【作者】 李乐；

【导师】 徐世元；

【作者基本信息】 南方医科大学 ， 麻醉学， 2013， 博士

【摘要】 糖尿病周围神经病(diabetic peripheral neuropathy, DPN)是最常见的糖尿病慢性并发症之一,威胁糖尿病患者的生命并影响其生活质量。糖尿病足部溃疡与DPN密切相关,病史长可最终导致截肢。此种并发症可能因长期高糖环境导致,也有报道高糖环境与周围及中枢神经的结构、功能异常相关。但至今DPN的发病机制仍不明确。糖尿病患者数量的增加导致DPN发病率逐年升高,此类病人需要区域神经阻滞下手术或镇痛的人数亦随之逐年增加。局部麻醉药广泛用于神经阻滞与镇痛已逾百年。局部麻醉药虽具有良好的安全性,但近年来关于其神经毒性的报道日渐增多,因而引起了麻醉医生的关注。但局部麻醉药神经毒性的具体发生机制还未完全阐明。高糖环境引起已存在或潜在的神经损伤可能增强局部麻醉药的神经毒性。研究DPN患者是否对局部麻醉药的神经毒性更加敏感及其机制,有利于防治糖尿病患者局部麻醉药中毒及选择局部麻醉药种类和剂量。体内外实验报道局部麻醉药与高糖可诱导细胞凋亡,以致细胞功能障碍甚至死亡。有学者发现线粒体功能障碍,可引起细胞内的活性氧族(reactive oxygen species, ROS)产生增多,而增多的ROS又可正反馈影响线粒体功能,同时ROS的爆发与内质网应激(endoplasmic reticulum stress, ERS)有关。线粒体功能障碍及内质网应激达到一定程度后,分别激活线粒体及内质网依赖的凋亡通路,最终导致细胞凋亡。本研究构建体外高糖神经损伤模型,研究高糖环境下的神经细胞是否对于局部麻醉药的神经毒性更加敏感,及其具体作用机制一是否与细胞凋亡,线粒体功能障碍以及内质网应激有关。另一方面,在活性氧抑制剂一银杏内酯B(Ginkgolide B, GB)作用下,探讨布比卡因引起线粒体功能障碍及内置网应激是否与ROS有关,以明确ROS下游靶点。第一部分建立体外高糖神经损伤模型目的在体外建立SH-SY5Y细胞株的高糖神经损伤模型,观测高糖环境对SH-SY5Y细胞活性及凋亡程度的影响。方法将SH-SY5Y细胞分为C组：无血清培养基处理24h；‘M1-5组：分别用溶有5,25,50,100,200mmol/L甘露醇的无血清培养基培养24h；G1-5组：分别用溶有5,25,50,100,200mmol/L无水葡萄糖(除培养基含有的葡萄糖外)的无血清培养基培养24h。Cell counting Kit-8(CCK-8)试剂盒检测细胞活性,流式细胞术(flow cytometry, FCM)检测细胞凋亡数。透射电镜观察SH-SY5Y细胞超微结构变化。计量资料均以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。不同浓度的甘露醇与葡萄糖对细胞活性及凋亡数采用两因素析因设计方差分析,单独效应分析时,组间比较采用t检验或单因素方差分析,多重比较采用LSD法,方差不齐时采用Welch法和Dunnett’s T3法,P<0.05为差异有统计学意义。结果细胞活性、凋亡数在不同浓度的葡萄糖组间(F=60.064,P=0.000；F=241.828,P=0.000)及甘露醇组间(F=24.661,P=0.000；F=88.191,P=0.000)的差异均具有统计学意义。有与对照组相比,不同浓度的葡萄糖均可引起SH-SY5Y细胞活力下降,浓度增高,细胞活性下降程度也随之增加。100mmol/L的葡萄糖对SH-SY5Y细胞活性的影响大于同浓度的甘露醇(t=-3.052,P=0.012),差异具有统计学意义。溶有25,50,100,200mmol/L葡萄糖培养基培养24h后,SH-SY5Y细胞的凋亡数均高于同浓度的甘露醇组(t=5.531,P=0.001；t=4.231,P=0.003；t=15.267,P=0.000,t=6.284,P=0.000),差异具有统计学意义,并在浓度为100mmol/L时,两组之间的凋亡细胞数目差异最大。结论葡萄糖对SH-SY5Y细胞有毒性作用,且随着浓度增加,细胞毒性相应增加。在较高浓度时(25,50,100,200mmol/L),葡萄糖对细胞凋亡的影响强于甘露醇,尤其是M4与G4之间,差别最为明显。推测,100mmol/L的葡萄糖的细胞毒性主要由代谢产生,而不是渗透压。故选择100mmol/L葡萄糖培养24h的SH-SY5Y细胞作为体外高糖神经损伤模型。第二部分高糖培养的SH-SY5Y细胞对布比卡因神经毒性更加敏感目的采用高糖培养的SH-SY5Y细胞,予以布比卡因作用,通过电镜观察亚细胞结构,测定细胞活力,细胞凋亡率,探讨高糖培养细胞是否对布比卡因伸进细胞毒性更加敏感。方法SH-SY5Y细胞100mmol/L高糖培养或者无血清培养基培养24h后,用含有不同浓度(0.25,0.5,1.0,2.0mmol/L)布比卡因的无血清培养基24h。CCK-8试剂盒检测细胞活性,FCM检测细胞凋亡数。选择合适的布比卡因浓度后,SH-SY5Y细胞离体培养,分为4组,即：对照组(Con组,无血清培养基培养细胞48h),布比卡因组(Bup组,无血清培养基培养24h+含有1mmol/L布比卡因的无血清培养基培养24h),高糖组(Glu组,含有100mmol/L葡萄糖培养24h+无血清培养基培养24h),高糖+布比卡因组(Glu+Bup组,含有100mmol/L葡萄糖培养24h+1mmol/L布比卡因的无血清培养基培养24h),透射电镜观察细胞亚细胞结构变化。计量资料均以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。高糖预处理与非高糖预处理之间的细胞活性及凋亡数采用两因素析因设计方差分析,单独效应分析时,组间比较采用t检验或单因素方差分析,多重比较采用LSD法,方差不齐时采用Welch法和Dunnett’s T3法,P<0.05为差异有统计学意义。结果细胞活性、凋亡数在不同浓度布比卡因组间(F=72.039,P=0.000；F=33.522,P=0.000)及高糖预处理后的布比卡因组间(F=72.039,P=0.004；F=72.039,P=60.832)的差异具有统计学意义。布比卡因可引起细胞活性下降以及细胞凋亡数增加,并随着布比卡因浓度增高,这一作用相应增强。高糖预处理较非高糖预处理的细胞,在0.25,0.5,1.0mmol/L布比卡因处理后,细胞活性下降(t=10.450,P=0.000；t=11.283,P=4.302；t=15.267,P=0.002)且凋亡细胞数目增加(t=-2.413,P=0.036；t=-3.647,P=0.004；t=-3.761,P=0.004),差异均具有统计学意义。选择1mmol/L作为后续实验实验的布比卡因浓度。电镜下观察SH-SY5Y细胞亚结构,较Con组,Glu组与Bup组细胞粗面内质网出现脱颗粒、肿胀等表现,线粒体出现水肿等形态学改变,Glu组细胞亚结构与Bup组类似,但程度轻,Glu+Bup组细胞亚细胞机构破坏较其他组更明显,基本细胞结构模糊。结论高糖预处理明显增加了布比卡因的神经毒性,导致细胞凋亡数也较非预处理组明显增加,并产生不同的细胞器结构的改变。提示高糖环境培养的细胞较正常培养的细胞对于布比卡因的神经毒性更加敏感,推测这种敏感性的增加可能与细胞凋亡增加及细胞器的结构功能改变有关。第三部分高糖培养的SH-SY5Y细胞在布比卡因作用下线粒体功能变化目的通过研究高糖培养的SH-SY5Y细胞在布比卡因作用下,细胞内的ROS水平,线粒体特异性产生的ROS,线粒体复合物Ⅰ、Ⅲ活性,线粒体膜电位的变化以及cleaved Caspase-3和HtrA2蛋白表达水平,以探讨高糖环境引起布比卡因神经毒性的增加是否与ROS产生、线粒体功能障碍有关。方法SH-SY5Y细胞离体培养,分为4组,即：对照组(Con组,无血清培养基培养细胞48h),布比卡因组(Bup组,无血清培养基培养24h+含有1mmol/L布比卡因的无血清培养基培养24h),高糖组(Glu组,含有100mmol/L葡萄糖培养24h+无血清培养基培养24h),高糖+布比卡因组(Glu+Bup组,含有含有100mmol/L葡萄糖培养24h+1mmol/L布比卡因的无血清培养基培养24h)。FCM分析细胞内ROS水平及线粒体特异性的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。分光光度计检测线粒体复合物Ⅰ和Ⅲ的活性。Western blot法检测cleaved Caspase-3和HtrA2蛋白表达水平。计量资料均以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。细胞内ROS水平,线粒体特异产生的ROS含量,线粒体膜电位,线粒体呼复合物Ⅰ和Ⅲ及cleaved Caspase-3和HtrA2蛋白表达水平组间比较采用单因素方差分析,多重比较采用LSD法,方差不齐时采用Welch法和Dunnett’s T3法,P<0.05为差异有统计学意义。结果细胞内及线粒体内的ROS水平、线粒体复合物Ⅰ和Ⅲ活性、JC-1聚合体/单体比值及HtrA2和Cleaved caspase-3蛋白表达水平在不同组间的差异均具有统计学意义(F=121.071；F=78.122；F=481.327,F=78.561；F=30.997；F=10.241,F=9.603；P均小于0.05)。Bup组及Glu组均较Con组细胞内的ROS产生水平增高(P=0.000,P=0.000),Glu+Bup组的水平高于Bup组(P=0.000),差异均具有统计学意义。线粒体特异性的ROS与细胞内ROS水平结果类似。FCM检测线粒体膜JC-1聚合体、单体比值显示,Bup组和Glu组分别为1.96±0.29和3.17±0.78均低于Con组(6.58±2.07)(P=0.000,P=0.002),而Glu+Bup组的比值(0.59+0.30)较Bup减低(P=0.047),差异具有统计学意义。布比卡因均可影响线粒体复合物Ⅰ和Ⅲ活性(P=0.000,P=0.000),高糖预处理后加强了布比卡因对于线粒体复合物Ⅰ和Ⅲ活性的影响(P=0.000,P=0.002)。Western blot显示,Bup组的HtrA2和Cleaved caspase-3蛋白表达水平均高于Con组(P=0.019,P=0.033),Glu+Bup组较Bup组表达增高(P=0.041,P=0.018),差异均具有统计学意义。结论高糖预处理可增强布比卡因引起的细胞内ROS水平的升高,伴随着线粒体特异性产生的ROS产生增多,此种增高可能由于由线粒体复合物Ⅰ和Ⅲ活性减低产生,增多的ROS引起线粒体膜电位去极化,导致凋亡蛋白表达,最终激活线粒体依赖的凋亡通路。第四部分高糖培养的SH-SY5Y细胞在布比卡因作用下产生内质网应激目的通过研究高糖培养的SH-SY5Y细胞在布比卡因作用下,细胞内Grp78和Caspase-12mRNA及蛋白水平的变化,以探讨高糖环境导致的布比卡因神经毒性增加是否与内质网应激(endoplasmic reticulum stress, ERS)有关。方法SH-SY5Y细胞离体培养,分为4组,即：对照组(Con组,无血清培养基培养细胞48h),布比卡因组(Bup组,无血清培养基培养24h+含有1mmol/L布比卡因的无血清培养基培养24h),高糖组(Glu组,含有100mmol/L葡萄糖培养24h+无血清培养基培养24h),高糖+布比卡因组(Glu+Bup组,含有含有100mmol/L葡萄糖培养24h+1mmol/L布比卡因的无血清培养基培养24h)。定量逆转录聚合酶链反应(quantitative reverse transcription polymerase chain reaction, qRT-PCR)检测细胞内Grp78和Caspase-12mRNA水平。Western blot检测Grp78和Caspase-12蛋白表达。计量资料均以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。Grp78和Caspase-12mRNA及蛋白水平采用单因素方差分析,多重比较采用LSD法,方差不齐时采用Welch法和Dunnett’s T3法,P<0.05为差异有统计学意义。结果不同处理组间Grp78和caspase-12mRNA及蛋白表达水平(F=15.503,F=11.525；F=8.864,F=29.639,P均小于0.05)的差异均具有统计学意义。与Con组相比,布比卡因及高糖环境均可引起Grp78的mRNA(P=0.010, P=0.006)及蛋白表达增高(P=0.000,P=0.001),高糖环境预处理可加强布比卡因对Grp78转录和翻译程度的影响(P=0.008,P=0.021),差异具有统计学意义。布比卡因及高糖环境对于Caspase-12蛋白表达的影响与对Grp78类似,但只有Glu+Bup组的Caspase-12的mRNA水平较Con组有所升高(P=0.009)。结论布比卡因可以导致ERS,反映在Grp78的蛋白及mRNA水平上,对于Caspase-12的mRNA影响不明显,提示只有当ERS进展到一定程度才可能激活内质网依赖的细胞凋亡通路。高糖环境可增强布比卡因激活ERS的作用,并激活内质网依赖的凋亡通路。这可能是高糖培养的神经细胞对于布比卡因神经毒性更加敏感的作用机制之一。第五部分银杏内酯B对布比卡因神经毒性的影响目的探讨抗氧化剂银杏内酯B (Ginkgolide B, GB)对布比卡因神经毒性的影响,研究线粒体功能障碍,内质网应激是否与ROS有关,寻找ROS的下游作用靶点。方法SH-SY5Y细胞离体培养,用不同浓度的GB(5,10,20,40μ mol/L)预处理6h后,予以1mmol/L布比卡因作用细胞24h,FCM检测细胞凋亡情况。分为4组,即：对照组(Con组,无血清培养基培养细胞30h),布比卡因组(Bup组,无血清培养基培养6h+含有1mmol/L布比卡因的无血清培养基培养24h),银杏内酯B组(GB组,含有有40μ mol/LGB的无血清培养基培养6h+无血清培养基培养24h),银杏内酯B+布比卡因组(GB+Bup组,含有有40μ mol/LGB的无血清培养基培养6h+含有1mmol/L布比卡因的无血清培养基培养24h)。FCM检测细胞内ROS水平,JC-1检测线粒体膜电位。分光光度计检测线粒体复合物Ⅰ和Ⅲ的活性。Western blot法检测cleaved Caspase-3, HtrA2, Grp78和Caspase-12蛋白表达水平。计量资料均以均数±标准差(x±s)表示,采用SPSS17.0统计软件分析。细胞凋亡情况,细胞ROS水平,线粒体呼吸链复合物Ⅰ和Ⅲ,线粒体膜电位变化,cleaved caspase-3, HtrA2, caspase-12和Grp78蛋白表达量组间比较采用单因素方差分析,多重比较采用LSD法,方差不齐时采用Welch法和Dunnett’s T3法,P<0.05为差异有统计学意义。结果不同处理组间细胞凋亡率的差异具有统计学意义(F=167.786,P=0.000)。FCM检测细胞凋亡,发现10,20,40μ mol/L GB均可减少由布比卡因引起的细胞凋亡(P=0.000,P=0.000,P=0.000),且GB浓度越高,这种保护作用越明显。选用40μ mol/L GB进行后续实验。ROS阳性率、线粒体呼吸链复合物Ⅰ和Ⅲ活性、红荧光与绿荧光的比值以及Caspase-3, HtrA2, Grp78和Caspase-12蛋白表达水平在不同处理组间的差异具有统计学意义(F=226.503；F=118.253,F=50.191；F=50.154；F=4.510,F=81.502；F=8.137,F=9.277；P均小于0.05)。GB+Bup组细胞内的ROS水平低于Bup组(P=0.000),而单纯GB处理对细胞内ROS水平无影响(P=0.101)。FCM检测线粒体膜JC-1聚合体/单体比值显示,Bup组为1.12±0.43,低于Con组(8.41±1.41)(P=0.000),而GB+Bup组的比值(3.55+0.71)较Bup组升高(P=0.004),差异均具有统计学意义。与Bup组比较,经GB预处理后的线粒体复合物Ⅰ和Ⅲ活性增强(P=0.004,P=0.004)。Western blot检测cleaved Caspase-3, HtrA2, Grp78和Caspase-12蛋白表达水平,Bup组均较Con组表达增加(P=0.012,P=0.000,P=0.001,P=0.001),GB预处理后,蛋白水平表达下降(P=0.032,P=0.001,P=0.038,P=0.016),差异具有显著性。结论抗氧化剂GB减少细胞内的ROS水平,减少布比卡因引起的细胞凋亡,对线粒体膜复合物Ⅰ和Ⅲ活性有有一定的保护作用,同时可防止因布比卡因引起的线粒体膜电位去极化。GB预处理减少了内质网应激特异性蛋白及线粒体凋亡通路特异蛋白的表达。提示降低胞内的ROS水平,可抑制布比卡因对于线粒体功能的破坏及对内质网应激的激活,最终导致细胞凋亡数减少,推测线粒体功能及内质网应激可能是增高细胞内ROS的作用靶点,且与布比卡因神经毒性有关。更多还原

【Abstract】 Diabetic peripheral neuropathy (DPN) is a life-threatening condition and the most common complication of diabetes, which affects at least50%of all diabetic patients in their lifetimes. Diabetic foot ulcers, frequently leading to the need for amputation, are common. Longstanding hyperglycemia results in such complications, and may result in functional and structural deficits in both the central and peripheral nervous systems. The precise pathogenesis of DPN remains unclear. The increasing incidence of diabetes has led to an increase in the number of patients presenting with DPN; and these patients frequently require regional anesthesia to manage end-organ complications.Although local anesthetics have traditionally been accepted as safe, they have also been shown to be neurotoxic. Recent studies have demonstrated that anesthetic related neurotoxicity occurs through the apoptotic pathway. The underlying molecular mechanisms for this observation are not clearly understood. The pre-existing or potential nerve cell injury induced by hyperglycemia may magnify the neurotoxicity of local anesthetics. To study whether patients with DPN is more sensitive to local anesthetics will prevent the neuro toxicity of local and regulate dose of local anesthetics.Apoptosis has been proposed as a possible mechanism for hyperglycemia or local anesthetic-induced neural dysfunction and cell death in both the in vitro and in vivo setting. The mitochondria dysfunction and endoplasmic reticulum (ER) stress may be the underlying mechanism of neurotoxicity induced by high glucose and bupivacaine associating with overproduction of ROS.In this study, model of hyperglycemia in vitro was established in order to examine the impact of high glucose on the neurotoxicity induced by bupivacaine and how high glucose modulates the bupivacaine toxicity in vitro. On the other hand, to research the effect of antioxidant, GB, on the neurotoxicity induced by bupivacaine, and the relationship between ROS and Mitochondria Dysfunction and ER stress.Section Ⅰ To establish model of hyperglycemia in vitroObjective To construct model of hyperglycemia in vitro in SH-SY5Ycell lines and observe the impact of hyperglycemia on cell viability and apoptosis. Methods Cells were divided into the C group:cells were incubated with serum-starved medium for24h; M1-5group:cells were with serum-free medium of increasing mannitol concentrations(5,25,50,100,200mmol/L) for24h; G1-5group cells were with serum-free medium of increasing glucose concentrations(5,25,50,100,200mmol/L) for24h (in addition to the glucose included in DMEM/F12medium). Cell viability and apoptosis were investigated with a CCK-8assay and flowcytometry, respectively.Values were expressed as the mean±standard deviation (SD), using SPSS17.0statistical software for statistical analysis. The apoptosis and cell viability assays were analyzed by Factorial design ANOVA. Multiple comparisons tests were performed by LSD. A p-value of less than0.05was considered to be statistically significant.Results There was significant differemce among M1-5groups (F=60.064, P=0.000; F=241.828,P=0.000) and G1-5groups (F=24.661, P=0.000; F=88.191, P=0.000) on cell viability and apoptotic rate. Apoptosis increased and cell viability decreased with higher concentrations of glucose or mannitol. There was significantly difference between100mmol/L glucose and mannotol on cell viability (t=-3.052, P=0.012). However, at each concentration, glucose had a significantly higher apoptotic effect than glucose, except5mM (t=5.531, P=0.001;t=4.231, P=0.003; t=15.267, P=0.000,t=6.284, P=0.000). This difference was apparent at a glucose concentration of100mM.Conclusions High glucose has toxicity effect on SH-SY5Y cells. This difference between glucose and mannitol group was apparent at a glucose concentration of100mM, suggesting that glucose toxicity is mainly determined by the metabolite effect. In a separate experiment, and after establishing the optimal glucose concentration to induce apoptosis, we tested bupivacaine’s apoptotic effect onSH-SY5Y cells that were exposed to100mM of glucose for24hours. Section II Cells incubated in hyperglycemia condition are more sensitive to bupivacaineObjective SH-SY5Y cells were observed with a transmission electron microscope, examed cell viability and apoptosis in order to find whether cells in hyperglycemia condition are more sensitive to bubivacaine.Methods SH-SY5Y cells were pre-treated with100mmol/L of glucose in vitro, to imitate DPN prior to administration of different concentrations bupivacaine(0.25,0.5,1.0,2.0mmol/L) or placebo. Cell viability and apoptosis were investigated with a CCK-8assay and flowcytometry, respectively. After establishing the optimal bupivacaine concentration to induce apoptosis, cells were assigned to four groups:(i) Control (Con):untreated;(ii) Bup:cells treated with1mM bupivacaine for24hours;(iii) Glu:cells treated with100mM glucose for24hours;(iv) Glu+Bup:cells treated with100mM glucose for24hours prior to bupivacaine administration for24hours. Cell was observed with a transmission electron microscope to find morphological changes of cells.Values were expressed as the mean±standard deviation (SD), using SPSS17.0statistical software for statistical analysis. The apoptosis and cell viability assays were analyzed by Factorial design ANOVA. Multiple comparisons tests were performed by LSD. A p-value of less than0.05was considered to be statistically significant.Results There was significant differemce among bupivacaine groups (F=72.039, P=0.000; F=33.522, P=0.000) and bupivacaine with glucose pretreatment groups (F=72.039, P=0.004; F=72.039, P=60.832) on cell viability and apoptotic rate.Apoptosis increased and cell viability decreased with higher concentrations of bupivacaine. Bupivacaine induced cell growth inhibition in a concentration-dependent manner. Hyperglycemic conditions enhanced cytotoxicity at each concentration except2mM(t=10.450,P=0.000;t=11.283,P=4.302;t=15.267, P=0.002). Hyperglycemic conditions increased bupivacaine-induced apoptosis (t-2.413, P=0.036;t=-3.647, P=0.004; t=-3.761, P=0.004). By utilizing the transmission electron microscope, normal SH-SY5Y cells were round and regular, with normal morphology of rough endoplasmic reticulum(rER) and mitochondria(Mt). After exposure to bupivacainefor24hours without glucose pretreatment, rER demonstrated degranulation and expansion related to diminished protein synthesis, and mitochondrial swelling. After treatment with glucose for24hours, rER demonstrated degranulation and expansion, and mild mitochondrial swelling. Cells in the bupivacaine with glucose pretreatment group showed a disintegrated structure.Conclusions Hyperglycemic conditions are synergistic with bupivacaine-induced apoptosis and cell injury in SH-SY5Y cells, suggesting cells incubated with high glucose may be more sensitive to local anesthetics accociating with apoptosis and organelle altered.Section Ⅲ The effect of bupivacaine on SH-SY5Y cells mitochondrial in hyperglycemic conditionsObjective To investigate whether the enhanced neurotoxicity of bupivacaine mediates the considerable increase of ROS and mitochondrial dysfunction.Methods Cells were assigned to four groups:(i) Control (Con):untreated;(ii) Bup:cells treated with1mM bupivacaine for24hours;(iii) Glu:cells treated with100mM glucose for24hours;(iv) Glu+Bup:cells treated with100mMglucose for24hours prior to bupivacaine administration for24hours. Intracellular ROS level and mitochondrially generated ROS were measured by FCM.5,5’,6,6’-tetrachloro-1,1’,3，3’-tetraethylbenzimidazole-carbocyanide iodine(JC-1) was employed to measure mitochondrial depolarization inSH-SY5Y cells. Mitochondrial complexes I and III activity were also studied. cleaved Caspase-3and HtrA2 expression were measured by Western blot.Values were expressed as the mean±standard deviation (SD), using SPSS17.0statistical software for statistical analysis. Data was analyzed by one-way ANOVA. Multiple comparisons tests were performed by LSD. A p-value of less than0.05was considered to be statistically significant.Results There was significant difference among groups on ROS levels, activities of complex I and III, ratios of mitochondrial membrane JC-1polymer/monomer and cleaved capsase-3and HtrA2protein levels (F=121.071; F=78.122; F=481.327, F-78.561; F=30.997; F=10.241, F=9.603; all P values<0.05). After treatment with100mM glucose or/and1mM bupivacaine, intracellular ROS increased(P=0.000, P=0.000), and the ROS levels of group pretreated with glucose significantly higher than that of non-pretreated group (P=0.000). The results of fluorescence intensity measured by FCM showed that the ratios of mitochondrial membrane JC-1polymer/monomer in Bup group and Glu group were1.96±0.29and3.16±0.78, respectively, which were lower than that of Con group (6.58±2.07)(P=0.000, P=0.002). And that in Glu+Bup group (0.59+0.30)was lower than that in Bup group, significantly (P=0.047). The decreased activities of complex I and III induced by bupivacaine were enhanced by high glucose pretreatment(P=0.000, P=0.002). Bupivacaine without glucose pretreatment resulted in significant increases both cleaved capsase-3and HtrA2protein levels (P=0.019, P=0.033). These levels were significantly higher in the pretreated group, as compared to untreated controls (P=0.041, P=0.018)Conclusions High glucose increased the intracellular ROS production induced by bupivacaine which may be associated with decrease in mitochondrial complex I and III activity. The increase of ROS production resulted in dissipation of the mitochondrial membrane potential, which activated mitochondrial-dependent apoptotic pathway.Section IVThe effect of bupivacaine on endoplasmic reticulum stress in hyperglycemic conditionsObjective To investigate whether the enhanced neurotoxicity of bupivacaine mediates endoplasmic reticulum stress.Methods Cells were assigned to four groups:(i) Control (Con):untreated;(ii) Bup:cells treated with1mM bupivacaine for24hours;(iii) Glu:cells treated with100mM glucose for24hours;(iv) Glu+Bup:cells treated with100mMglucose for24hours prior to bupivacaine administration for24hours. Grp78and caspase-12expression were measured by qRT-PCR and Western blot, representing ER stress.Values were expressed as the mean±standard deviation (SD), using SPSS17.0statistical software for statistical analysis. Data was analyzed by one-way ANOVA. Multiple comparisons tests were performed by LSD. A p-value of less than0.05was considered to be statistically significant.Results There was significant difference among groups on the level of Grp78and caspse-12mRNA and protein (F=15.503, F=11.525; F=8.864, F=29.639, all P values<0.05). Bubivacaine and high glucose increased the level of Grp78mRNA (P=0.010, P=0.006) and protein (P=0.000, P=0.001) compared with control group. Glucose pretreatment enhanced the influence on Grp78mRNA and protein (P=0.008, P=0.021). The effect of bupivacine and high glucose on caspase-12protein is like that of Grp78,but only glucose pretreatment with bupivaine increased caspase-12mRNA compared with that in control group (P=0.009)Conclusion Bupivacaine may result in ER stress, which could be enhanced by hyperglycemia. ER stress accociating with apoptosis may be the underlying mechanism of bupivacaine neurotoxicity. Section V The effect of Ginkgolide B on bupivacaine neurotoxicityObjective To explore the protective effect of ginkgolide B on bupivacaine induced apoptosis by examing intracellular ROS level, mitochondrial function and ER stress.Methods SH-SY5Y cells were pre-treated with different concentrations(5,10,20,40μmol/L) of GB in vitro, prior to administration of1mmol/L bupivacaine. Cell apoptosis were investigated with flowcytometry. In addition, mitochondrial membrane potential, reactive oxygen species(ROS), mitochondrially generated ROS, mitochondrial complexes I and III activity were studied, in order to explore the molecular mechanism of bupivacaine-induced mitochondrial injury。 Grp78and caspase-12expression Western blot, representing endoplasmic ER stress.Values were expressed as the mean±standard deviation (SD), using SPSS17.0statistical software for statistical analysis. Data was analyzed by one-way ANOVA. Multiple comparisons tests were performed by LSD.A p-value of less than0.05was considered to be statistically significant.Results There was significant difference among groups on apoptotic rate (F=167.786, P=0.000).10,20,40μ mol/L GB decreased the apoptotic cells induced by bupivacaine (P=0.000, P=0.000, P=0.000) in a dose-dependent manner.40μ mol/L GB was chosed for follow-up study. There were significant difference among groups (F=226.503; F=118.253, F=50.191; F=50.154; F=4.510,F=81.502; F=8.137, F=9.277; all P values<0.05) in level of ROS, mitochondrial complex I and III activity, JC-1polymer/monomer and Cleaved Caspase-3, HtrA2, Grp78and Caspase-12protein. The level of ROS in GB+Bup group was lower than that in Bup group (P=0.000). Mitochondrial membrane potential expressed as JC-1polymer/monomer, that in Bup group (1.12±0.43) was lower than that in Con group (8.41±1.41)(P=0.000), the ratio(3.55+0.71) in GB+Bup group much higher than Bup group (P=0.004). Compared with Bup group, GB pretreatment attenuated the decreased in mitochondrial complex I and III activity induced by bupivacaine (P=0.004, P=0.004). Cleaved Caspase-3, HtrA2, Grp78and Caspase-12protein were measured by Western blot, Bupivacaine increased the level of these proteins (P=0.012, P=0.000, P=0.001, P=0.001), which could be relieved by GB(P=0.032, P=0.001, P=0.038, P=0.016)Conclusion Antioxidant, GB, decreased ROS production induced by bupivacaine. Decrease in ROS production may be resulted from incease of aomplex I and III activity, and inhibit mitochondrial potential depolarization and ER stress, associating with reduction of apoptosis. That is suggesting ROS overproduction induced by bupivacaine may be responsible for mitochondrial dysfunctiong and ER stress.更多还原