| 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. |
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