neurons [J]. NeuroMolecular Medicine,2009,11(1):28-42.
[69]LU J, XU S Y, ZHANG Q G, et al. Bupivacaine induces reactive oxygen species production via activation of the AMP-activated protein kinase-dependent pathway [J]. Pharmacology,2011,87(3-4):12.1-9.
[70]LU S C. Regulation of glutathione synthesis [J]. Mol Aspects Med,2009, 30(1-2):42-59.
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