Curcumin Attenuates Bupivacaine-induced Neurotoxicity In SH-SY5Y Cells By Enhancing PRDX3 Expression Via Activation Of The Akt Signaling Pathway

Local anesthetics are necessary for the regional anesthesia and pain management, but they have the potential neurotoxicity even at normal clinical dose. For example, bupivacaine is widely used for epidural anesthesia, nerve blockade, and postoperative analgesia in clinical patients and has been shown to induce neural dysfunction and apoptosis in vitro. Recent studies have demonstrated that the neuronal cell injury induced by local anesthetics is at least in part associated with oxidative stress that leads to failure of cellular homeostasis, involving decline of mitochondrial membrane potential (△ψm), accumulation of reactive oxygen species (ROS), and ultimately liberation of cytochrome c and activation of the mitochondrial-dependent apoptosis pathway. Although the exact mechanism of bupivacaine-induced neurotoxicity remains elusive, it is also important to develop a means to prevent the neuronal cell injury.Curcumin, has a variety potential therapeutic effects on numerous pathologic disorders, including cognitive deficits, neuroinflammation, and plaque pathology in Alzheimer’s disease models. Some studies showed that curcumin is associated with the cellular antioxidant defense system and may elevate the expression of Peroxiredoxin 3. Peroxiredoxin 3 (Prdx3), one peroxidase in the six isoforms of PRDX family in mammalian cells, is located in the matrix of mitochondria and plays an important role in oxidative stress reaction. It is reported that the decrease of Prdx3 may abate the capability of mitochondria to neutralize the production of harmful reactive oxygen species (ROS) derived from physiological reaction, suggesting that Prdx3 provides cytoprotection from oxidative stress as a mitochondria-specific ROS-scavenging protein. Therefore, we think that curcumin may attenuate mitochondrial-dependent apoptosis in SH-SY5Y cells by enhancing Prdx3 expression.Moreover, the neuronal cell injury induced by bupivacaine is an inducer of mitochondrial-dependent apoptosis that activates specific signaling. For example, in vitro studies have shown that activation of threonine-serine protein kinase B (Akt) was involved in the apoptosis of human proximal tubular cells induced by bupivacaine.It is known that the threonine-serine protein kinase B (Akt) plays an important role in the protection against various stimuli that cause cell injury both in vitro and in vivo. Our findings showed that the bupivacaine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the curcumin treatment increased the levels of Akt phosphorylation. More significantly, the pharmacological inhibition of Akt abolished the cytoprotective effect of curcumin against bupivacaine-induced cell injury. Our data suggest that pretreating SH-SY5Y cells with curcumin provides a protective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway.To test our hypothesis, we investigated the effects of curcumin on bupivacaine-induced neuronal injury in SH-SY5Y cells. We observed that pretreatment with curcumin significantly attenuated bupivacaine-induced neuronal damage. Curcumin prevented the decline of mitochondrial membrane potential, reduced the Bax/Bcl-2 ratio at the protein level and increased the levels of phospho-Akt in the cells treated with bupivacaine. More significantly, a blockade of Akt activation abolished the curcumin-induced protection. Collectively, our findings suggest that curcumin attenuates bupivacaine-induced neuronal injury by, at least in part, activating an Akt-dependent mechanism.Chapter 1 To establish model of bupivacaine-induced neurtoxicity in vitroObjective To construct model of bupivacaine-induced neurtoxicity in vitro in SH-SY5Ycell lines and observe the impact of bupivacaine on cell viability and apoptosis.Methods Cells were divided into the C group:cells were incubated with serum-starved medium for 24h; Bupo.5-Bup2.5 group cells were with serum-free medium of increasing bupivacaine concentrations (0.5,1.0,1.5,2.0,2.5 mM) for 24h (in DMEM/F12 medium). Cell viability and apoptosis were investigated with a MTT and TUNEL assay, respectively.Statistical Values were expressed as the mean±standard deviation (SD), using SPSS 17.0 statistical 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 than 0.05 was considered to be statistically significant.Results There was significant difference among Bup1.0～Bup2.5 groups (P<0.05) and C group on cell viability and apoptotic rate. Apoptosis increased and cell viability decreased with higher concentrations of bupivacaine. The LD was 1.47 mM, so we select the concentration(1.5 mM) to complete the following experiments.Conclusions Bupivacaine has toxicity effect on SH-SY5Y cells. This difference between C and Bupi.o-Bup2.5 groups was apparent. In a separate experiment, and after establishing the concentration to induce apoptosis, we tested bupivacaine’s apoptotic effect on SH-SY5Y cells that were exposed to 1.5 mM of bupivacaine for 24 hours.Chapter 2 Curcumin attenuated bupivacaine-induced apoptosis in the SH-SY5Y cellsObjective To test whether curcumin has a neuroprotective effect on bupivacaine-induced neurtoxicity in vitro in SH-SY5Ycell lines and observe the impact of curcumin on cell viability and apoptosis.Methods Cells were divided into the C group:cells were incubated with serum-starved medium for 24h; Curo.5-Curio groups cells treated with (0.5,1.0,2.0,5.0,10 uM) curcumin for 24 h prior to 1.5 mM bupivacaine exposure for 24h. Cell viability and apoptosis were investigated with a MTT and TUNEL assay, respectively.Statistical Values were expressed as the mean±standard deviation (SD), using SPSS 17.0 statistical 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 than 0.05 was considered to be statistically significant.Results There was significant difference among Cur0.5～Cur10 groups (P<0.05) and C group on cell viability and apoptotic rate. Apoptosis increased and cell viability decreased with 1 and 2 μM concentrations of curcumin. We select the concentration(1 μM) to complete the following experiments.Conclusions Curcumin has neuroprotective effect on bupivacaine-induced neurtoxicity in SH-SY5Y cells. This difference between C and Cur1.0～Cur2.0 groups was apparent. We select the concentration(1.5 mM) to complete the following experiments.Chapter 3 Curcumin attenuated bupivacaine-induced neurotoxicity in SH-SY5Y cells via activation of the Akt signaling pathwayObjective To investigate the levels of activation of the Akt signaling pathway and the effect of Akt inhibition by triciribine on cell injury following bupivacaine and curcumin treatment.Methods Cells were divided into four groups:(i) untreated controls (Con), (ii) cells treated with 1.5 mM bupivacaine for 24 h (Bup), (iii) cells pretreated with 1μM curcumin for 24 h (Cur), and (iv) cells treated with 1μM curcumin for 24 h prior to 1.5 mM bupivacaine exposure for 24 h (Cur+Bup). The Akt-specific inhibitor triciribine (1μM) was added to the cell culture 30 min before curcumin was added, and then, cells were exposed to bupivacaine for 24 h in the presence or absence of curcumin. Cell viability and apoptosis were investigated with a MTT and TUNEL assay, respectively. Mitochondrial membrane potential and the level of ROS were detected by flow cytometry. The expression levels of Akt, p-Akt, Bax, Bcl-2, and cleaved caspase-9 protein were detected by western blot. And we examined Prdx3 protein and mRNA expression levels by western blot and qRT-PCR.Statistical Values were expressed as the mean±standard deviation (SD), using SPSS 17.0 statistical 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 than 0.05 was considered to be statistically significant.Results Curcumin prevented bupivacaine-decreased Akt phosphorylation and inhibition of Akt abolished the protection of curcumin against bupivacaine-induced cell apoptosis. Curcumin pretreatment prevented loss of mitochondrial membrane potential (△ψm) induced by bupivacaine and inhibition of Akt abolished the protection of curcumin against bupivacaine-induced loss of △ψm in SH-SY5Y cells.Conclusions Curcumin pretreatment attenuates the bupivacaine-induced neurtoxicity in SH-SY5Y cells.The neuroprotection of curcumin is related to the activation of Akt signaling pathway.Chapter 4 Curcumin attenuates bupivacaine-induced neurotoxicity in SH-SY5Y cells by enhancing Prdx3 expression.Objective To test whether Prdx3 protein was related to the neuroprotection of curcumin on bupivacaine-induced neurtoxicity in vitro.Methods Prdx3 gene in SH-SY5Y cells was silenced by RNA interference.Real-time PCR was applied to detect the expression of Prdx3 at mRNA level in SH-SY5Y cells.West blot was applied to detect the expression of Prdx3、Bcl-2、Bax and caspase-9 at protein level.In addition, TUNEL assay were used to measure the cell apoptosis.Statistical Values were expressed as the meanistandard deviation (SD), using SPSS 17.0 statistical 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 than 0.05 was considered to be statistically significant.Results There was no significant difference among group C and group Bup (P> 0.05) on Prdx3 expression; There was significant difference among group C and group Bup on Bcl-2 expression; There was significant difference among group C and group Bup on the expression of casepase-9.Conclusions We think that curcumin may attenuate mitochondrial-dependent apoptosis in SH-SY5Y cells by enhancing Prdx3 expression.