左旋布比卡因经线粒体CI~-通道和p38MAPK途径致细胞凋亡
摘要
局部麻醉药简称局麻药,为临床区域阻滞和疼痛治疗中的常用药物,但该类药物存在直接或间接的神经毒性作用,这早已被其发展史及药理与毒理学研究所证实。随着神经阻滞麻醉的广泛应用,局麻药引起的神经毒性反应逐渐引起了临床工作者的重视,相关的研究报道也逐渐增多。许多研究发现这种神经毒性与局麻药引起的神经细胞凋亡有关,但其确切机制仍未完全阐明。
研究发现,神经细胞凋亡时产生特征性形态改变,在DNA降解之前,线粒体膜功能发生紊乱,内膜跨膜电位消失,线粒体内蛋白酶活化物释放,激发各种凋亡相关的代谢变化。细胞内活性氧簇(Reactive oxygen species, ROS)的爆发是导致神经细胞急性损伤的主要因素之一。ROS在神经细胞内蓄积时,可破坏细胞内氧化还原动态平衡,损伤线粒体膜。此外,ROS亦可直接与蛋白、脂质、核酸等发挥作用使其失去功能,起中间信使的作用,进一步引发神经损伤。研究表明,布比卡因可使Schwann细胞内ROS爆发,触发细胞凋亡。
细胞凋亡主要经过两条信号转导途径。其一是死亡受体途径,细胞表面的死亡受体通过其细胞外结构域与相应的死亡配体结合,将细胞外凋亡信号传入细胞内,激活caspase-8,而后激活caspase-3,从而启动细胞凋亡；其二是线粒体途径,氧自由基、钙超载等刺激因素可使线粒体通透性增加,线粒体膜电位下降,释放细胞色素C和凋亡诱导因子(Apoptosis-inducing factor, AIF),进而激活caspase-9和caspase-3,导致细胞凋亡。细胞凋亡与线粒体通透性转运孔(Mitochondrial permeability transition pore, mPTP)开放有关。mPTP由电压依赖性阴离子通道(Voltage dependent anion channel, VDAC)、腺苷酸运输体(Adenine nueleotide translocator, ANT)和亲环素D (Cyclophilin D, Cyp-D)构成。Cl-可以从开放的VDAC通道进入线粒体内,并能引起线粒体膜损伤和膜电位下降,导致分布在线粒体膜间的细胞色素C、AIF等促凋亡分子释放。研究表明,Cl-通道阻断剂(4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium, DIDS)可特异性阻断VDAC通道,抑制ROS产生、减少细胞凋亡和坏死。
丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)是一类丝氨酸／苏氨酸残基的蛋白激酶,是细胞信号转导的重要途径。p38丝裂原活化蛋白激酶(p38Mitogen-Activated Protein Kinase, p38MAPK)是丝裂原活化蛋白激酶家族重要成员之一,许多应激性刺激如H202等均能使p38MAPK信号途径激活。近年来的研究发现,p38MAPK信号途径通过控制多种转录因子的基因表达活性,影响多种细胞因子的产生,调节一氧化氮和细胞骨架蛋白的合成,参与应激条件下细胞的免疫调节、炎症反应和细胞凋亡等过程。Lirk等发现p38MAPK抑制剂4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyddyl)-1H-imidazole (SB203580)可抑制利多卡因诱发的神经细胞轴突退变。
本科研小组前期研究发现,布比卡因通过干扰氧化磷酸化和抑制线粒体呼吸链复合物I使ATP生成减少,AMPK持续激活,引起ROS的爆发性产生并导致细胞凋亡。进一步的研究发现,线粒体的阴离子通道VDAC和p38MAPK可能与布比卡因诱导的细胞凋亡密切相关。布比卡因可通过使细胞内ROS增多,一方面直接激活胞浆p38MAPK途径,引起细胞凋亡；一方面导致线粒体的VDAC通道开放,Cl-进入线粒体增多,线粒体基质肿胀和膜电位下降,线粒体的通透性进一步增高,释放出细胞色素C, AIF等促凋亡因子引起细胞凋亡,同时释放出更多的ROS,进一步激活p38MAPK蛋白,最终导致细胞凋亡。
左旋布比卡因(Lvobupivacaine, LB)是一种长效酰胺类局麻药,理化性质和麻醉效能与布比卡因相似,两药的心血管和神经系统毒性反应一直是临床高度关注的问题。研究已证明,LB为布比卡因的左旋体,其心血管毒性显著低于布比卡因,但两药的神经毒性比较,目前仍存在争议。LB诱发的神经毒性是否与ROS产生和介导有关?LB是否也通过激活p38MAPK途径和线粒体Cl-通道诱发神经细胞凋亡?目前尚不清楚。
本研究拟先从细胞水平,应用细胞生物学和分子生物学手段,比较不同浓度LB诱发SH-SY5Y细胞产生ROS和细胞凋亡的作用,并通过使用Cl-通道阻断剂DIDS及p38MAPK拮抗剂SB203580,以揭示LB引发细胞凋亡的通路是否与线粒体Cl-通道被激活和p38MAPK信号转导通路有关。然后通过整体动物实验,研究不同浓度LB对大鼠脊神经ROS增多和细胞凋亡的影响,同时也通过使用DIDS和SB203580来探讨LB致大鼠脊髓神经细胞的凋亡是否与线粒体Cl-通道被激活和p38MAPK信号转导通路有关,以验证细胞实验的结果并探讨体外与体内实验的异同性,为临床中防治LB引起的神经毒性提供一定的理论依据。
第1章不同浓度左旋布比卡因诱导SH-SY5Y细胞产生ROS和细胞凋亡
目的在细胞水平探讨不同浓度LB诱发SH-SY5Y细胞产生ROS的水平与细胞凋亡的关系。
方法以SH-SY5Y细胞为研究对象,分为6组：Ommol/L LB组(对照组)、0.5mmol/L LB组(LBo.5组)、1mmol/LLB组(LB1.0组)、1.5mmol/LLB组(LB1.5组)、2mmol/L LB组(LB2.0组)、2.5mmol/L LB组(LB2.5组)。各组细胞在含0、0.5、1.0、1.5、2.0、2.5mM LB的培养液中分别孵育1、2、3、4、5、6h,观察细胞形态,流式细胞仪检测细胞内ROS平均荧光强度。各组细胞在含不同浓度LB的培养液中孵育4h,用CCK-8法检测细胞活力,并采用FCM检测细胞凋亡。
计量资料以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。不同药物处理组在各时间点测得的ROS水平比较采用析因设计资料的方差分析。细胞活力及凋亡率均采用完全随机单因素方差分析,组间比较采用LSD法；若方差不齐采用welch校正法,组间比较采用Dunnett's T3法。
结果流式细胞术检测结果表明,细胞经不同浓度LB处理后,各浓度组细胞内ROS水平逐渐增高,3-4h达高峰,尤以LB2.0组4h的峰值最高(209.04±1.15),之后ROS水平出现下降趋势。LB0.5、LB1.0、LB1.5和LB20各处理组的ROS含量随着LB浓度的增加而增加,但LB2.5组ROS含量在2h后均低于LB2.0组；CCK-8法检测经不同浓度LB处理4h后的细胞活力,细胞活力呈现出剂量依赖性的降低,LB为2.5mM时细胞活力最低,为(23.15±1.60)%；流式细胞仪检测到LB各组间细胞凋亡率有统计学差异(P=0.000),其中LB为2mM的调亡率最高,(为32.57±1.59)。
结论LB可引起SH-SY5Y细胞呈浓度依赖性的ROS升高、caspase-3活化和细胞凋亡增多、活力降低。2mMLB在4h细胞内ROS产生最多,细胞凋亡率最高,呈典型的细胞凋亡现象。
第2章左旋布比卡因经线粒体Cl-通道和p38MAPK信号转导通路致SH-SY5Y细胞凋亡
目的在细胞水平探讨LB致SH-SY5Y细胞ROS增多,对细胞线粒体Cl-通道和p38MAPK的影响,以揭示线粒体C1-通道和p38MAPK信号转导通路在LB致SH-SY5Y细胞凋亡中的作用。
方法将SH-SY5Y细胞随机分为4组：无预处理组(non-pretreated组)、DIDS组、SB203580组、DIDS+SB203580组。DIDS组、SB203580组和DIDS+SB203580组细胞分别经50μmol/L DIDS、10μmol/L SB203580、50μmol/L DIDS+10μmol/L SB203580预处理30min后用含2.0mmol/LLB的培养液处理,non-pretreated组用含2mmol/LLB的培养液处理。各组细胞经LB处理4h用流式细胞术检测细胞内ROS水平,四氯四乙基苯并咪唑基羰花青碘化物(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbocyanide iodine, JC-1)检测线粒体膜电位,Western blot法检测p38MAPK和p-p38MAPK蛋白表达水平,激光共聚焦显微镜检测线粒体内Cl-荧光强度,CCK-8法检测细胞活力,FCM和Hochest33258检测细胞凋亡率。同时,未用LB处理的各组细胞在相同时间点检测上述相同的指标作为对照。
计量资料以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。细胞ROS含量、Cl-荧光强度、线粒体膜电位变化、细胞活力、细胞凋亡率、p38和p-p38MAPK蛋白表达量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LB培养4h后,DIDS组和DIDS+SB203580组细胞内ROS水平低于non-pretreated组(P<0.01)。流式细胞术检测线粒体膜JC-1荧光强度比值显示,DIDS组和DIDS+SB203580组分别为0.82±0.01和0.7±0.01,均高于non-pretreated组(0.52±0.01),差异均有统计学意义(P<0.01)。激光共聚焦显微镜检测线粒体内Cl-浓度显示,DIDS组和DIDS+SB203580组的Cl-浓度均低于non-pretreated组,差异有统计学意义(P<0.01). Western blot显示,DIDS组和DIDS+SB203580组的p-p38MAPK蛋白表达水平均低于non-pretreated组。MTT检测显示,DIDS组和SB203580组的细胞活力高于non-pretreated组(P<0.01),但均低于DIDS+SB203580组(P<0.01)。流式细胞术检测细胞凋亡率显示,LB处理后DIDS组、DIDS+SB203580组、SB203580组和non-pretreated组分别为23.4±1.58%、12.73±0.96%、24.53±0.93%和38.17±0.22%。Hoechst33258染色法检测显示,经LB处理后DIDS组、DIDS+SB203580组、SB203580组和Non-pretreated组的凋亡率分别为22.88±1.13%、12.45±0.74%、24.68±0.85%和37.8±0.99%。提示经LB处理后DIDS组.DIDS+SB203580组和SB203580组的凋亡率均低于non-pretreated组(P<0.01),DIDS+SB203580组的凋亡率低于SB203580组(P<0.01)。
结论LB可通过影响线粒体C1-通道和激活p38MAPK途径导致细胞凋亡。LB引起SH-SY5Y细胞ROS产生增多后,不但直接活化p38MAPK途径,引起细胞凋亡,并且导致线粒体VDAC通道开放,Cl-流入线粒体内,线粒体膜功能受损和膜电位下降,产生和释放更多的ROS,进一步激活p38MAPK蛋白,导致细胞凋亡。
第3章不同浓度左旋布比卡因对大鼠脊髓神经细胞ROS产量和细胞凋亡的影响
目的在整体动物水平比较不同浓度LB对大鼠脊髓神经细胞ROS产量和细胞凋亡的影响,评价不同浓度LB对大鼠脊髓神经损伤的程度。
方法成年健康雄性SD大鼠60只,体重220-250g。随机分成5组,每组12只：正常对照组(C组)、溶媒组(D组)、0.5%LB组(LB0.5组)、2.0%LB组(LB2.0组)和5.0%LB组(LB5.0组)。C组、D组、LB0.s组、LB2.0组和LB5.o组分别鞘内注射生理盐水、2%二甲基亚砜(DMSO)、0.5%、2.0%、5.0%LB20μl。给完药后,采用随机数字表法,将每组大鼠分出6只(n=6)做感觉运动功能观察,分别于鞘内给药前(基础状态)、给药后10min、20min、30min、1h、2h、4h、8h、1d、2d、3d测定甩尾反应潜伏期TFL并用最大效应百分比MPE表示,并进行后肢运动功能MF评分。3d后大鼠用4%多聚甲醛灌注,取脊髓L4-L5节段,行HE染色病理切片,免疫组化检测caspase-3阳性细胞数以及TUNEL法原位检测细胞凋亡。每组大鼠另外分出的6只(n=6)不做感觉运动功能观察,给药1d后即用生理盐水灌注,取脊髓L4-L5节段,ELISA检测脊髓ROS水平。
采用SPSS17.0统计学软件进行分析,计量资料以均数±标准差(x±s)表示。MPE值、运动功能评分比较采用重复测量设计的方差分析。脊髓ROS水平、caspase-3的阳性细胞计数和TUNEL凋亡细胞计数比较采用单因素方差分析,多重比较采用Bonferroni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
1.各组大鼠鞘内注药后MPE的比较,C组和D组的MPE不随时间变化,且在任意时间点处两组的MPE值均无统计学差异。各组内的不同时点进行比较显示,LB0.s组、LB2.0组和LBs.o组MPE在鞘内注药后10min均达到高峰后呈下降趋势,LB0.5组和LB2.o组分别于注药后4h和8h恢复至基线水平,而LB5.o组则于2d方恢复至基线水平。
2.各组大鼠鞘内注药后MF评分的比较,显示C组和D组的MF评分不随时间变化,且在任意时间下两组的MF值均无统计学差异。各组内的不同时间点进行比较显示,LBo.s组、LB2.o组和LBs.o组MF评分在鞘内注药后10min均达到高峰后呈下降趋势,LB0.5组在20min MF基本恢复；LB2.o组在1h MF基本恢复,而LBs.o组则在4hMF恢复。
3.HE染色病理切片观察,C组、D组和LBo.s组脊髓神经元胞体形态和结构基本未见异常；LB2.o组脊髓背角浅层神经元结构有不同程度的破坏,可见细胞核裂解；LBs.o组脊髓背角浅层神经元凋亡最为明显,凋亡细胞单个散在分布于组织中,可见凋亡小体。
4.各组大鼠脊髓ROS检测显示,C组和D组大鼠脊髓ROS水平没有明显变化。同C组比较,LBo.s组、LB2.o组和LBs.o组大鼠脊髓ROS水平明显升高,差异有统计学意义(P<0.01)。LBo.s组、LB2.o组和LBs.o组任意两组之间差异均有统计学意义(均有P<0.01)。
5.各组大鼠IHC检测显示,C组和D组大鼠脊髓神经组织caspase-3阳性细胞数没有明显变化。同C组比较,LB0.5组、LB2.o组和LBs.o组大鼠脊髓背角浅层caspase-3阳性细胞数明显增加,差异有统计学意义(P<0.01)。LBo.s组、LB2.o组和LBs.0组任意两组之间差异均有统计学意义(均有P<0.01)。
6.各组大鼠脊髓TUNEL细胞阳性率检测显示,C组和D组大鼠脊髓神经组织TUNEL阳性细胞数没有明显变化。同C组比较,LBo.s组、LB2.o组和LBs.o组大鼠脊髓背角浅层TUNEL阳性细胞数明显增加,差异有统计学意义(P<0.01)。LBo.s组、LB2.o组和LBs.o组任意两组之间差异均有统计学意义(均有P<0.01),
结论以2%DMSO作为溶媒对大鼠脊髓神经组织没有神经损伤和神经毒性作用；HE染色病理切片、IHC染色caspase-3阳性细胞和TUNEL标记凋亡细胞发现,大鼠脊髓LB损伤后细胞凋亡主要发生在背角浅层神经细胞；LB可引起大鼠脊髓神经组织浓度依赖性的ROS增加,脊髓背角浅层caspase-3和TUNEL阳性细胞率增加,提示大鼠脊髓背角浅层神经细胞凋亡增加,造成神经损伤,导致一定程度的感觉功能障碍,浓度越高感觉功能恢复延迟越明显。
第4章SB203580和DIDS对大鼠脊神经细胞左旋布比卡因损伤后细胞凋亡的影响
目的在整体动物水平研究SB203580和DIDS对大鼠脊髓神经细胞LB损伤后细胞凋亡的影响,探讨LB神经毒性的作用机制。
方法成年健康雄性SD大鼠,体重220-250g。取鞘内置管成功大鼠48只,随机分成4组,每组12只。LB组、DIDS+LB组、SB203580+LB组、(DIDS+SB203580)+LB组。LB组用微量注射器缓慢鞘内注射0.9%生理盐水20μL,2h之后用微量注射器缓慢(10’)鞘内注射5.0%LB20μl; DIDS+LB组经股静脉以程控微量泵给予14mg/Kg的DIDS [4ml/(Kg-h)]2h,之后鞘内缓慢注射5.0%LB20μl; SB203580+LB组鞘内注射0.1%SB203580(溶于2%DMSO)20μl,2h之后鞘内缓慢注射5.0%LB20μl;(DIDS+SB203580)+LB组先鞘内注射0.1%SB20358020μ1,之后经股静脉以程控微量泵给予14mg/Kg的DIDS2h,然后鞘内缓慢注射5.0%LB20μl。给完药后,采用随机数字表法,将每组动物分出6只(n=6)做感觉运动功能观察,大鼠分别于鞘内给药后30min、1h、2h、4h、8h、12h、16h、1d、2d、3d测定甩尾反应潜伏期TFL并用最大效应百分比MPE表示,并进行后肢运动功能MF评分。3d后大鼠用4%多聚甲醛灌注,取脊髓L4-L5节段,行HE染色病理切片,免疫组化检测caspase-3、caspase-9阳性细胞数以及TUNEL法原位检测细胞凋亡。每组大鼠另外分出的6只(n=6)不做感觉运动功能观察,给药1d后即用生理盐水灌注,取脊髓L4-L5节段,ELISA检测脊髓ROS水平,Western blot检测p38MAPK和p-p38MAPK的蛋白表达。
另取鞘内置管成功大鼠48只,随机分成4组,每组12只。NS组、DIDS+NS组、SB203580+NS组、(DIDS+SB203580)+NS组。NS组用微量注射器缓慢鞘内注射NS20μL,2h之后再用微量注射器缓慢(10’)鞘内注射NS20μL; DIDS+NS组经股静脉以程控微量泵给予14mg/Kg的DIDS [4ml/(Kg-h)]2h,之后鞘内缓慢注射NS20μ1; SB203580+NS组鞘内注射0.1%SB203580(溶于2%DMSO)20μl,2h之后鞘内缓慢注射NS20μl;(DIDS+SB203580)+NS组先鞘内注射0.1%SB20358020μ,之后经股静脉以程控微量泵给予14mg/Kg的DIDS2h,然后鞘内缓慢注射NS20μl。给完药后,采用随机数字表法,将每组动物分出6只(n=6)做感觉运动功能观察3d后处死取材,6只(n=6)则给药1d后即处死取材。未鞘内注射LB的各组大鼠在相同时间点检测上述相同的指标作为对照。
采用SPSS17.0统计学软件进行分析,计量资料以均数±标准差(x±s)表示。MPE值、运动功能评分比较采用重复测量设计的方差分析。脊髓ROS水平、caspase-3、caspase-9的阳性细胞计数、p38MAPK和p-p38MAPK的蛋白表达水平比较、凋亡细胞计数比较采用单因素方差分析,多重比较采用Bonferroni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
1.各组大鼠鞘内注药后MPE各组内的不同时点进行比较,LB组DIDS+LB组、SB203580+LB组和(DIDS+SB203580)+LB组MPE在鞘内注药后30min-2h均处于高峰平台而后呈下降趋势,DIDS+LB组、SB203580+LB组和(DIDS+SB203580)+LB组的恢复比LB组快,而(DIDS+SB203580)+LB组的恢复又比DIDS+LB组和SB203580+LB组快,四组均于注药后2d方恢复至基线水平。
2.各组大鼠鞘内注药后MF评分的比较显示,各组大鼠后肢运动功能的恢复在各时间点没有显著性差异,LB组、DIDS+LB组、SB203580+LB组和(DIDS+SB203580)+LB组MF均在4h恢复。
3.HE染色病理切片观察,LB组脊髓组织神经元胞体形态和结构破坏明显,胞浆尼氏小体有减少、破裂,可见细胞核裂解；DIDS+LB组和SB203580+LB组神经元结构有不同程度的破坏；(DIDS+SB203580)+LB组则损伤较轻,胞浆尼氏小体形态尚完整,胞核和胞浆比例也较前三组正常。
4.未鞘内注射LB的各组大鼠脊髓ROS水平差异没有统计学意义(F=0.058,P=0.981),提示此时各组间ROS水平没有差别；鞘内注射LB后各组大鼠脊髓ROS水平差异显著(F=321.229,P=0.000),提示此时各组间ROS水平不同,DIDDS+LB组、(DIDS+SB203580)+LB组ROS水平低于SB203580+LB组和LB组,差异均有统计学意义(P<0.01)。而鞘内注射LB组大鼠脊髓组织ROS水平均较未注射组水平显著升高(均有P<0.01)。
5.未鞘内注射LB的各组大鼠脊髓caspase-3阳性细胞数差异没有统计学意义(F=0.024,P=0.995)；鞘内注射LB后各组大鼠脊髓caspase-3阳性细胞数差异显著(F=21.735,P=0.000),提示此时各组间caspase-3阳性细胞数不同,(DIDS+SB203580)+LB组caspase-3阳性细胞计数低于其余三组,差异均有统计学意义(P<0.01)。而鞘内注射LB组大鼠脊髓背角浅层神经组织caspase-3阳性细胞数均较未注射组显著升高(均有P<0.01)。
6.未鞘内注射LB的各组大鼠脊髓神经组织caspase-9阳性细胞数差异没有统计学意义(F=0.019,P=0.996)；鞘内注射LB后各组大鼠脊髓背角浅层神经组织caspase-9阳性细胞数差异显著(F=66.166,P=0.000),提示此时各组间caspase-9阳性细胞数不同,DIDS+LB组、(DIDS+SB203580)+LB组均低于SB203580+LB组和LB组,差异均有统计学意义(P<0.01)。而鞘内注射LB组大鼠脊髓背角浅层caspase-9阳性细胞数均较未注射组显著升高(均有P<0.01)。
7.未鞘内注射LB的各组大鼠脊髓p38MAPK蛋白表达水平差异没有统计学意义(F=0.941,P=0.439),鞘内注射LB各组间p38MAPK蛋白表达水平的差异无统计学意义(F=0.936,P=0.442)；未鞘内注射LB的各组大鼠脊髓p-p38MAPK蛋白表达水平差异没有统计学意义(F=0.306,P=0.757)；鞘内注射LB后各组大鼠脊髓p-p38MAPK蛋白表达水平差异显著(F=243.117,P=0.000),提示此时各组间p-p38MAPK蛋白表达水平不同,(DIDS+SB203580)+LB组、SB203580+LB组p-p38MAPK蛋白表达水平低于DIDS+LB组和LB组,差异有统计学意义(P<0.05)。而鞘内注射LB组大鼠脊髓p-p38MAPK蛋白表达水平均较未注射组显著升高(均有P<0.01)。
8.未鞘内注射LB的各组大鼠脊髓TUNEL阳性细胞数差异没有统计学意义(P=0.170,P=0.951)；鞘内注射LB后各组大鼠脊髓背角浅层TUNEL阳性细胞数差异显著(F=23.998,P=-0.000),提示此时各组间TUNEL阳性细胞数不同,(DIDS+SB203580)+LB组TUNEL阳性细胞计数低于其余三组,差异均有统计学意义(P<0.01)。而鞘内注射LB组大鼠脊髓背角浅层TUNEL阳性细胞数均较未注射组显著升高(均有P<0.01)。
结论LB可能通过干扰氧化磷酸化和抑制线粒体呼吸链复合物I使ATP生成减少,引发大鼠脊髓神经细胞内ROS爆发性增多,直接激活p38MAPK途径,引起细胞凋亡；并可导致线粒体C1-通道开放,线粒体膜功能受损而产生和释放更多的ROS,促使p38MAPK蛋白激活导致细胞凋亡。同时也可能因为内外膜剥离造成线粒体损伤,释放出细胞色素C和促凋亡诱导因子等,激活caspase-9基因,进而激活caspase-3基因形成活化复合物,最终引起细胞凋亡。因此,可认为LB引起的大鼠脊髓神经毒性机制可能与线粒体的C1-通道和p38MAPK信号转导途径有关。
Local anesthetics are commonly used for local anesthesia and pain therapy, but they have the potential neurotoxicity. With the wide application in nerve block anesthesia, reports increase gradually hat local anesthetics cause neurotoxicity, and clinical workers pay high attention to this phenomenon. Many research found that this neurotoxicity caused by local anesthetics cause was associated with cell apoptosis, but the exact mechanism is still not fully understood.
Research found that the mitochondrial membrane function change, inner membrane potential disappeared and protease activation thing in the mitochondria released, and arouse all kinds of apoptosis related metabolic changes, then the apoptotic cells were induced to produce characteristic form change and DNA degradation. Burst of reactive oxygen species (ROS) is one of the main factors causing the nerve cells acute injury. ROS accumulation can affect oxidoreduction balance and damage the mitochondrial membrane in nerve cells, In addition, ROS can directly affect protein, lipid, and nucleic acid make them lose function. Moreover, ROS can also be a messenger, further cause nerve damage.
Cell apoptosis pathways consist of two signal transduction pathways. One is the death receptor pathway. The death receptor of cell surface binds the death ligand by the ectodomain, introduces the death signal into cell, and activates caspase-8and caspase-3, leading to apoptosis. The other is mitochondria pathway. Stimulating factors, such as oxygen free radical and calcium overload, increase the permeability of mitochondria, decrease mitochondria membrane potential, release cytochrome C and apoptosis-inducing factor(AIF), and subsequently activate caspase-9and caspase-3, leading to apoptosis. Cell apoptosis is related to the opening of mitochondrial permeability transition pore(mPTP). The mPTP is composed of voltage dependent anion channel(VDAC), adenine nueleotide translocator(ANT) and cyclophilin D(Cyp-D). The influx of chloridion (Cl-) into mitochondria can occur through the open VDAC and Can cause mitochondfial membrane damage and collapse the membrane potential, leading to release of pro-apoptotic molecule such as cytochrome C and AIF. Accordingly, the increase in intracellular ROS induced by bupivacaine prompts the opening of VDAC, causing the influx of Cl-into mitochondria, increase of mitoehondrial osmotic pressure, swell of matrix, decrease of mitochondrial membrane potential and augmentation of permeability, and release of pro-apoptotic factors, which leads to cell apoptosis.
P38mitogen-activated protein kinase (MAPK) is an important member of MAPK family. The p38MAPK signaling pathway is activated by different stimuli associated with stress, such as H2O2, TNF-a, lipopolysaccharide and hyperosmotic fluid. Previous studies demonstrated that the p38MAPK signaling pathway controls much gene expression activity of transcription factor, affects the production of many cytokines, and regulates the synthesis of NO and cytoskeleton protein. p38MAPK participates in immunological regulation, inflammatory reaction and cell apoptosis under the circumstance of stress. Studies found that peripheral nerve toxicity of lidocaine is related to the activation of p38MAPK. Lirk found that p38MAPK inhibitor4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1Hmidazole (SB203580) Can inhibit neurocyte axon degeneration.
In previous studies, Our team found bupivacaine may interfere with oxidative phosphorylation and inhibit the complex I of the mitochondrial respiratory chain, decrease the production of ATP, lead to single phosphoric acid active adenosine protein kinase (AMP-activated protein kinase, AMPK) continuous activation, and cause the mitochondria create and release more ROS in the cells, then cause cell damage and apoptosis through the mechanisms of ROS-induced-ROS release, further study showed bupivacaine may induce nerve cell apoptosis by via p38MAPK and mitochondrial Cl-channel pathway.
Levobupivacaine (LB) is a novel, long-acting amide local anesthetics. Due to similar physical and chemical properties and anesthetic potency to that of bupivacaine with less and lower toxicity in cardiovascular system and central nervous system, it shows a wide promising application potential in clinic and becomes a hot study project to investigate its mechanism in research field. Whether Levobupivacaine induced neurotoxicity is related to ROS production and mediation, or Levobupivacaine also induced neuronal apoptosis through activation of the p38MAPK pathway and mitochondrial Cl-channel, is still not clear,
In this study, we compared the effects of SH-SY5Y cells producing ROS and apoptosis induced by different concentrations of levobupivacaine via cell biology and molecular biology methods at the cellular level. The chloride channel blocker (44'the-diisothiocyanatostilbene-2,2'-disulfonic acid disodium DIDS) and p38MAPK antagonist SB203580were used to explore whether the apoptosis triggered by levobupivacaine is related to the p38MAPK signaling pathway and mitochondrial chloride ion channel activation. The further observation focused on the effects of different concentrations of levobupivacaine to the ROS increment and apoptosis of rat spinal nerve cells at the whole animal level. Meanwhile, the SB203580and DIDS were also used to investigate whether the nerve cells apoptosis in L4-5spinal cord segment of rats triggered by levobupivacaine is related to the p38MAPK signaling pathway and mitochondrial chloride ion channel activation. By studying mitochondria and p38MAPK pathways on spinal nerve cells apoptosis induced by Levobupivacaine, our study might provide a theoretical basis to prevent from neurotoxicity induced by levobupivacaine or develop an effective pharmaceutical treatment to relieve it.
Chapter1Levobupivacaine induce ROS generation and apoptosis in SH-SY5Y cell
Objective To explore the relationship of ROS level and apoptosis induced by different concentrations of levobupivacaine in SH-SY5Y cells
Method We culture SH SY5Y cell as the research object, treated by0,0.5,1,1.5,2,2.5mM levobupivacaine culture medium for1,2,3,4,5,6h, respectively, observe the cell morphology, detect the cell vitality with CCK-8method, Hochest33258detect the changes of apoptosis morphology, detect intracellular average ROS fluorescence intensity and cell apoptosis rate using flow cytometry instrument.
Statistical the measurement data were record mean±standard deviation, SPSS13.0statistics software was used to analysis the date. Two factors factorial analysis of variance was used to analysiscellular ROS level. The cell vitality, cell apoptosis rate and the comparison between groups was analysis by single factor analysis of variance (one-way ANOVA) in single effect analysis, multiple comparisons was analysis using LSD method for the homogeneity of variance and Welch or Dunnett's T3for the unequal variances were adopted when separate effect analysis as necessary, P<0.05for the difference was statistically significant.
Results Flow cytometry results showed that the intracellular ROS level gradually increased and peaked at4h,decreased at5and6h after the cells treated by different concentration of levobupivacaine. Compared to untreated group, intracellular ROS leveln of the groups after levobupivacaine treatment1,2,3,4,5, and6h were higher,the difference was statistically significant(P<0.01).; detect the cell vitality after treated by different concentration of levobupivacaine after4h using CCK-8method, the cell viability present a dose dependent decrease cell survival, the cell survival rate was the lowest when the LB is2.5mM as (23.15±1.60)%, the results show statistical difference among the groups processed by different LB (F=2043.476, P=0.000); the apoptosis rate detected by flow cytometry instrument was statistically difference among each group(F=614.342, P=0.000), and the apoptosis rate was highest when LB was2mM; SH SY5Y cells was observed highly agglutinated and showed strong blue fluorescence after treated with different concentrations of LB using Hoechst33258dyeing, the number of cells present strong blue fluorescence nucleus was most especially in LB=2mM.
Conclusion Levobupivacaine can cause SH-SY5Y cells ROS dependence apoptosis in SH-SY5Y cells, and the cells apoptosis rate was the highest when LB=2mM, levobupivacaine can also cause dose dependent cell survival rate decreased in SH-SY5Y cells.
Chapter2Reserch on Levobupivacaine induces type SH-SY5Y apoptosis via Cl-of moyochondria and p38MAPK signal conduction pathway.
Objective To discuss the effect on Cl-pathway of moyochondria and p38MAPK caused by increased ROS of SH-SY5Y which result from LB, revealing the functions of Cl-of moyochondria and p38MAPK signal conduction pathway in the process that LB induces type SH-SY5Y apoptosis.
Methods SH-SY5Y cells randomly divided into four groups:DIDS group, SB203580group, DIDS+SB203580group and non-pretreated group. Cells of DIDS group, SB203580group and DIDS+SB203580group were pretreated respectively with50μmol/L DIDS,10μmol/L SB203580, and50μmol/L DIDS plus10μmol/L SB20358030min prior to the treatment with the culture medium containing2mmol/L levobupivacaine and cells of non-pretreated group treated with the culture medium containing2mmol/L levobupivacaine. After4hours levobupivacaine treatment, intracellular ROS level was measured by FCM, the mitochondrial membrane potential was measured by5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzirnidazole-carbocyanideiodine(JC-1), the p38and p-p38MAPK protein was examined by western blots, and mitochondrial Cl-concentration was measured by laser scanning con_focal microScope.4h after levobupivaeaine treatment, the cell viability was examined by MTT assay, and cell apoptosis was examined by FCM. Meanwhile, the same indicators were also be tested in the cells of each group untreated with levobupivacaine at the same time to be as contrast.
Measurement data were presented as mean+standard deviation. SPSS13.0statistical software was used for analysis. Factorial design ANOVA was used to analyze the data from ROS measurement, Cl-fluorescence intensity mitochondrial membrane potential assay, cells survival rate assay, cell apoptosis rate assay, p38and p-p38MAPK protein expression. Comparisons among groups 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 After levobupivacaine treatment, the ROS levels of DIDS group and DIDS+SB203580group were lower than that of non-pretreated group(<0.01). The results of fluorescence intensity measured by FCM showed that the ratios of mitochondrial membrane JC-1polymer/monomer in DIDS group and DIDS+SB203580group were0.82+0.01and0.79±0.01respectively, which were higher than that of non-pretreated group(0.52±0.01). The difference had statistical significance(P<0.01). Mitochondrial Cl-concentration measured by laser scanning confocal microscope showed that the Cl-concentration of DIDS group and DIDS+SB203580group were lower than that of non-pmtreated group(P<0.01). Western blot showed that the p-p38MAPK protein expression of DIDS group and DIDS+SB203580group were lower than that of non-pretreated group(P<0.01). CCK-8assay revealed that the survival rates of DIDS and SB203580groups were higher than that of non-pretreated group(P<0.01), but lower than that of DIDS+SB203580group (P<0.01). Cells apoptosis rates of group DIDS, DIDS+SB203580, SB203580and non-pretreated were23.42±1.58%,12.73±0.96%,24.53±0.93%and38.17±0.22%, which were measured by FCM after levobupivacaine treatment. The apoptosis rate of SB203580group was lower than that of non-pretreated group(P<0.01), but there were no distinct difference in ROS levels and the ratios of JC-1polymer/monomer fluorescence intensity between the SB203580group and non-pretreated group (P<0.05). Hoechst33258staining assay revealed that the apoptosis rates of group DIDS, DIDS+SB203580, SB203580and non-pretreated were22.88±1.13%,12.45±0.74%,24.68±0.85%and37.87±0.99%.
Conclusion These findings indicate that the mitochondrial VDAC channel and activated p38MAPK pathway effected by levobupicavmne probably causes apoptosis. Increased intracellular ROS induced by Levobupivacaine opens mitochondrial VDAC channel and results in an influx of Cl-into mitochondria, which seems to be responsible for the mitochondrial depolarization, permeability enlargment of the mitochondria and the p38MAPK activation.
Chapter3Effects of different concentrations of levobupivacaine on ROS increasement and cell apoptosis of spinal nerve in rats
Objective To compare the effects of the ROS increasement and cell apoptosis of spinal nerve in rats with different concentrations of levobupivacaine and to investigate the extent of spinal nerve injury.
Methods After inserted intrathecal catheter, adult healthy male SD rats (weight220～250g) were divided into five groups (n=6):normal control group (C), the solvent group (D),0.5%levobupivacaine group (LB0.5),2.0%levobupivacaine the group (LB2.0) and5.0%levobupivacaine group (LB5.0) using a random number table. Group C, D, LB0.5, LB2.0and LB5.0were injected with saline, dimethyl sulfoxide,0.5%,2.0%,5.0%levobupivacaine20μl via intrathecal catheter respectively. Rats in each group were observed and recorded tail flick response latency (TFL) in the percentage of the maximum effect (MPE) and hind limb motor function score (MF) before intrathecal administration (basal state), after administration of lOmin,20min,30min,1h,2h,4h,8h,1d,2d,3d. After3d, rats were perfused with paraformaldehyde, and L4-5spinal cord segments were cut for pathological observation. The ROS level of nerve tissue was detected, and caspase-3and apoptosis of nerve cells were identified via immunohistochemical assay and TUNEL assay respectively.
All of the data were analyzed using SPSS13.0statistical software and were expressed as mean±standard deviation (x±s). The data among the TFL groups before and after catheter were compared using one-way ANOVA, and paired t-test was used between before and after catheter. The MPE values were analyzed via repeated measures analysis of variance. Caspase-3-positive cell counts and apoptotic cell counts were compared via single-factor analysis of variance, and the Bonferroni method (homogeneity of variance) or Dunnett's T3method (unequal variances) was used in multiple comparisons. Motor function scores were expressed with median (10,90bit point median percentile), and Kruskal-Wallis test was used among groups. P <0.05was considered statistically significant.
Result
1. Comparison of MPE among groups in rats after intrathecal injection shows MPE were different among groups (F=14220.696, P=0.000), and there was significant MPE change (F=5358.641, P=0.000) after injection with MPE positively correlating to time. Also, an interaction between the groups and the time was observed (F=1410.001, P=0.000). The results showed the differences among groups differed in time points. Further separate effects analysis found that there was no significant difference of MPE (P>0.05) between Group C and Group D at all time points. When compared within each group at different time points, MPE in Group LB0.5, Group LB2.0and Group LB5.0achieved their peak and downward in10min after the intrathecal injection. The MPE in Group LB0.5and Group LB2.0returned to baseline levels at4h and8h after injection, respectively, and Group LB5.0returned to baseline levels after2d.
2. The comparison of MF scores showed no motor dysfunction in Group C and Group D after intrathecal injection. The motor function was significantly different after lOmin-2h intrathecal injection in each group according to the mean rank analysis. The MF scores were significantly increased among Group LB0.5, LB2.0and LB5.0than that of Group C and Group D. The MF score of Group LB0.5resumed in20min, LB2.0resumed in one hour MF, and LB5.0resumed in4h.
3. Group C, D and LB0.5shows intact neuronal cell morphology and structure of spinal cord tissue in H&E. Group LB2.0shows varying degrees of damage with nuclei lyses in the neuronal structures of spinal cord tissue. Group LB5.0shows obvious spinal cord neuronal apoptosis with scattered apoptotic cells and apoptotic bodies.
4. The comparison of ROS levels in each spinal cord tissues of rats shows there were no significant changes in Group C and Group D. The ROS levels in Groups LB0.5, LB2.0and LB5.0increased with the increase of drug concentration. The difference was statistically significant (P<0.01), with a ROS level of192.93±10.05in Group LB5.0.
5. The Immunohistochemical positive cells counts of caspase-3shows no statistically significant among Group C, D and LB0.5in the spinal cord nerve tissue of rats. However, the caspase-3positive cells were significantly (P<0.01) increased in Group LB2.0and Group LB5.0compared with Group C.
6. The TUNEL positive cells percentage detection shows no statistically significant among Group C, D and LB0.5in the spinal cord nerve tissue of rats. However, the TUNEL positive cells percentage was significantly (P<0.01) increased in Group LB2.0and Group LB5.0compared with Group C.
Conclusion Levobupivacaine can cause a concentration-dependent ROS increase and induce apoptosis in the rat spinal cord nerve tissue, resulting in nerve damage and sensory dysfunction at a certain extent. The higher of concentration of Levobupivacaine, the more delayed sensory function recovery was observed.
Chapter4Effects of SB203580and DIDS on spinal nerve cells apoptosis induced by levobupivacaine in rat
Objective To investigate the effect of SB203580and DIDS on apoptosis in rat spinal nerve injured by levobupivacaine and to explore the related spinal neurotoxicity mechanism.
Methods After inserted intrathecal catheter, adult healthy male SD rats (weight220～250g) were divided into four groups (n=6):Group LB, Group DIDS+LB, Group SB203580+LB, Group (DIDS+SB203580)+LB. Group LB were slowly intrathecal injected with20μl of0.9%saline via micro-syringe, and then slowly (10') injected with20μl of5.0%levobupivacaine via micro-syringe after2hours. Group DIDS+LB were given14mg/kg of DIDS through the femoral vein via the program-controlled micro-pump [4ml/(Kg-h)] within2hours, and then slowly injected with20μl of5.0%levobupivacaine. Group SB203580+LB were intrathecal injected with20μl of0.1%SB203580(dissolved in2%DMSO) within2hours, and then slowly injected with20μl of5.0%levobupivacaine. Group (DIDS+SB203580)+LB were intrathecal injected with20μl of0.1%SB203580, then given14mg/kg of DIDS through the femoral vein via the program-controlled micro-pump within2hours, and then slowly injected with20μl of5.0%levobupivacaine. Six rats in each group were separated using a random number table and the sensory-motor function was observed. The tail flick response latency (TFL) were observed and recorded using the maximum percentage effect (MPE) and hind limb motor function score (MF) after30min,1h,2h,4h,8h,12h,16h, Id,2d,3d of intrathecal administration. After3d, rats were perfused with paraformaldehyde, and L4-5spinal cord segments were cut for pathological observation, immunohistochemical detection of caspase-3, caspase-9positive cells and TUNEL in situ detection of apoptosis. The other six rats in each group were perfused with paraformaldehyde, and L4-5spinal cord segments were cut for pathological observation, ELISAL detection of he ROS level of nerve tissue, and Western blot analysis of p38MAPK and p-p38MAPK protein expression.
Using SPSS13.0software for statistical treatment, measurement data were expressed as mean±standard deviation. Comparisons among groups TFL were made by analysis of variance (ANOVA) and paired-sample T test. MPE were made by analysis of Repeated Measures. p-p38MAPK and p38MAPK expression, caspase-3、 caspase-9positive cell counts and apoptotic cell counts were made by analysis of variance (ANOVA). MF scores are presented as median (10th—90th percentiles) and were compared using the Kruskal-Wallis test followed by the Mann-Whitney u-test. P<0.05was considered significant.
Result
1. Comparison of MPE among groups in rats after intrathecal injection shows MPE were different among groups (F=143.324, P=0.000), and there was significant MPE change (F=130.948, P=0.000) after injection with MPE positively correlating to time. Also, an interaction between the groups and the time was observed (F=179.714, P=0.000). The results showed the differences among groups differed in time points. When compared within each group at different time points, MPE in Group LB, Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB achieved their peak in30min and downward after4-8h after the intrathecal injection. The MPE in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB recovered at12h-ld after injection, which is faster than that of Group LB. The recovery of Group (DIDS+SB203580)+LB is faster than that of Group DIDS+LB and Group SB203580+LB. all the groups returned to baseline levels after2d.
2. The comparison of MF scores of rats showed there was no significant difference of the hind limb motor function recovery at each time point among each groups after intrathecal injection. The MF scores in different groups were recovered in4hours.
3. Histopathological observation found that the neurons morphology and structural were damaged, cytoplasmic Nissl body decreased and cracked with nucleus lyses in Group LB. The neuronal structures in Group DIDS+LB and Group SB203580+LB show varying degrees of damage. The neuronal structures in Group (DIDS+SB203580)+LB were Less damaged; the cytoplasmic Nissl body morphology was still intact and the ratio of nucleus to cytoplasm was normal when compared with the first three groups.
4. The comparison of ROS levels in each spinal cord tissues of rats shows no statistically significant differences (P>0.05) between Group DIDS+LB and Group (DIDS+SB203580)+LB. There were also no statistically significant differences (P>0.05) between Group SB203580+LB and Group LB. The ROS levels in Groups DIDS+LB and Group (DIDS+SB203580)+LB were less than that in Group SB203580+LB and Group LB, and the difference was statistically significant (P<0.05).
5. The Caspase-3positive cell counts in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that in Group LB (P<0.05). The Caspase-3positive cell counts in Group DIDS+LB, Group SB203580+LB were higher than that in Group (DIDS+SB203580)+LB, the difference was statistically significant (P<0.05).
6. The Caspase-9positive cell counts showed no significant difference (P>0.05) between Group SB203580+LB and Group LB. It was also no significant difference (P>0.05) between Group DIDS+LB and Group (DIDS+SB203580)+LB. The Caspase-9positive cell counts in Group DIDS+LB and Group (DIDS+SB203580)+LB were lower than that in Group SB203580+LB and Group LB (P<0.05).
7. The p-p38MAPK expression level is significant different (F=234.117, P=0.000) among groups. The p-p38MAPK expression levels in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that in Group LB (P<0.05). The p-p38MAPK expression levels in Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that of in Group DIDS+LB (P <0.05). The p38MAPK expression level showed no differences (F=0.936, P=0.442) among groups.
8. The apoptosis rate in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that in Group LB (P<0.05). The apoptosis rate in Group DIDS+LB, Group SB203580+LB were higher than that in Group (DIDS+SB203580)+LB, the difference was statistically significant (P<0.05).
Conclusion Levobupivacaine may interfere with oxidative phosphorylation and inhibit mitochondrial respiratory chain complex I, which decreased the production of ATP and triggered explosive increasement of ROS in the rat spinal cord nerve cells. On the one hand, the direct activation of p38MAPK pathway can induce apoptosis; On the other hand the mitochondrial Cl-channel opening and mitochondrial membrane dysfunction may lead to the production and release of more ROS, which activate p38MAPK protein and result in apoptosis. Mitochondrial Damage may also be caused by the peeling of inner and outer membrane, which may release cytochrome C and apoptosis-inducing factors. Thus it may activate caspase-9and caspase-3to form an activated complex, and ultimately induce apoptosis. Therefore, the mechanism of neurotoxicity caused by levobupivacaine in the rat spinal cord may relate to mitochondrial Cl-channels and p38MAPK signal transduction pathway.
研究发现,神经细胞凋亡时产生特征性形态改变,在DNA降解之前,线粒体膜功能发生紊乱,内膜跨膜电位消失,线粒体内蛋白酶活化物释放,激发各种凋亡相关的代谢变化。细胞内活性氧簇(Reactive oxygen species, ROS)的爆发是导致神经细胞急性损伤的主要因素之一。ROS在神经细胞内蓄积时,可破坏细胞内氧化还原动态平衡,损伤线粒体膜。此外,ROS亦可直接与蛋白、脂质、核酸等发挥作用使其失去功能,起中间信使的作用,进一步引发神经损伤。研究表明,布比卡因可使Schwann细胞内ROS爆发,触发细胞凋亡。
细胞凋亡主要经过两条信号转导途径。其一是死亡受体途径,细胞表面的死亡受体通过其细胞外结构域与相应的死亡配体结合,将细胞外凋亡信号传入细胞内,激活caspase-8,而后激活caspase-3,从而启动细胞凋亡；其二是线粒体途径,氧自由基、钙超载等刺激因素可使线粒体通透性增加,线粒体膜电位下降,释放细胞色素C和凋亡诱导因子(Apoptosis-inducing factor, AIF),进而激活caspase-9和caspase-3,导致细胞凋亡。细胞凋亡与线粒体通透性转运孔(Mitochondrial permeability transition pore, mPTP)开放有关。mPTP由电压依赖性阴离子通道(Voltage dependent anion channel, VDAC)、腺苷酸运输体(Adenine nueleotide translocator, ANT)和亲环素D (Cyclophilin D, Cyp-D)构成。Cl-可以从开放的VDAC通道进入线粒体内,并能引起线粒体膜损伤和膜电位下降,导致分布在线粒体膜间的细胞色素C、AIF等促凋亡分子释放。研究表明,Cl-通道阻断剂(4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium, DIDS)可特异性阻断VDAC通道,抑制ROS产生、减少细胞凋亡和坏死。
丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)是一类丝氨酸／苏氨酸残基的蛋白激酶,是细胞信号转导的重要途径。p38丝裂原活化蛋白激酶(p38Mitogen-Activated Protein Kinase, p38MAPK)是丝裂原活化蛋白激酶家族重要成员之一,许多应激性刺激如H202等均能使p38MAPK信号途径激活。近年来的研究发现,p38MAPK信号途径通过控制多种转录因子的基因表达活性,影响多种细胞因子的产生,调节一氧化氮和细胞骨架蛋白的合成,参与应激条件下细胞的免疫调节、炎症反应和细胞凋亡等过程。Lirk等发现p38MAPK抑制剂4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyddyl)-1H-imidazole (SB203580)可抑制利多卡因诱发的神经细胞轴突退变。
本科研小组前期研究发现,布比卡因通过干扰氧化磷酸化和抑制线粒体呼吸链复合物I使ATP生成减少,AMPK持续激活,引起ROS的爆发性产生并导致细胞凋亡。进一步的研究发现,线粒体的阴离子通道VDAC和p38MAPK可能与布比卡因诱导的细胞凋亡密切相关。布比卡因可通过使细胞内ROS增多,一方面直接激活胞浆p38MAPK途径,引起细胞凋亡；一方面导致线粒体的VDAC通道开放,Cl-进入线粒体增多,线粒体基质肿胀和膜电位下降,线粒体的通透性进一步增高,释放出细胞色素C, AIF等促凋亡因子引起细胞凋亡,同时释放出更多的ROS,进一步激活p38MAPK蛋白,最终导致细胞凋亡。
左旋布比卡因(Lvobupivacaine, LB)是一种长效酰胺类局麻药,理化性质和麻醉效能与布比卡因相似,两药的心血管和神经系统毒性反应一直是临床高度关注的问题。研究已证明,LB为布比卡因的左旋体,其心血管毒性显著低于布比卡因,但两药的神经毒性比较,目前仍存在争议。LB诱发的神经毒性是否与ROS产生和介导有关?LB是否也通过激活p38MAPK途径和线粒体Cl-通道诱发神经细胞凋亡?目前尚不清楚。
本研究拟先从细胞水平,应用细胞生物学和分子生物学手段,比较不同浓度LB诱发SH-SY5Y细胞产生ROS和细胞凋亡的作用,并通过使用Cl-通道阻断剂DIDS及p38MAPK拮抗剂SB203580,以揭示LB引发细胞凋亡的通路是否与线粒体Cl-通道被激活和p38MAPK信号转导通路有关。然后通过整体动物实验,研究不同浓度LB对大鼠脊神经ROS增多和细胞凋亡的影响,同时也通过使用DIDS和SB203580来探讨LB致大鼠脊髓神经细胞的凋亡是否与线粒体Cl-通道被激活和p38MAPK信号转导通路有关,以验证细胞实验的结果并探讨体外与体内实验的异同性,为临床中防治LB引起的神经毒性提供一定的理论依据。
第1章不同浓度左旋布比卡因诱导SH-SY5Y细胞产生ROS和细胞凋亡
目的在细胞水平探讨不同浓度LB诱发SH-SY5Y细胞产生ROS的水平与细胞凋亡的关系。
方法以SH-SY5Y细胞为研究对象,分为6组：Ommol/L LB组(对照组)、0.5mmol/L LB组(LBo.5组)、1mmol/LLB组(LB1.0组)、1.5mmol/LLB组(LB1.5组)、2mmol/L LB组(LB2.0组)、2.5mmol/L LB组(LB2.5组)。各组细胞在含0、0.5、1.0、1.5、2.0、2.5mM LB的培养液中分别孵育1、2、3、4、5、6h,观察细胞形态,流式细胞仪检测细胞内ROS平均荧光强度。各组细胞在含不同浓度LB的培养液中孵育4h,用CCK-8法检测细胞活力,并采用FCM检测细胞凋亡。
计量资料以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。不同药物处理组在各时间点测得的ROS水平比较采用析因设计资料的方差分析。细胞活力及凋亡率均采用完全随机单因素方差分析,组间比较采用LSD法；若方差不齐采用welch校正法,组间比较采用Dunnett's T3法。
结果流式细胞术检测结果表明,细胞经不同浓度LB处理后,各浓度组细胞内ROS水平逐渐增高,3-4h达高峰,尤以LB2.0组4h的峰值最高(209.04±1.15),之后ROS水平出现下降趋势。LB0.5、LB1.0、LB1.5和LB20各处理组的ROS含量随着LB浓度的增加而增加,但LB2.5组ROS含量在2h后均低于LB2.0组；CCK-8法检测经不同浓度LB处理4h后的细胞活力,细胞活力呈现出剂量依赖性的降低,LB为2.5mM时细胞活力最低,为(23.15±1.60)%；流式细胞仪检测到LB各组间细胞凋亡率有统计学差异(P=0.000),其中LB为2mM的调亡率最高,(为32.57±1.59)。
结论LB可引起SH-SY5Y细胞呈浓度依赖性的ROS升高、caspase-3活化和细胞凋亡增多、活力降低。2mMLB在4h细胞内ROS产生最多,细胞凋亡率最高,呈典型的细胞凋亡现象。
第2章左旋布比卡因经线粒体Cl-通道和p38MAPK信号转导通路致SH-SY5Y细胞凋亡
目的在细胞水平探讨LB致SH-SY5Y细胞ROS增多,对细胞线粒体Cl-通道和p38MAPK的影响,以揭示线粒体C1-通道和p38MAPK信号转导通路在LB致SH-SY5Y细胞凋亡中的作用。
方法将SH-SY5Y细胞随机分为4组：无预处理组(non-pretreated组)、DIDS组、SB203580组、DIDS+SB203580组。DIDS组、SB203580组和DIDS+SB203580组细胞分别经50μmol/L DIDS、10μmol/L SB203580、50μmol/L DIDS+10μmol/L SB203580预处理30min后用含2.0mmol/LLB的培养液处理,non-pretreated组用含2mmol/LLB的培养液处理。各组细胞经LB处理4h用流式细胞术检测细胞内ROS水平,四氯四乙基苯并咪唑基羰花青碘化物(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbocyanide iodine, JC-1)检测线粒体膜电位,Western blot法检测p38MAPK和p-p38MAPK蛋白表达水平,激光共聚焦显微镜检测线粒体内Cl-荧光强度,CCK-8法检测细胞活力,FCM和Hochest33258检测细胞凋亡率。同时,未用LB处理的各组细胞在相同时间点检测上述相同的指标作为对照。
计量资料以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。细胞ROS含量、Cl-荧光强度、线粒体膜电位变化、细胞活力、细胞凋亡率、p38和p-p38MAPK蛋白表达量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LB培养4h后,DIDS组和DIDS+SB203580组细胞内ROS水平低于non-pretreated组(P<0.01)。流式细胞术检测线粒体膜JC-1荧光强度比值显示,DIDS组和DIDS+SB203580组分别为0.82±0.01和0.7±0.01,均高于non-pretreated组(0.52±0.01),差异均有统计学意义(P<0.01)。激光共聚焦显微镜检测线粒体内Cl-浓度显示,DIDS组和DIDS+SB203580组的Cl-浓度均低于non-pretreated组,差异有统计学意义(P<0.01). Western blot显示,DIDS组和DIDS+SB203580组的p-p38MAPK蛋白表达水平均低于non-pretreated组。MTT检测显示,DIDS组和SB203580组的细胞活力高于non-pretreated组(P<0.01),但均低于DIDS+SB203580组(P<0.01)。流式细胞术检测细胞凋亡率显示,LB处理后DIDS组、DIDS+SB203580组、SB203580组和non-pretreated组分别为23.4±1.58%、12.73±0.96%、24.53±0.93%和38.17±0.22%。Hoechst33258染色法检测显示,经LB处理后DIDS组、DIDS+SB203580组、SB203580组和Non-pretreated组的凋亡率分别为22.88±1.13%、12.45±0.74%、24.68±0.85%和37.8±0.99%。提示经LB处理后DIDS组.DIDS+SB203580组和SB203580组的凋亡率均低于non-pretreated组(P<0.01),DIDS+SB203580组的凋亡率低于SB203580组(P<0.01)。
结论LB可通过影响线粒体C1-通道和激活p38MAPK途径导致细胞凋亡。LB引起SH-SY5Y细胞ROS产生增多后,不但直接活化p38MAPK途径,引起细胞凋亡,并且导致线粒体VDAC通道开放,Cl-流入线粒体内,线粒体膜功能受损和膜电位下降,产生和释放更多的ROS,进一步激活p38MAPK蛋白,导致细胞凋亡。
第3章不同浓度左旋布比卡因对大鼠脊髓神经细胞ROS产量和细胞凋亡的影响
目的在整体动物水平比较不同浓度LB对大鼠脊髓神经细胞ROS产量和细胞凋亡的影响,评价不同浓度LB对大鼠脊髓神经损伤的程度。
方法成年健康雄性SD大鼠60只,体重220-250g。随机分成5组,每组12只：正常对照组(C组)、溶媒组(D组)、0.5%LB组(LB0.5组)、2.0%LB组(LB2.0组)和5.0%LB组(LB5.0组)。C组、D组、LB0.s组、LB2.0组和LB5.o组分别鞘内注射生理盐水、2%二甲基亚砜(DMSO)、0.5%、2.0%、5.0%LB20μl。给完药后,采用随机数字表法,将每组大鼠分出6只(n=6)做感觉运动功能观察,分别于鞘内给药前(基础状态)、给药后10min、20min、30min、1h、2h、4h、8h、1d、2d、3d测定甩尾反应潜伏期TFL并用最大效应百分比MPE表示,并进行后肢运动功能MF评分。3d后大鼠用4%多聚甲醛灌注,取脊髓L4-L5节段,行HE染色病理切片,免疫组化检测caspase-3阳性细胞数以及TUNEL法原位检测细胞凋亡。每组大鼠另外分出的6只(n=6)不做感觉运动功能观察,给药1d后即用生理盐水灌注,取脊髓L4-L5节段,ELISA检测脊髓ROS水平。
采用SPSS17.0统计学软件进行分析,计量资料以均数±标准差(x±s)表示。MPE值、运动功能评分比较采用重复测量设计的方差分析。脊髓ROS水平、caspase-3的阳性细胞计数和TUNEL凋亡细胞计数比较采用单因素方差分析,多重比较采用Bonferroni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
1.各组大鼠鞘内注药后MPE的比较,C组和D组的MPE不随时间变化,且在任意时间点处两组的MPE值均无统计学差异。各组内的不同时点进行比较显示,LB0.s组、LB2.0组和LBs.o组MPE在鞘内注药后10min均达到高峰后呈下降趋势,LB0.5组和LB2.o组分别于注药后4h和8h恢复至基线水平,而LB5.o组则于2d方恢复至基线水平。
2.各组大鼠鞘内注药后MF评分的比较,显示C组和D组的MF评分不随时间变化,且在任意时间下两组的MF值均无统计学差异。各组内的不同时间点进行比较显示,LBo.s组、LB2.o组和LBs.o组MF评分在鞘内注药后10min均达到高峰后呈下降趋势,LB0.5组在20min MF基本恢复；LB2.o组在1h MF基本恢复,而LBs.o组则在4hMF恢复。
3.HE染色病理切片观察,C组、D组和LBo.s组脊髓神经元胞体形态和结构基本未见异常；LB2.o组脊髓背角浅层神经元结构有不同程度的破坏,可见细胞核裂解；LBs.o组脊髓背角浅层神经元凋亡最为明显,凋亡细胞单个散在分布于组织中,可见凋亡小体。
4.各组大鼠脊髓ROS检测显示,C组和D组大鼠脊髓ROS水平没有明显变化。同C组比较,LBo.s组、LB2.o组和LBs.o组大鼠脊髓ROS水平明显升高,差异有统计学意义(P<0.01)。LBo.s组、LB2.o组和LBs.o组任意两组之间差异均有统计学意义(均有P<0.01)。
5.各组大鼠IHC检测显示,C组和D组大鼠脊髓神经组织caspase-3阳性细胞数没有明显变化。同C组比较,LB0.5组、LB2.o组和LBs.o组大鼠脊髓背角浅层caspase-3阳性细胞数明显增加,差异有统计学意义(P<0.01)。LBo.s组、LB2.o组和LBs.0组任意两组之间差异均有统计学意义(均有P<0.01)。
6.各组大鼠脊髓TUNEL细胞阳性率检测显示,C组和D组大鼠脊髓神经组织TUNEL阳性细胞数没有明显变化。同C组比较,LBo.s组、LB2.o组和LBs.o组大鼠脊髓背角浅层TUNEL阳性细胞数明显增加,差异有统计学意义(P<0.01)。LBo.s组、LB2.o组和LBs.o组任意两组之间差异均有统计学意义(均有P<0.01),
结论以2%DMSO作为溶媒对大鼠脊髓神经组织没有神经损伤和神经毒性作用；HE染色病理切片、IHC染色caspase-3阳性细胞和TUNEL标记凋亡细胞发现,大鼠脊髓LB损伤后细胞凋亡主要发生在背角浅层神经细胞；LB可引起大鼠脊髓神经组织浓度依赖性的ROS增加,脊髓背角浅层caspase-3和TUNEL阳性细胞率增加,提示大鼠脊髓背角浅层神经细胞凋亡增加,造成神经损伤,导致一定程度的感觉功能障碍,浓度越高感觉功能恢复延迟越明显。
第4章SB203580和DIDS对大鼠脊神经细胞左旋布比卡因损伤后细胞凋亡的影响
目的在整体动物水平研究SB203580和DIDS对大鼠脊髓神经细胞LB损伤后细胞凋亡的影响,探讨LB神经毒性的作用机制。
方法成年健康雄性SD大鼠,体重220-250g。取鞘内置管成功大鼠48只,随机分成4组,每组12只。LB组、DIDS+LB组、SB203580+LB组、(DIDS+SB203580)+LB组。LB组用微量注射器缓慢鞘内注射0.9%生理盐水20μL,2h之后用微量注射器缓慢(10’)鞘内注射5.0%LB20μl; DIDS+LB组经股静脉以程控微量泵给予14mg/Kg的DIDS [4ml/(Kg-h)]2h,之后鞘内缓慢注射5.0%LB20μl; SB203580+LB组鞘内注射0.1%SB203580(溶于2%DMSO)20μl,2h之后鞘内缓慢注射5.0%LB20μl;(DIDS+SB203580)+LB组先鞘内注射0.1%SB20358020μ1,之后经股静脉以程控微量泵给予14mg/Kg的DIDS2h,然后鞘内缓慢注射5.0%LB20μl。给完药后,采用随机数字表法,将每组动物分出6只(n=6)做感觉运动功能观察,大鼠分别于鞘内给药后30min、1h、2h、4h、8h、12h、16h、1d、2d、3d测定甩尾反应潜伏期TFL并用最大效应百分比MPE表示,并进行后肢运动功能MF评分。3d后大鼠用4%多聚甲醛灌注,取脊髓L4-L5节段,行HE染色病理切片,免疫组化检测caspase-3、caspase-9阳性细胞数以及TUNEL法原位检测细胞凋亡。每组大鼠另外分出的6只(n=6)不做感觉运动功能观察,给药1d后即用生理盐水灌注,取脊髓L4-L5节段,ELISA检测脊髓ROS水平,Western blot检测p38MAPK和p-p38MAPK的蛋白表达。
另取鞘内置管成功大鼠48只,随机分成4组,每组12只。NS组、DIDS+NS组、SB203580+NS组、(DIDS+SB203580)+NS组。NS组用微量注射器缓慢鞘内注射NS20μL,2h之后再用微量注射器缓慢(10’)鞘内注射NS20μL; DIDS+NS组经股静脉以程控微量泵给予14mg/Kg的DIDS [4ml/(Kg-h)]2h,之后鞘内缓慢注射NS20μ1; SB203580+NS组鞘内注射0.1%SB203580(溶于2%DMSO)20μl,2h之后鞘内缓慢注射NS20μl;(DIDS+SB203580)+NS组先鞘内注射0.1%SB20358020μ,之后经股静脉以程控微量泵给予14mg/Kg的DIDS2h,然后鞘内缓慢注射NS20μl。给完药后,采用随机数字表法,将每组动物分出6只(n=6)做感觉运动功能观察3d后处死取材,6只(n=6)则给药1d后即处死取材。未鞘内注射LB的各组大鼠在相同时间点检测上述相同的指标作为对照。
采用SPSS17.0统计学软件进行分析,计量资料以均数±标准差(x±s)表示。MPE值、运动功能评分比较采用重复测量设计的方差分析。脊髓ROS水平、caspase-3、caspase-9的阳性细胞计数、p38MAPK和p-p38MAPK的蛋白表达水平比较、凋亡细胞计数比较采用单因素方差分析,多重比较采用Bonferroni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
1.各组大鼠鞘内注药后MPE各组内的不同时点进行比较,LB组DIDS+LB组、SB203580+LB组和(DIDS+SB203580)+LB组MPE在鞘内注药后30min-2h均处于高峰平台而后呈下降趋势,DIDS+LB组、SB203580+LB组和(DIDS+SB203580)+LB组的恢复比LB组快,而(DIDS+SB203580)+LB组的恢复又比DIDS+LB组和SB203580+LB组快,四组均于注药后2d方恢复至基线水平。
2.各组大鼠鞘内注药后MF评分的比较显示,各组大鼠后肢运动功能的恢复在各时间点没有显著性差异,LB组、DIDS+LB组、SB203580+LB组和(DIDS+SB203580)+LB组MF均在4h恢复。
3.HE染色病理切片观察,LB组脊髓组织神经元胞体形态和结构破坏明显,胞浆尼氏小体有减少、破裂,可见细胞核裂解；DIDS+LB组和SB203580+LB组神经元结构有不同程度的破坏；(DIDS+SB203580)+LB组则损伤较轻,胞浆尼氏小体形态尚完整,胞核和胞浆比例也较前三组正常。
4.未鞘内注射LB的各组大鼠脊髓ROS水平差异没有统计学意义(F=0.058,P=0.981),提示此时各组间ROS水平没有差别；鞘内注射LB后各组大鼠脊髓ROS水平差异显著(F=321.229,P=0.000),提示此时各组间ROS水平不同,DIDDS+LB组、(DIDS+SB203580)+LB组ROS水平低于SB203580+LB组和LB组,差异均有统计学意义(P<0.01)。而鞘内注射LB组大鼠脊髓组织ROS水平均较未注射组水平显著升高(均有P<0.01)。
5.未鞘内注射LB的各组大鼠脊髓caspase-3阳性细胞数差异没有统计学意义(F=0.024,P=0.995)；鞘内注射LB后各组大鼠脊髓caspase-3阳性细胞数差异显著(F=21.735,P=0.000),提示此时各组间caspase-3阳性细胞数不同,(DIDS+SB203580)+LB组caspase-3阳性细胞计数低于其余三组,差异均有统计学意义(P<0.01)。而鞘内注射LB组大鼠脊髓背角浅层神经组织caspase-3阳性细胞数均较未注射组显著升高(均有P<0.01)。
6.未鞘内注射LB的各组大鼠脊髓神经组织caspase-9阳性细胞数差异没有统计学意义(F=0.019,P=0.996)；鞘内注射LB后各组大鼠脊髓背角浅层神经组织caspase-9阳性细胞数差异显著(F=66.166,P=0.000),提示此时各组间caspase-9阳性细胞数不同,DIDS+LB组、(DIDS+SB203580)+LB组均低于SB203580+LB组和LB组,差异均有统计学意义(P<0.01)。而鞘内注射LB组大鼠脊髓背角浅层caspase-9阳性细胞数均较未注射组显著升高(均有P<0.01)。
7.未鞘内注射LB的各组大鼠脊髓p38MAPK蛋白表达水平差异没有统计学意义(F=0.941,P=0.439),鞘内注射LB各组间p38MAPK蛋白表达水平的差异无统计学意义(F=0.936,P=0.442)；未鞘内注射LB的各组大鼠脊髓p-p38MAPK蛋白表达水平差异没有统计学意义(F=0.306,P=0.757)；鞘内注射LB后各组大鼠脊髓p-p38MAPK蛋白表达水平差异显著(F=243.117,P=0.000),提示此时各组间p-p38MAPK蛋白表达水平不同,(DIDS+SB203580)+LB组、SB203580+LB组p-p38MAPK蛋白表达水平低于DIDS+LB组和LB组,差异有统计学意义(P<0.05)。而鞘内注射LB组大鼠脊髓p-p38MAPK蛋白表达水平均较未注射组显著升高(均有P<0.01)。
8.未鞘内注射LB的各组大鼠脊髓TUNEL阳性细胞数差异没有统计学意义(P=0.170,P=0.951)；鞘内注射LB后各组大鼠脊髓背角浅层TUNEL阳性细胞数差异显著(F=23.998,P=-0.000),提示此时各组间TUNEL阳性细胞数不同,(DIDS+SB203580)+LB组TUNEL阳性细胞计数低于其余三组,差异均有统计学意义(P<0.01)。而鞘内注射LB组大鼠脊髓背角浅层TUNEL阳性细胞数均较未注射组显著升高(均有P<0.01)。
结论LB可能通过干扰氧化磷酸化和抑制线粒体呼吸链复合物I使ATP生成减少,引发大鼠脊髓神经细胞内ROS爆发性增多,直接激活p38MAPK途径,引起细胞凋亡；并可导致线粒体C1-通道开放,线粒体膜功能受损而产生和释放更多的ROS,促使p38MAPK蛋白激活导致细胞凋亡。同时也可能因为内外膜剥离造成线粒体损伤,释放出细胞色素C和促凋亡诱导因子等,激活caspase-9基因,进而激活caspase-3基因形成活化复合物,最终引起细胞凋亡。因此,可认为LB引起的大鼠脊髓神经毒性机制可能与线粒体的C1-通道和p38MAPK信号转导途径有关。
Local anesthetics are commonly used for local anesthesia and pain therapy, but they have the potential neurotoxicity. With the wide application in nerve block anesthesia, reports increase gradually hat local anesthetics cause neurotoxicity, and clinical workers pay high attention to this phenomenon. Many research found that this neurotoxicity caused by local anesthetics cause was associated with cell apoptosis, but the exact mechanism is still not fully understood.
Research found that the mitochondrial membrane function change, inner membrane potential disappeared and protease activation thing in the mitochondria released, and arouse all kinds of apoptosis related metabolic changes, then the apoptotic cells were induced to produce characteristic form change and DNA degradation. Burst of reactive oxygen species (ROS) is one of the main factors causing the nerve cells acute injury. ROS accumulation can affect oxidoreduction balance and damage the mitochondrial membrane in nerve cells, In addition, ROS can directly affect protein, lipid, and nucleic acid make them lose function. Moreover, ROS can also be a messenger, further cause nerve damage.
Cell apoptosis pathways consist of two signal transduction pathways. One is the death receptor pathway. The death receptor of cell surface binds the death ligand by the ectodomain, introduces the death signal into cell, and activates caspase-8and caspase-3, leading to apoptosis. The other is mitochondria pathway. Stimulating factors, such as oxygen free radical and calcium overload, increase the permeability of mitochondria, decrease mitochondria membrane potential, release cytochrome C and apoptosis-inducing factor(AIF), and subsequently activate caspase-9and caspase-3, leading to apoptosis. Cell apoptosis is related to the opening of mitochondrial permeability transition pore(mPTP). The mPTP is composed of voltage dependent anion channel(VDAC), adenine nueleotide translocator(ANT) and cyclophilin D(Cyp-D). The influx of chloridion (Cl-) into mitochondria can occur through the open VDAC and Can cause mitochondfial membrane damage and collapse the membrane potential, leading to release of pro-apoptotic molecule such as cytochrome C and AIF. Accordingly, the increase in intracellular ROS induced by bupivacaine prompts the opening of VDAC, causing the influx of Cl-into mitochondria, increase of mitoehondrial osmotic pressure, swell of matrix, decrease of mitochondrial membrane potential and augmentation of permeability, and release of pro-apoptotic factors, which leads to cell apoptosis.
P38mitogen-activated protein kinase (MAPK) is an important member of MAPK family. The p38MAPK signaling pathway is activated by different stimuli associated with stress, such as H2O2, TNF-a, lipopolysaccharide and hyperosmotic fluid. Previous studies demonstrated that the p38MAPK signaling pathway controls much gene expression activity of transcription factor, affects the production of many cytokines, and regulates the synthesis of NO and cytoskeleton protein. p38MAPK participates in immunological regulation, inflammatory reaction and cell apoptosis under the circumstance of stress. Studies found that peripheral nerve toxicity of lidocaine is related to the activation of p38MAPK. Lirk found that p38MAPK inhibitor4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1Hmidazole (SB203580) Can inhibit neurocyte axon degeneration.
In previous studies, Our team found bupivacaine may interfere with oxidative phosphorylation and inhibit the complex I of the mitochondrial respiratory chain, decrease the production of ATP, lead to single phosphoric acid active adenosine protein kinase (AMP-activated protein kinase, AMPK) continuous activation, and cause the mitochondria create and release more ROS in the cells, then cause cell damage and apoptosis through the mechanisms of ROS-induced-ROS release, further study showed bupivacaine may induce nerve cell apoptosis by via p38MAPK and mitochondrial Cl-channel pathway.
Levobupivacaine (LB) is a novel, long-acting amide local anesthetics. Due to similar physical and chemical properties and anesthetic potency to that of bupivacaine with less and lower toxicity in cardiovascular system and central nervous system, it shows a wide promising application potential in clinic and becomes a hot study project to investigate its mechanism in research field. Whether Levobupivacaine induced neurotoxicity is related to ROS production and mediation, or Levobupivacaine also induced neuronal apoptosis through activation of the p38MAPK pathway and mitochondrial Cl-channel, is still not clear,
In this study, we compared the effects of SH-SY5Y cells producing ROS and apoptosis induced by different concentrations of levobupivacaine via cell biology and molecular biology methods at the cellular level. The chloride channel blocker (44'the-diisothiocyanatostilbene-2,2'-disulfonic acid disodium DIDS) and p38MAPK antagonist SB203580were used to explore whether the apoptosis triggered by levobupivacaine is related to the p38MAPK signaling pathway and mitochondrial chloride ion channel activation. The further observation focused on the effects of different concentrations of levobupivacaine to the ROS increment and apoptosis of rat spinal nerve cells at the whole animal level. Meanwhile, the SB203580and DIDS were also used to investigate whether the nerve cells apoptosis in L4-5spinal cord segment of rats triggered by levobupivacaine is related to the p38MAPK signaling pathway and mitochondrial chloride ion channel activation. By studying mitochondria and p38MAPK pathways on spinal nerve cells apoptosis induced by Levobupivacaine, our study might provide a theoretical basis to prevent from neurotoxicity induced by levobupivacaine or develop an effective pharmaceutical treatment to relieve it.
Chapter1Levobupivacaine induce ROS generation and apoptosis in SH-SY5Y cell
Objective To explore the relationship of ROS level and apoptosis induced by different concentrations of levobupivacaine in SH-SY5Y cells
Method We culture SH SY5Y cell as the research object, treated by0,0.5,1,1.5,2,2.5mM levobupivacaine culture medium for1,2,3,4,5,6h, respectively, observe the cell morphology, detect the cell vitality with CCK-8method, Hochest33258detect the changes of apoptosis morphology, detect intracellular average ROS fluorescence intensity and cell apoptosis rate using flow cytometry instrument.
Statistical the measurement data were record mean±standard deviation, SPSS13.0statistics software was used to analysis the date. Two factors factorial analysis of variance was used to analysiscellular ROS level. The cell vitality, cell apoptosis rate and the comparison between groups was analysis by single factor analysis of variance (one-way ANOVA) in single effect analysis, multiple comparisons was analysis using LSD method for the homogeneity of variance and Welch or Dunnett's T3for the unequal variances were adopted when separate effect analysis as necessary, P<0.05for the difference was statistically significant.
Results Flow cytometry results showed that the intracellular ROS level gradually increased and peaked at4h,decreased at5and6h after the cells treated by different concentration of levobupivacaine. Compared to untreated group, intracellular ROS leveln of the groups after levobupivacaine treatment1,2,3,4,5, and6h were higher,the difference was statistically significant(P<0.01).; detect the cell vitality after treated by different concentration of levobupivacaine after4h using CCK-8method, the cell viability present a dose dependent decrease cell survival, the cell survival rate was the lowest when the LB is2.5mM as (23.15±1.60)%, the results show statistical difference among the groups processed by different LB (F=2043.476, P=0.000); the apoptosis rate detected by flow cytometry instrument was statistically difference among each group(F=614.342, P=0.000), and the apoptosis rate was highest when LB was2mM; SH SY5Y cells was observed highly agglutinated and showed strong blue fluorescence after treated with different concentrations of LB using Hoechst33258dyeing, the number of cells present strong blue fluorescence nucleus was most especially in LB=2mM.
Conclusion Levobupivacaine can cause SH-SY5Y cells ROS dependence apoptosis in SH-SY5Y cells, and the cells apoptosis rate was the highest when LB=2mM, levobupivacaine can also cause dose dependent cell survival rate decreased in SH-SY5Y cells.
Chapter2Reserch on Levobupivacaine induces type SH-SY5Y apoptosis via Cl-of moyochondria and p38MAPK signal conduction pathway.
Objective To discuss the effect on Cl-pathway of moyochondria and p38MAPK caused by increased ROS of SH-SY5Y which result from LB, revealing the functions of Cl-of moyochondria and p38MAPK signal conduction pathway in the process that LB induces type SH-SY5Y apoptosis.
Methods SH-SY5Y cells randomly divided into four groups:DIDS group, SB203580group, DIDS+SB203580group and non-pretreated group. Cells of DIDS group, SB203580group and DIDS+SB203580group were pretreated respectively with50μmol/L DIDS,10μmol/L SB203580, and50μmol/L DIDS plus10μmol/L SB20358030min prior to the treatment with the culture medium containing2mmol/L levobupivacaine and cells of non-pretreated group treated with the culture medium containing2mmol/L levobupivacaine. After4hours levobupivacaine treatment, intracellular ROS level was measured by FCM, the mitochondrial membrane potential was measured by5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl benzirnidazole-carbocyanideiodine(JC-1), the p38and p-p38MAPK protein was examined by western blots, and mitochondrial Cl-concentration was measured by laser scanning con_focal microScope.4h after levobupivaeaine treatment, the cell viability was examined by MTT assay, and cell apoptosis was examined by FCM. Meanwhile, the same indicators were also be tested in the cells of each group untreated with levobupivacaine at the same time to be as contrast.
Measurement data were presented as mean+standard deviation. SPSS13.0statistical software was used for analysis. Factorial design ANOVA was used to analyze the data from ROS measurement, Cl-fluorescence intensity mitochondrial membrane potential assay, cells survival rate assay, cell apoptosis rate assay, p38and p-p38MAPK protein expression. Comparisons among groups 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 After levobupivacaine treatment, the ROS levels of DIDS group and DIDS+SB203580group were lower than that of non-pretreated group(<0.01). The results of fluorescence intensity measured by FCM showed that the ratios of mitochondrial membrane JC-1polymer/monomer in DIDS group and DIDS+SB203580group were0.82+0.01and0.79±0.01respectively, which were higher than that of non-pretreated group(0.52±0.01). The difference had statistical significance(P<0.01). Mitochondrial Cl-concentration measured by laser scanning confocal microscope showed that the Cl-concentration of DIDS group and DIDS+SB203580group were lower than that of non-pmtreated group(P<0.01). Western blot showed that the p-p38MAPK protein expression of DIDS group and DIDS+SB203580group were lower than that of non-pretreated group(P<0.01). CCK-8assay revealed that the survival rates of DIDS and SB203580groups were higher than that of non-pretreated group(P<0.01), but lower than that of DIDS+SB203580group (P<0.01). Cells apoptosis rates of group DIDS, DIDS+SB203580, SB203580and non-pretreated were23.42±1.58%,12.73±0.96%,24.53±0.93%and38.17±0.22%, which were measured by FCM after levobupivacaine treatment. The apoptosis rate of SB203580group was lower than that of non-pretreated group(P<0.01), but there were no distinct difference in ROS levels and the ratios of JC-1polymer/monomer fluorescence intensity between the SB203580group and non-pretreated group (P<0.05). Hoechst33258staining assay revealed that the apoptosis rates of group DIDS, DIDS+SB203580, SB203580and non-pretreated were22.88±1.13%,12.45±0.74%,24.68±0.85%and37.87±0.99%.
Conclusion These findings indicate that the mitochondrial VDAC channel and activated p38MAPK pathway effected by levobupicavmne probably causes apoptosis. Increased intracellular ROS induced by Levobupivacaine opens mitochondrial VDAC channel and results in an influx of Cl-into mitochondria, which seems to be responsible for the mitochondrial depolarization, permeability enlargment of the mitochondria and the p38MAPK activation.
Chapter3Effects of different concentrations of levobupivacaine on ROS increasement and cell apoptosis of spinal nerve in rats
Objective To compare the effects of the ROS increasement and cell apoptosis of spinal nerve in rats with different concentrations of levobupivacaine and to investigate the extent of spinal nerve injury.
Methods After inserted intrathecal catheter, adult healthy male SD rats (weight220～250g) were divided into five groups (n=6):normal control group (C), the solvent group (D),0.5%levobupivacaine group (LB0.5),2.0%levobupivacaine the group (LB2.0) and5.0%levobupivacaine group (LB5.0) using a random number table. Group C, D, LB0.5, LB2.0and LB5.0were injected with saline, dimethyl sulfoxide,0.5%,2.0%,5.0%levobupivacaine20μl via intrathecal catheter respectively. Rats in each group were observed and recorded tail flick response latency (TFL) in the percentage of the maximum effect (MPE) and hind limb motor function score (MF) before intrathecal administration (basal state), after administration of lOmin,20min,30min,1h,2h,4h,8h,1d,2d,3d. After3d, rats were perfused with paraformaldehyde, and L4-5spinal cord segments were cut for pathological observation. The ROS level of nerve tissue was detected, and caspase-3and apoptosis of nerve cells were identified via immunohistochemical assay and TUNEL assay respectively.
All of the data were analyzed using SPSS13.0statistical software and were expressed as mean±standard deviation (x±s). The data among the TFL groups before and after catheter were compared using one-way ANOVA, and paired t-test was used between before and after catheter. The MPE values were analyzed via repeated measures analysis of variance. Caspase-3-positive cell counts and apoptotic cell counts were compared via single-factor analysis of variance, and the Bonferroni method (homogeneity of variance) or Dunnett's T3method (unequal variances) was used in multiple comparisons. Motor function scores were expressed with median (10,90bit point median percentile), and Kruskal-Wallis test was used among groups. P <0.05was considered statistically significant.
Result
1. Comparison of MPE among groups in rats after intrathecal injection shows MPE were different among groups (F=14220.696, P=0.000), and there was significant MPE change (F=5358.641, P=0.000) after injection with MPE positively correlating to time. Also, an interaction between the groups and the time was observed (F=1410.001, P=0.000). The results showed the differences among groups differed in time points. Further separate effects analysis found that there was no significant difference of MPE (P>0.05) between Group C and Group D at all time points. When compared within each group at different time points, MPE in Group LB0.5, Group LB2.0and Group LB5.0achieved their peak and downward in10min after the intrathecal injection. The MPE in Group LB0.5and Group LB2.0returned to baseline levels at4h and8h after injection, respectively, and Group LB5.0returned to baseline levels after2d.
2. The comparison of MF scores showed no motor dysfunction in Group C and Group D after intrathecal injection. The motor function was significantly different after lOmin-2h intrathecal injection in each group according to the mean rank analysis. The MF scores were significantly increased among Group LB0.5, LB2.0and LB5.0than that of Group C and Group D. The MF score of Group LB0.5resumed in20min, LB2.0resumed in one hour MF, and LB5.0resumed in4h.
3. Group C, D and LB0.5shows intact neuronal cell morphology and structure of spinal cord tissue in H&E. Group LB2.0shows varying degrees of damage with nuclei lyses in the neuronal structures of spinal cord tissue. Group LB5.0shows obvious spinal cord neuronal apoptosis with scattered apoptotic cells and apoptotic bodies.
4. The comparison of ROS levels in each spinal cord tissues of rats shows there were no significant changes in Group C and Group D. The ROS levels in Groups LB0.5, LB2.0and LB5.0increased with the increase of drug concentration. The difference was statistically significant (P<0.01), with a ROS level of192.93±10.05in Group LB5.0.
5. The Immunohistochemical positive cells counts of caspase-3shows no statistically significant among Group C, D and LB0.5in the spinal cord nerve tissue of rats. However, the caspase-3positive cells were significantly (P<0.01) increased in Group LB2.0and Group LB5.0compared with Group C.
6. The TUNEL positive cells percentage detection shows no statistically significant among Group C, D and LB0.5in the spinal cord nerve tissue of rats. However, the TUNEL positive cells percentage was significantly (P<0.01) increased in Group LB2.0and Group LB5.0compared with Group C.
Conclusion Levobupivacaine can cause a concentration-dependent ROS increase and induce apoptosis in the rat spinal cord nerve tissue, resulting in nerve damage and sensory dysfunction at a certain extent. The higher of concentration of Levobupivacaine, the more delayed sensory function recovery was observed.
Chapter4Effects of SB203580and DIDS on spinal nerve cells apoptosis induced by levobupivacaine in rat
Objective To investigate the effect of SB203580and DIDS on apoptosis in rat spinal nerve injured by levobupivacaine and to explore the related spinal neurotoxicity mechanism.
Methods After inserted intrathecal catheter, adult healthy male SD rats (weight220～250g) were divided into four groups (n=6):Group LB, Group DIDS+LB, Group SB203580+LB, Group (DIDS+SB203580)+LB. Group LB were slowly intrathecal injected with20μl of0.9%saline via micro-syringe, and then slowly (10') injected with20μl of5.0%levobupivacaine via micro-syringe after2hours. Group DIDS+LB were given14mg/kg of DIDS through the femoral vein via the program-controlled micro-pump [4ml/(Kg-h)] within2hours, and then slowly injected with20μl of5.0%levobupivacaine. Group SB203580+LB were intrathecal injected with20μl of0.1%SB203580(dissolved in2%DMSO) within2hours, and then slowly injected with20μl of5.0%levobupivacaine. Group (DIDS+SB203580)+LB were intrathecal injected with20μl of0.1%SB203580, then given14mg/kg of DIDS through the femoral vein via the program-controlled micro-pump within2hours, and then slowly injected with20μl of5.0%levobupivacaine. Six rats in each group were separated using a random number table and the sensory-motor function was observed. The tail flick response latency (TFL) were observed and recorded using the maximum percentage effect (MPE) and hind limb motor function score (MF) after30min,1h,2h,4h,8h,12h,16h, Id,2d,3d of intrathecal administration. After3d, rats were perfused with paraformaldehyde, and L4-5spinal cord segments were cut for pathological observation, immunohistochemical detection of caspase-3, caspase-9positive cells and TUNEL in situ detection of apoptosis. The other six rats in each group were perfused with paraformaldehyde, and L4-5spinal cord segments were cut for pathological observation, ELISAL detection of he ROS level of nerve tissue, and Western blot analysis of p38MAPK and p-p38MAPK protein expression.
Using SPSS13.0software for statistical treatment, measurement data were expressed as mean±standard deviation. Comparisons among groups TFL were made by analysis of variance (ANOVA) and paired-sample T test. MPE were made by analysis of Repeated Measures. p-p38MAPK and p38MAPK expression, caspase-3、 caspase-9positive cell counts and apoptotic cell counts were made by analysis of variance (ANOVA). MF scores are presented as median (10th—90th percentiles) and were compared using the Kruskal-Wallis test followed by the Mann-Whitney u-test. P<0.05was considered significant.
Result
1. Comparison of MPE among groups in rats after intrathecal injection shows MPE were different among groups (F=143.324, P=0.000), and there was significant MPE change (F=130.948, P=0.000) after injection with MPE positively correlating to time. Also, an interaction between the groups and the time was observed (F=179.714, P=0.000). The results showed the differences among groups differed in time points. When compared within each group at different time points, MPE in Group LB, Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB achieved their peak in30min and downward after4-8h after the intrathecal injection. The MPE in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB recovered at12h-ld after injection, which is faster than that of Group LB. The recovery of Group (DIDS+SB203580)+LB is faster than that of Group DIDS+LB and Group SB203580+LB. all the groups returned to baseline levels after2d.
2. The comparison of MF scores of rats showed there was no significant difference of the hind limb motor function recovery at each time point among each groups after intrathecal injection. The MF scores in different groups were recovered in4hours.
3. Histopathological observation found that the neurons morphology and structural were damaged, cytoplasmic Nissl body decreased and cracked with nucleus lyses in Group LB. The neuronal structures in Group DIDS+LB and Group SB203580+LB show varying degrees of damage. The neuronal structures in Group (DIDS+SB203580)+LB were Less damaged; the cytoplasmic Nissl body morphology was still intact and the ratio of nucleus to cytoplasm was normal when compared with the first three groups.
4. The comparison of ROS levels in each spinal cord tissues of rats shows no statistically significant differences (P>0.05) between Group DIDS+LB and Group (DIDS+SB203580)+LB. There were also no statistically significant differences (P>0.05) between Group SB203580+LB and Group LB. The ROS levels in Groups DIDS+LB and Group (DIDS+SB203580)+LB were less than that in Group SB203580+LB and Group LB, and the difference was statistically significant (P<0.05).
5. The Caspase-3positive cell counts in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that in Group LB (P<0.05). The Caspase-3positive cell counts in Group DIDS+LB, Group SB203580+LB were higher than that in Group (DIDS+SB203580)+LB, the difference was statistically significant (P<0.05).
6. The Caspase-9positive cell counts showed no significant difference (P>0.05) between Group SB203580+LB and Group LB. It was also no significant difference (P>0.05) between Group DIDS+LB and Group (DIDS+SB203580)+LB. The Caspase-9positive cell counts in Group DIDS+LB and Group (DIDS+SB203580)+LB were lower than that in Group SB203580+LB and Group LB (P<0.05).
7. The p-p38MAPK expression level is significant different (F=234.117, P=0.000) among groups. The p-p38MAPK expression levels in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that in Group LB (P<0.05). The p-p38MAPK expression levels in Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that of in Group DIDS+LB (P <0.05). The p38MAPK expression level showed no differences (F=0.936, P=0.442) among groups.
8. The apoptosis rate in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that in Group LB (P<0.05). The apoptosis rate in Group DIDS+LB, Group SB203580+LB were higher than that in Group (DIDS+SB203580)+LB, the difference was statistically significant (P<0.05).
Conclusion Levobupivacaine may interfere with oxidative phosphorylation and inhibit mitochondrial respiratory chain complex I, which decreased the production of ATP and triggered explosive increasement of ROS in the rat spinal cord nerve cells. On the one hand, the direct activation of p38MAPK pathway can induce apoptosis; On the other hand the mitochondrial Cl-channel opening and mitochondrial membrane dysfunction may lead to the production and release of more ROS, which activate p38MAPK protein and result in apoptosis. Mitochondrial Damage may also be caused by the peeling of inner and outer membrane, which may release cytochrome C and apoptosis-inducing factors. Thus it may activate caspase-9and caspase-3to form an activated complex, and ultimately induce apoptosis. Therefore, the mechanism of neurotoxicity caused by levobupivacaine in the rat spinal cord may relate to mitochondrial Cl-channels and p38MAPK signal transduction pathway.
引文
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