Curcumin Attenuated Bupivacaine-Induced Neurotoxicitybin SH-SY5Y Cells Via Activation of the Akt Signaling Pathway

You-Ling Fan1,2 • Heng-Chang Li1,3 • Wei Zhao1 • Hui-Hua Peng2 •
Fang Huang2 • Wei-Hang Jiang2 • Shi-Yuan Xu1
Received: 14 December 2015 / Revised: 6 April 2016 / Accepted: 11 May 2016
© Springer Science+Business Media New York 2016

Abstract

Bupivacaine is widely used for regional anes- thesia, spinal anesthesia, and pain management. However, bupivacaine could cause neuronal injury. Curcumin, a low molecular weight polyphenol, has a variety of bioactivities and may exert neuroprotective effects against damage induced by some stimuli. In the present study, we tested whether curcumin could attenuate bupivacaine-induced neurotoxicity in SH-SY5Y cells. Cell injury was evaluated by examining cell viability, mitochondrial damage and apoptosis. We also investigated 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. Our findings showed that the bupiva- caine treatment could induce neurotoxicity. Pretreatment of the SH-SY5Y cells with curcumin significantly attenuated bupivacaine-induced neurotoxicity. Interestingly, the cur- cumin treatment increased the levels of Akt phosphoryla- tion. 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 pro- tective effect on bupivacaine-induced neuronal injury via activation of the Akt signaling pathway.

Keywords Local anesthetics · Curcumin · Neurotoxicity ·
Akt · SH-SY5Y cells

Introduction

Local anesthetics are necessary for regional anesthesia in surgical procedures and pain management. However, they may have potential neurotoxicity and cause postoperative neurological complications, such as persistent lumbosacral neuropathy and cauda equine syndrome. For example, bupivacaine, an amide-type local anesthetic, is widely used for epidural anesthesia, nerve blockade and postoperative analgesia in clinical patients. It has been shown to trigger a complex cascade response leading to neuronal apoptosis in vitro [1–4]. Recent studies have demonstrated that bupivacaine-induced neurotoxicity involves the inactiva- tion of the AKT signaling pathway, decline of mitochon- drial membrane potential (Dwm), accumulation of reactive oxygen species (ROS) and disruption of calcium home- ostasis. Although the exact mechanism of this local anes- thetic toxicity remains largely unknown, there is an urgent need to develop strategies to prevent neuronal cell injury. There are compelling data indicating that the threonine- serine protein kinase B (Akt) signaling pathway plays an cause neural dysfunction and apoptosis [5]. For example, studies have shown that the activation of Akt attenuates neuronal apoptosis induced by glutamate in vitro [6]. The in vivo activation of Akt significantly decreases neuronal apoptosis in the retinas of diabetic rats [7]. Ding’s group reported on the cytoprotective effect of the Akt signaling pathway in bupivacaine-treated mouse neuroblastoma neuro 2a (N2a) cells [8, 9]. Conversely, the cytoprotective effect of Akt can be abolished when the Akt signaling pathway is inhibited by RNA interference [10, 11]. Taken together, these results suggest that the activation of the Akt-dependent pathway possibly protects against bupiva- caine-induced neuronal injury in SH-SY5Y cells.
Curcumin, a biologically active component extracted from the rhizome of turmeric (Curcuma longa), has mul- tiple biological actions, including anti-apoptotic, anti-in- flammatory, antioxidant, and anti-proliferative effects. It has been shown to protect normal organs, such as the liver, kidneys, oral mucosa and heart, from drug toxicity [12]. Recent studies have demonstrated that curcumin possesses neuroprotective effects in different neuronal cell lines and tissues. For example, it has also been shown that curcumin protects PC12 cells against beta-amyloid-induced toxicity
[13] and prevents hippocampal cell death due to excito- toxicity [14]. In addition, a recent study reported that curcumin exhibited neuroprotective effects via the activa- tion of Akt cascades in rodent cortical neurons [15]. Thus, we speculate that it is possible that curcumin could atten- uate bupivacaine-induced neurotoxicity by activating the Akt signaling pathway.
To test our hypothesis, we investigated the effects of curcumin on bupivacaine-induced neuronal injury in SH- SY5Y cells. Our findings suggest that curcumin attenuates bupivacaine-induced neuronal injury by, at least in part, activating an Akt-dependent mechanism.

Materials and Methods

Materials
SH-SY5Y cells were purchased from the Shanghai Insti- tutes for Biological Sciences (Shanghai, China). Bupiva- caine hydrochloride (purity 99.9 %) and curcumin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Curcumin was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution of 10 mM. It was further diluted with medium for cell treatments. The final DMSO concentration was \0.1 %. For other reagents, we used Dulbecco’s modified Eagle medium (DMEM)/F12 and fetal bovine serum (Gibco, USA), 5,50, 6,60-tetrachloro-1,10, 3,30-te- traethyl tetraethyl benzimidazolyl carbocyanine iodide (JC- 1), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide (MTT), 20,70-dichlorofluorescein diacetate (DCFH-DA), mitochondrial isolation agent and a mito- chondrial storage solution (all from Beyotime, China). We also used monoclonal antibodies to cleave caspase-9. Monoclonal antibodies for Bcl-2 and Bax were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained from Proteintech Group (Chicago, IL). Primary antibodies for total Akt (Akt) and phospho-Akt (p-Akt) were obtained from Cell Sig- naling Technology (Beverly, MA, USA). Triciribine was obtained from Kangcheng Biotech (Shanghai, China). All reagents were obtained from commercial suppliers and were of standard biochemical quality.

Cell Culture
SH-SY5Y cells were maintained at 37 °C in 5 % CO2 in DMEM/F12 medium, supplemented with 15 % fetal bovine serum, 100 U/mL penicillin, and 100 g/mL strep- tomycin. The medium was changed every 2 days.
Drug TreatmentCells were divided into four groups: (1) untreated controls (Con), (2) cells treated with 1.5 mM bupivacaine for 24 h (Bup), (3) cells pretreated with 1 lM curcumin for 24 h (Cur), and (4) cells treated with 1 lM curcumin for 24 h prior to 1.5 mM bupivacaine exposure for 24 h (Cur ? Bup). For Akt inhibition experiments, the Akt-specific inhibitor triciribine (1 lM) 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.

MTT Assay
The effect of bupivacaine on the number of viable SH-SY5Y cells was determined by an MTT assay. Cells were seeded onto 96-well plates at 5 9 103 cells/well with 100 lL of culture medium and treated with 0.5, 1.0, 1.5, 2.0, 2.5 mM bupivacaine for 24 h. Treated cells were incubated with 20 lL MTT at 37 °C for 4 h, the medium was removed, and 150 lL DMSO was added to dissolve the formazan crystals produced from the MTT by viable cells. The optical density (OD) of the homogenous purple formazan/DMSO solutions was measured at 570 nm on a spectrophotometer (Bio-Tek Instruments, Winooski, VT, USA).
In another set of experiments, the effect of bupivacaine on the number of viable SH-SY5Y cells pretreated with various concentrations of curcumin for 24 h was also determined by the MTT assay. To determine the working range of curcumin, cells were pretreated with 1, 2, 5, 10 lM curcumin for 24 h or subjected to a control media prior to treatment with 1.5 mM bupivacaine for 24 h (the half-maximal neurotoxic dose according to our above experiments). Cells were then treated as described previously.

Apoptosis Detection
To determine the apoptotic morphology of cells, the ter- minal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay was applied using In Situ Cell Death Detection Kit (Boster Company, Wuhan, China) in the SH-SY5Y cells according to the manufac- turer’s protocol. After rinsing with PBS, cells were visu- alized using Converter-POD with 0.03 % DAB. The ratio of TUNEL positive cells to total cells under a light microscope was calculated as apoptotic rate (Leica DM 3000). Five fields were chosen randomly in each group.

Mitochondrial Membrane Potential Assay
Mitochondrial membrane potential (Dwm) depolarization, an early step in the mitochondrial apoptosis cascade, was measured fluorometrically using JC-1. Briefly, cells cul- tured in 6-well plates and incubated with a JC-1 staining solution (5 lg/mL) at 37 °C for 20 min. They were then rinsed twice with PBS. Mitochondrial membrane potential was estimated by measuring the fluorescence ratio of free JC-1 monomers (green) to JC-1 aggregates in mitochondria (red) by dual emission fluorescence microscopy (Nikon ECLIPSE TE2000-u, Japan) and flow cytometry. Mito- chondrial depolarization was indicated by an increase in the proportion of cells emitting green fluorescence.

Western Blot Analysis
Total protein was harvested from SH-SY5Y cells with a lysis buffer after incubating as described previously [8, 9]. After centrifugation, protein concentrations were determined by a bicinchoninic acid (BCA) protein assay kit (Beyotime, Haimen, China). Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, elec- trotransferred to polyvinylidene difluoride (PVDF) mem- branes. Membranes were blocked with 5 % nonfat dry milk in Tris-buffered saline and then immunoblotted with anti- Akt (1:1000), anti-p-Akt (1:1000), anti-Bax (1:1000), anti- Bcl-2 (1:1000), anti-cleaved caspase-9 (1:500), or anti- GAPDH antibody (1:1000, as the gel loading control) overnight at 4 °C. All antibodies were diluted in Tris–HCl buffered saline containing 5 % nonfat dry milk and 0.1 % Tween-20. After they were rinsed, the immunolabeled membranes were incubated with horseradish peroxidase (HRP) conjugated anti-rabbit immunoglobulin (1:1000) for 1 h. Specific proteins were detected by enhanced chemilu- minescence and exposure to X-ray film. Bands were quan- tified by scanning the films. The expression levels of Akt, p-Akt, Bax, Bcl-2, and cleaved caspase-9 protein were normalized to GAPDH.

Statistical Analysis
All experimental data were expressed as the mean ± SD. A comparison between the groups was performed using a one-way analysis of variance (ANOVA). Tukey’s proce- dure for multiple range tests was performed. P \ 0.05 was considered to be significant.

Results

Bupivacaine Inhibited Cell Viability and Induced Apoptosis of the SH-SY5Y Cells We incubated the SH-SY5Y cells with bupivacaine at different concentrations (0.5, 1, 1.5, 2, 2.5 mM). Cell viability was evaluated by the MTT assay, and apoptosis was visualized using the TUNEL assay. As shown in Fig. 1a, bupivacaine decreased the viability of the SH- SY5Y cells in a dose-dependent manner. The proportion of apoptotic cells increased with the increase in bupivacaine treatment concentration, as shown in Fig. 1b and c. The measured LD50 of bupivacaine was 1.477 mM. Therefore, we selected 1.5 mM bupivacaine for the following experiments.

Curcumin Attenuated Bupivacaine-Induced SH-SY5Y Cell Injury
We evaluated whether curcumin protects SH-SY5Y cells from the bupivacaine challenge. The cell viability was evaluated by the MTT assay. As shown in Fig. 2a, 1 and 2 lM curcumin significantly attenuated bupivacaine-de- creased cell viability. When the concentrations increased to 5 and 10 lM, the cytoprotective effect of curcumin was absent. No significant difference was found between the benefits of curcumin at 1 and 2 lM. However, the cyto- toxic effect of curcumin at 2 lM was significantly increased compared with that of the untreated control group. Therefore, we selected 1 lM curcumin in the fol- lowing experiments.

Curcumin Attenuated Bupivacaine-Induced Apoptosis in the SH-SY5Y Cells
We examined whether the curcumin pretreatment could decrease bupivacaine-induced apoptosis using the TUNEL assay. As shown in Fig. 2b and c, the proportion of apoptotic cells significantly decreased in the Cur ? Bup group (approximately 20 %) compared with the Bup group (approximately 32 %). Moreover, the presence of apoptosis was confirmed by the western blot analysis. As shown in Fig. 4a, c and d, western blots demonstrated that cleaved caspase-9 expression was significantly decreased and the ratio of Bcl-2/Bax was significantly elevated in the Cur ? Bup group compared with the Bup group.

Fig. 1 Proliferation effects of bupivacaine on SH-SY5Y cells and bupivacaine induced apoptosis of the SH-SY5Y cells. a Cells were incubated in the presence or absence of various concentrations of bupivacaine for 24 h. Cell growth was determined by MTT assay. b Summarized data show apoptotic rate as detected by TUNEL assay. c The effect of bupivacaine on cells apoptosis as detected by TUNEL assays. Values are the mean ± SD of n = 6, *P \ 0.05 versus control group

Curcumin Prevented Loss of Dwm Induced by Bupivacaine
Loss of mitochondrial membrane potential is an early and critical step in mitochondria-mediated apoptotic signaling. Therefore, we measured the mitochondrial membrane poten- tial in the SH-SY5Y cells by flow cytometry following the JC- 1 staining. As shown in Fig. 3, the mitochondrial membrane potential was significantly decreased in the SH-SY5Y cells exposed to 1.5 mM bupivacaine, while curcumin pretreatment significantly attenuated the bupivacaine-decreased Dwm. These results suggest that curcumin may prevent apoptosis by preserving mitochondrial function and by preventing the activation of mitochondrial-dependent apoptosis.

Curcumin Prevented Bupivacaine-Decreased Akt Phosphorylation
Many studies have shown Akt signaling involvement in the regulation of neural survival [5, 7]. Therefore, we inves- tigated the levels of p-Akt and total Akt (Akt) in the cells treated with bupivacaine for 24 h in the presence and absence of curcumin. As shown in Fig. 4a and b, the bupivacaine treatment significantly decreased the level of phosphorylated Akt and ratio of p-Akt/Akt compared with the untreated control cells. More importantly, the pre- treatment with curcumin could effectively increase the level of phosphorylated Akt and ratio of p-Akt/Akt com- pared with the Bup group.

Fig. 2 Curcumin attenuated bupivacaine-induced cell injury and apoptosis in SH-SY5Y cells. a Cells were treated with 1.5 mM bupivacaine for 24 h and/or pretreated with various concentrations of curcumin (0.5, 1, 2, 5 and 10 lM) for 24 h before exposure to bupivacaine. Cell growth was determined by MTT assay. b Summa- rized data show apoptotic rate as detected by TUNEL assay. c The effect of curcumin on bupivacaine-induced apoptosis as detected by TUNEL assays. Con: untreated SH-SY5Y cells. Cur: SH-SY5Y cells exposed to 1 mM curcumin for 24 h. Bup: SH-SY5Y cells exposed to
1.5 mM bupivacaine for 24 h. Cur ? Bup: SH-SY5Y cells treated with 1 lM curcumin for 24 h prior to 1.5 mM bupivacaine exposure for 24 h. Values are the mean ± SD of n = 6, *P \ 0.05 versus control group, #P \ 0.05 versus Bup group

Fig. 3 Curcumin pretreatment prevented loss of mitochondrial membrane potential (Dwm) induced by bupivacaine and inhibition of Akt abolished the protection of curcumin against bupivacaine- induced loss of Dwm in SH-SY5Y cells. a Dwm were detected by flow cytometry. Con: untreated SH-SY5Y cells. Bup: SH-SY5Y cells exposed to 1.5 mM bupivacaine for 24 h. Cur ? Bup: SH-SY5Y cells treated with 1 lM curcumin for 24 h prior to 1.5 mM bupivacaine exposure for 24 h. Cur ? Bup ? Tri: Triciribine (1 lM) 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 of curcumin. b Dwm expressed as the ratio of JC-1 polymer over monomer. Values are the mean ± SD of n = 6 (*P \ 0.05; #P \ 0.01)
Inhibition of Akt Abolished the Protection of Curcumin Against Bupivacaine-Induced Cell Apoptosis To determine whether curcumin-exerted cytoprotection against bupivacaine-induced cell apoptosis is mediated through an Akt-dependent mechanism, we used the specific Akt inhibitor triciribine before the cells were treated with and without curcumin. Figure 4a, c and d showed that cleaved caspase-9 expression was significantly increased and the ratio of Bcl-2/Bax was significantly decreased in the Cur ? Bup ? Tri group compared with the Cur ? Bup group.

Fig. 4 Curcumin prevented bupivacaine-decreased Akt phosphory- lation and inhibition of Akt abolished the protection of curcumin against bupivacaine-induced cell apoptosis. a The western blot bands show p-Akt, total Akt, cleaved caspase-9, Bax and Bcl-2 protein expression in SH-SY5Y cells. b, c and d Date of the ratio of p-Akt over total Akt, Bcl-2 over Bax and cleaved caspase-9 over GADPH. Values are the mean ± SD of n = 6 (*P \ 0.05; #P \ 0.01)
Inhibition of Akt Abolished the Protection of Curcumin Against Bupivacaine-Induced Loss of Dwm As shown in Fig. 3, the mitochondrial membrane potential was significantly decreased in the Cur ? Bup ? Tri group compare with the Cur ? Bup group. These data suggest that triciribine abrogated the cytoprotective effect of cur- cumin against bupivacaine-induced apoptosis via the pre- vention of mitochondrial dysfunction.

Discussion

In the present study, we demonstrated the protective effects of curcumin in bupivacaine-induced neurotoxicity. Pre- treatment with 1 lM curcumin significantly increased Akt phosphorylation in bupivacaine-treated cells. Significantly, the inhibition of the Akt signaling pathway abrogated the cytoprotective effect of curcumin against bupivacaine-induced cell death. Our results suggest that curcumin attenuates bupivacaine-induced neuronal injury through an Akt-dependent mechanism. Curcumin could be a potential neuroprotectant against neurotoxicity caused by local anesthetics used clinically.
Human neuroblastoma cell lines (e.g., SH-SY5Y) are different from true neuronal cells and have obvious traits of tumor cells. Therefore, SH-SY5Y cells are widely used for studying the neurotoxicity of local anesthetics because they can simulate the biological characteristics of neurons. Thus, SH-SY5Y cells were employed for our in vitro model of neuronal injury. Bupivacaine, at a concentration of 1.5 mM, which is equal to 0.045 %, was used in this study. Although this concentration is not within the range of concentrations that is used clinically, 1.5 mM bupiva- caine significantly induced neuronal damage, as evidenced by the morphological changes and MTT assay results. Our results are consistent with previous reports that bupiva- caine could trigger neuronal cell injury mainly by inducing necrosis and apoptosis [16].
Curcumin has been used for thousands of years in Chinese medicine and is generally considered to be safe. In in vivo studies, the pharmacological active concentrations of curcumin ranged from 0.1 to 10 mg/mL [17, 18]. In this study, we observed that 1 lM (approximately 0.4 mg/mL) of curcumin did not have any cytotoxicity and prevented bupivacaine toxicity when the SH-SY5Y cells was pre- treated with it for 24 h. We also observed that pretreatment with 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. When the concentrations increased to 2, 5 or 10 lM, we found that the neuropro- tective benefits of curcumin on bupivacaine-induced cell injury was decreased or even absent. The data suggest that the protection of curcumin on bupivacaine toxicity is dose dependent. A possible reason is that a high concentration of curcumin would have been considerably excitotoxic to the SH-SY5Y cells. Studies found that the cytotoxic effect of curcumin was mediated by various mechanisms including decrease in the cellular HSP70 protein level and activation of caspase-3, calpain, intracellular calcium signaling, mitochondrial permeability, oxidative stress [19, 20]. However, further research is required to determine the relative contribution of the cytotoxic effect of curcumin. Collectively, our results indicate that pretreatment with a low concentration of curcumin for 24 h will be beneficial in preventing bupivacaine-induced neurotoxicity.
It is known that apoptotic cells usually have a significant depolarization of the mitochondrial membrane potential compared with normal cells [21]. The loss of Dwm is a critical early event in the mitochondria-associated apoptosis cascade, which leads to the transformation of mitochondrial permeability and the release of cytochrome c and other pro- apoptotic factors into the cytosol [22, 23]. Therefore, pre- serving mitochondrial function could prevent the activation of mitochondrial-dependent apoptosis. In the present study, we observed that curcumin attenuated the bupivacaine-in- duced loss of Dwm. Furthermore, we observed that Akt inhibition significantly abolished the protective effects of curcumin against the bupivacaine-induced loss of Dwm. Collectively, our results indicated that the Akt-mediated stabilization of Dwm loss contributed to the cytoprotective effect of curcumin against the bupivacaine challenge.
A number of studies have demonstrated that Akt is a pivotal kinase downstream from the PI3-kinases and plays a critical role in the survival and death pathway of neurons under different cellular stress conditions [10, 24–26]. Pre- vious reports have shown that bupivacaine decreases the phosphorylation levels of Akt and results in cell damage in Na2 cells, human renal cells and mouse C2C12 myoblast cells. In contrast, increasing the phosphorylation levels of Akt attenuates bupivacaine-induced injury [8, 9, 27]. In the present study, we observed that bupivacaine decreases the phosphorylation levels of Akt and results in cell apoptosis. This was consistent with a previous report that bupiva- caine-induced apoptosis of human proximal tubular cells was associated with the inhibition of activities of Akt [28]. Triciribine, a specific and highly selective inhibitor for Akt phosphorylation, has no effect on the phosphorylation of known upstream activators of Akt. Thus, we used tri- ciribine to investigate the role of Akt in the present study. According to the results of some reports, we found that 1 lM triciribine does not affect the experiment results, such as cell viability, bupivacaine-induced nuclear con- densation, decline of mitochondrial potential and apoptosis [5, 9]. So the cells treated with triciribine alone have not been included in our study. We observed that the blockade of Akt activation diminished the cytoprotective effect of curcumin against the bupivacaine challenge. Taken toge- ther, our results suggest that activation of Akt signaling mediates the neuroprotection of curcumin against bupiva- caine-induced neuronal death.
In summary, our results demonstrate that the pretreat- ment of the SH-SY5Y cells with curcumin exhibited a protective effect on bupivacaine-induced neurotoxicity. This cytoprotective effect was mediated, at least in part, by the activation of the Akt signaling pathway. If our in vitro findings from transformed neuroblastoma cells could be applied to neurons in vivo, then this study might give credence to the use of curcumin as a potential treatment of bupivacaine-induced neurotoxicity.

Acknowledgments
This study was supported by Grants from the National Natural Science Foundation of China (Nos. 81471272, 81271390). None of the authors have financial relationships with biotechnology manufacturers, pharmaceutical companies, or other commercial entities with an interest in the subject matter or materials discussed in the manuscripts.

References

1. Li L, Zhang QG, Lai LY, Wen XJ, Zheng T, Cheung CW, Zhou SQ, Xu SY (2013) Neuroprotective effect of ginkgolide B on bupivacaine-induced apoptosis in SH-SY5Y cells. Oxid Med Cell Longev. doi:10.1155/2013/159864
2. Lelkes E, Unsworth BR, Lelkes PI (2001) Reactive oxygen species, apoptosis and altered NGF-induced signaling in PC12 pheochromocytoma cells cultured in elevated glucose: an in vitro cellular model for diabetic neuropathy. Neurotox Res 3:189–203
3. Lu J, Xu SY, Zhang QG, Lei HY (2011) Bupivacaine induces reactive oxygen species production via activation of the AMP activated protein kinase-dependent pathway. Pharmacology 87:121–129
4. Arai Y, Kondo T, Tanabe K et al (2002) Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells. J Biol Chem 277:18986–18993
5. Wang Z, Shen J, Wang J, Lu T, Li C, Zhang X, Liu L, Ding Z (2010) Lithium attenuats bupivacaine- induced neurotoxicity in vitro through phosphatidylinositol-3-kinase/threonineserine protein kinase B-and extracellular signal regulated kinase-de- pendent mechanisms. Neurotoxicology 31:101–112
6. Dasari VR, Veeravalli KK, Saving KL, Gujrati M, Fassett D, Klopfenstein JD, Dinh DH, Rao JS (2008) Neuroprotection by cord blood stem cells against glutamate-induced apoptosis is mediated by Akt pathway. Neurobiol Dis 32:486–498
7. Kim YH, Kim YS, Park CH, Chung IY, Yoo JM, Kim JG, Lee BJ, Kang SS, Cho GJ, Choi WS (2008) Protein kinase C-delta mediates neuronal apoptosis in the retinas of diabetic rats via the Akt signaling pathway. Diabetes 57:2181–2190
8. Ma R, Wang X, Lu C, Li C, Cheng Y, Ding G, Liu L, Ding Z (2010) Dexamethasone attenuated bupivacaine-induced neuron injury in vitro through a threonine-serine protein kinase B-de- pendent mechanism. Neuroscience 167:329–342
9. Wang X, Zhang X, Cheng Y, Li C, Zhang W, Liu L, Ding Z (2010) a-Lipoic acid prevents bupivacaine-induced neuron injury in vitro through a PI3K/Akt-dependent mechanism. Neurotoxi- cology 31:101–112
10. Nair VD, Olanow CW (2008) Differential modulation of Akt/ glycogen synthase kinase-3beta pathway regulates apoptotic and cytoprotective signaling responses. J Biol Chem 283:15469–15478
11. Liu L, Zhang XJ, Jiang SR, Ding ZN, Ding GX, Huang J, Cheng YL (2007) Heat shock protein 27 regulates oxidative stress-in- duced apoptosis in cardiomyocytes: mechanisms via reactive oxygen species generation and Akt activation. Chin Med J Engl 120:2271–2277
12. Goel A, Aggarwal BB (2010) Curcumin the golden spice from Indian saffron, is a chemosensitizer and radiosensitizer for tumors and chemoprotector and radioprotector for normal organs. Nutr Cancer 62:919–930
13. Park SY, Kim HS, Cho EK, Kwon BY, Phark S, Hwang KW, Sul D (2008) Curcumin protected PC12 cells against beta-amyloid- induced toxicity through the inhibition of oxidative damage and tau hyperphosphorylation. Food Chem Toxicol 46:2881–2887
14. Shin HJ, Lee JY, Son E, Lee DH, Kim HJ, Kang SS, Cho GJ, Choi WS, Roh GS (2007) Curcumin attenuates the kainic acid- induced hippocampal cell death in the mice. Neurosci Lett 416:49–54
15. Wang R, Li YH, Xu Y, Li YB, Wu HL, Guo H et al (2010) Curcumin produces neuroprotective effects via activating brain- derived neurotrophic factor/TrkB-dependent MAPK and PI-3 K cascades in rodent cortical neurons. Prog Neuropsychopharmacol Biol Psychiatry 34:147–153
16. Perez-Castro R, Patel S, Garavito-Aguilar ZV, Rosenberg A, Recio-Pinto E, Zhang J, Blanck TJ, Xu F (2009) Cytotoxicity of local anesthetics in human neuronal cells. Anesth Analg 108:997–1007
17. Esatbeyoglu T, Huebbe P, Ernst IM, Chin D, Wagner AE, Rimbach G (2012) Curcumin—from molecule to biological function. Angew Chem Int Ed Engl 51:5308–5332
18. Gupta SC, Sung B, Kim JH, Prasad S, Li S, Aggarwal BB (2012) Multi targeting by turmeric, the golden spice: from kitchen to clinic. Mol Nutr Food Res 57:1510–1528
19. Hollborn M, Chen R, Wiedemann P, Reichenbach A, Bringmann A, Kohen L (2013) Cytotoxic Effects of Curcumin in Human Retinal Pigment Epithelial Cells. PLoS ONE 8(3):e59603. doi:10. 1371/journal.pone.0059603
20. Hung CM, Su YH, Lin HY, Lin JN, Liu LC, Ho CT, Way TD (2012) Demethoxycurcumin modulates prostate cancer cell pro- liferation via AMPK-induced down-regulation of HSP70 and EGFR. J Agric Food Chem 60:8427–8434
21. Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssiere JL, Petit PX, Kroemer G (1995) Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med 181:1661–1672
22. Hagen TM, Moreau R, Suh JH, Visioli F (2002) Mitochondrial decay in the aging rat heart: evidence for improvement by dietary supple-mentation with acetyl-L-carnitine and/or lipoic acid. Ann N Y Acad Sci 959:491–507
23. Su CC, Lin YP, Cheng YJ, Huang JY, Chuang WJ, Shan YS, Yang BC (2007) Phosphatidylinositol 3-kinase/Akt activation by integrin-tumor matrix interaction suppresses Fas-mediated apoptosis in T cells. J Immunol 179:4589–4597
24. Chung H, Seo S, Moon M, Park S (2008) Phosphatidylinositol-3- kinase/Akt/glycogen synthase kinase-3 beta and ERK1/2 path- ways mediate NSC 154020 protective effects of acylated and unacylated ghrelin against oxygen-glucose deprivation-induced apoptosis in primary rat cortical neuronal cells. J Endocrinol 198:511–521
25. Datta SR, Brunet A, Greenberg ME (1999) Cellular survival: a play in three Akts. Genes Dev 13:2905–2927
26. Vauzour D, Vafeiadou K, Rice-Evans C, Williams RJ, Spencer JP (2007) Activation of pro-survival Akt and ERK1/2 signalling path-ways underlie the anti-apoptotic effects of flavanones in cortical neurons. J Neurochem 103:1355–1367
27. Maurice JM, Gan Y, Ma FX, Chang YC, Hibner M, Huang Y (2010) Bupivacaine causes cytotoxicity in mouse C2C12 myo- blast cells: involvement of ERK and Akt signaling pathways. Acta Pharmacol Sin 31:493–500
28. Lee HT, Xu H, Siegel CD, Krichevsky IE (2003) Local anes- thetics induce human renal cell apoptosis. Am J Nephrol 23:129–139