Apelin-13 Protects PC12 Cells Against Methamphetamine-Induced Oxidative Stress,Autophagy and Apoptosis

Methamphetamine (METH) is a potent psychomotor stimulant that has a high potential for abuse in humans. In addition, it is neurotoxic, especially in dopaminergic neurons. Long-lasting exposure to METH causes psychosis and increases the risk of Parkinson’s disease. Apelin-13 is a novel endogenous ligand which studies have shown that may have a neuroprotective effect. Therefore, we hypothesized that Apelin-13 might adequately prevent METH-induced neurotoxicity via the inhibition of apoptotic, autophagy, and ROS responses. In this study, PC12 cells were exposed to both METH (0.5, 1, 2, 3, 4, 6 mmol/L) and Apelin-13 (0.5, 1.0, 2.0, 4.0, 8.0 μmol/L) in vitro for 24 h to measure determined dose, and then downstream pathways were measured to investigate apoptosis, autophagy, and ROS responses. The results have indicated that Apelin-13 decreased the apoptotic response post-METH exposure in PC12 cells by increasing cell viability, reducing apoptotic rates. In addition, the study has revealed Apelin-13 decreased gene expression of Beclin-1 by Real-Time PCR and LC3-II by western blotting in METH-induced PC12 cells, which demonstrated autophagy is reduced. In addition, this study has shown that Apelin-13 reduces intracellular ROS of METH-induced PC12 cells. These results support Apelin-13 to be investigated as a potential drug for treatment of neurodegenerative diseases. It is suggested that Apelin-13 is beneficial in reducing oxidative stress, which may also play an important role in the regulation of METH-triggered apoptotic response. Hence, these data indicate that Apelin-13 could potentially alleviate METH-induced neurotoxicity via the reduction of oxidative damages, apoptotic, and autophagy cell death.

     

Betaine Protects Against Rotenone-Induced Neurotoxicity in PC12 Cells

Rotenone is an inhibitor of mitochondrial complex I-induced neurotoxicity in PC12 cells and has been widely studied to elucidate the pathogenesis of Parkinson’s disease. We investigated the neuroprotective effects of betaine on rotenone-induced neurotoxicity in PC12 cells. Betaine inhibited rotenone-induced apoptosis in a dose-dependent manner, with cell viability increasing from 50 % with rotenone treatment alone to 71 % with rotenone plus 100-μM betaine treatment. Flow cytometric analysis demonstrated cell death in the rotenone-treated cells to be over 50 %; the number of live cells increased with betaine pretreatment. Betaine pretreatment of PC12 cells attenuated rotenone-mediated mitochondrial dysfunction, including nuclear fragmentation, ATP depletion, mitochondrial membrane depolarization, caspase-3/7 activation, and reactive oxygen species production. Western blots demonstrated activation of caspase-3 and caspase-9, and their increased expression levels in rotenone-treated cells; betaine decreased caspase-3 and caspase-9 expression levels and suppressed their activation. Together, these results suggest that betaine may serve as a neuroprotective agent in the treatment of neurodegenerative diseases.     

Gualou Guizhi Granule Protects Against Oxidative Injury by Activating Nrf2/ARE Pathway in Rats and PC12 Cells

Stroke involves numerous pathophysiological processes and oxidative stress is considered as a main cellular event in its pathogenesis. The nuclear factor erythroid-2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway plays a key role in inducing phase II detoxifying enzymes and antioxidant proteins and is now considered as a interesting therapeutic target for the treatment of stroke. The objective of this study is to investigate the protective effect of Gualou Guizhi granule (GLGZG) against oxidative stress and explore the protective mechanism of the Nrf2/ARE pathway. In vivo, administration of GLGZG in a rat model of focal cerebral ischemia significantly suppressed oxidative injury by increasing the activity of superoxide dismutase and glutathione level and decreasing reactive oxygen species and malondialdehyde levels. Western blot analysis showed that GLGZG induced nuclear translocation of Nrf2, and combined with real-time PCR results, which indicated that GLGZG up-regulated the Nrf2/ARE pathway. In addition, in cultured PC12 cells, GLGZG protected against H₂O₂ induced oxidative injury and activated the Nrf2/ARE pathway. All the results demonstrated that GLGZG in the management of cerebral ischemia and H₂O₂ induced oxidative injury may be associated with activation of Nrf2/ARE signaling pathway.     

Fasudil Mesylate Protects PC12 Cells from Oxidative Stress Injury via the Bax-Mediated Pathway

We previously reported that fasudil mesylate (FM) improves neurological deficit and neuronal damage in rats with ischemia following middle cerebral artery occlusion and reperfusion in vivo. In this study, the properties of FM on hydrogen peroxide (H₂O₂)-induced oxidative stress insult in cultured PC12 cells as well as the underlying mechanisms were investigated in vitro. Pretreatment with FM (5, 10 μM) prior to H₂O₂ exposure significantly elevated cell viability, reduced cell apoptosis by MTT assay, LDH assay, Hoechst 33258 dye staining, and FM also decreased the accumulation of reactive oxygen species (ROS) by DCFH-DA staining and NBT test. Furthermore, FM also reversed the upregulation of Bax/Bcl-2 ratio, the downstream cascade following ROS. FM protected PC12 cells from oxidative stress insult via down-regulating the Bax/Bcl-2 ratio. These findings indicate that a direct effect of fasudil mesylate on PC12 cells may be partly responsible for its protective effect against oxidative stress injury.     

CrmA Protects Against Apoptosis and Ceramide Formation in PC12 Cells

TNF- activated caspase 8 and caspase 3 in PC12 cells, leading to cell death by apoptosis (DNA fragmentation). TNF- caspase activation and cell killing were blocked by transfection and overexpression of the viral protein CrmA, which specifically inhibits caspase 8. CrmA was also able to block the TNF--induced increase in ceramide formation in PC12 cells. Conversely, if caspase 8 was activated by light-activated Rose Bengal, there was an increase in both ceramide and caspase 3–mediated apoptosis, which was blocked by CrmA overexpression. This suggested that caspase 8 increases ceramide either by increasing its synthesis or by activating sphingomyelinase. Since fumonisin B1 did not block and sphingomyelin decreased when ceramide increased, we concluded that activation of sphingomyelinase is the most likely mechanism. The Rose Bengal activation of caspase 8 and increased ceramide formation was blocked with IETD-CHO, to show that reactive oxygen species (also generated by Rose Bengal) were not responsible for the observed increase in ceramide. Thus in PC12 pheochromocytoma cells, ceramide appears to amplify the death signal and there appears to be a sequence of events: TNF; TRADD, pro-caspase 8, caspase 8, sphingomyelinase, ceramide, caspase 3, apoptosis.     

Estradiol Protects Against Oxidative Stress Induced by Chronic Variate Stress

Neurochemical gender-specific effects have been observed following chronic stress. The aim of this study was to verify the effects of chronic variable stress on free radical production (evaluated by DCF test), lipoperoxidation (evaluated by TBARS levels), and total antioxidant reactivity (TAR) in three distinct structures of brain: hippocampus, cerebral cortex and hypothalamus of female rats, and to evaluate whether the replacement with estradiol in female rats exerts neuroprotection against oxidative stress. Results demonstrate that chronic stress had a structure-specific effect upon lipid peroxidation, since TBARS increased in hypothalamus homogenates of stressed animals, without alterations in the other structures analyzed. Estradiol replacement was able to counteract this effect. In hippocampus, estradiol induced a significant increase in TAR. No differences in DCF levels were observed. In conclusion, the hypothalamus is more susceptible to oxidative stress in female rats submitted to chronic variable stress, and this effect is prevented by estradiol treatment.     

Protective Effects of Selol Against Sodium Nitroprusside-Induced Cell Death and Oxidative Stress in PC12 Cells

Selol is an organic selenitetriglyceride formulation containing selenium at +4 oxidation level that can be effectively incorporated into catalytic sites of of Se-dependent antioxidants. In the present study, the potential antioxidative and cytoprotective effects of Selol against sodium nitroprusside (SNP)-evoked oxidative/nitrosative stress were investigated in PC12 cells and the underlying mechanisms analyzed. Spectrophoto- and spectrofluorimetic methods as well as fluorescence microscopy were used in this study; mRNA expression was quantified by real-time PCR. Selol dose-dependently improved the survival and decreased the percentage of apoptosis in PC12 cells exposed to SNP. To determine the mechanism of this protective action, the effect of Selol on free radical generation and on antioxidative potential was evaluated. Selol offered significant protection against the elevation of reactive oxidative species (ROS) evoked by SNP. Moreover, this compound restored glutathione homeostasis by ameliorating the SNP-evoked disturbance of GSH/GSSG ratio. The protective effect exerted by Selol was associated with the prevention of SNP-mediated down-regulation of antioxidative enzymes: glutathione peroxidase (Se-GPx), glutathione reductase (GR), and thioredoxin reductase (TrxR). Finally, GPx inhibition significantly abolished the cytoprotective effect of Selol. In conclusion, these results suggest that Selol effectively protected PC12 cells against SNP-induced oxidative damage and death by adjusting free radical levels and antioxidant system, and suppressing apoptosis. Selol could be successfully used in the treatments of diseases that involve oxidative stress and resulting apoptosis.     

Ceruloplasmin Protects Against Rotenone-Induced Oxidative Stress and Neurotoxicity

To clarify the neuroprotective property of ceruloplasmin and the pathogenesis of aceruloplasminemia, we generated ceruloplasmin-deficient (CP ^−/−) mice on the C57BL/10 genetic background and further treated them with a mitochondrial complex I inhibitor, rotenone. There was no iron accumulation in the brains of CP ^−/− mice at least up to 60 weeks of age. Without rotenone treatment, CP ^−/− mice showed slight motor dysfunction compared with CP ^+/+ mice, but there were no detectable differences in the levels of oxidative stress markers between these two groups. A low dose of rotenone did not affect the mitochondrial complex I activity in our mice, however, it caused a significant change in motor behavior, neuropathology, or the levels of oxidative stress markers in CP ^−/− mice, but not in CP ^+/+ mice. Our data support that ceruloplasmin protects against rotenone-induced oxidative stress and neurotoxicity, probably through its antioxidant properties independently of its function of iron metabolism.     

自噬抑制剂3-MA上调H2O2损伤的PC12细胞氧化应激水平

为探究自噬抑制剂6-氨基-3-甲基腺嘌呤（3-methyladenine,3-MA）对损伤细胞氧化应激水平的影响,将3-MA作用于H2O2诱导的PC12细胞损伤模型,以自噬增强剂雷帕霉素（rapamycin,Rap）作为对照,探讨自噬与氧化应激的关系。测定线粒体的膜电位和细胞内的活性氧（reactive oxygen species, ROS）与丙二醛(malondialdehyde, MDA)含量,以及超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)活性,评价损伤细胞的氧化应激状态。单丹(磺)酰戊二胺（monodansylcadaverine,MDC）染色,观察损伤细胞的自噬情况。蛋白质印迹分析损伤细胞中的自噬相关蛋白质LC3-II/LC3-I比值变化。实验结果显示：与正常组相比,H2O2损伤细胞的ROS水平上升到正常组的141%,MDA含量增加(P<0.001);CAT与SOD酶活力显著降低(P<0.001),差异均有统计学意义,证明损伤细胞氧化应激水平增加;MDC染色结果表明,H2O2组自噬明显增加。Western印迹结果表明,LC3-II/LC3-I值显著升高（P<0.05）;与损伤组相比,3-MA组MDC染色结果表明,自噬水平降低。Western印迹结果表明,LC3-II/LC3-I值下降;细胞内ROS水平升高,增加到正常组的208%。MDA含量增加(P<0.001),CAT、SOD酶活力降低(P<0.001)。综上结果表明,自噬抑制剂可增加H2O2诱导的PC12细胞损伤模型的氧化应激水平,增加细胞凋亡。     

Hydroxysafflor Yellow A Protects PC12 Cells Against the Apoptosis Induced by Oxygen and Glucose Deprivation

Hydroxysafflor yellow A (HSYA) was reported neuroprotective under several ischemic models in vivo. In this study, the direct effect of HSYA against oxygen–glucose deprivation (OGD) inducing acute neuronal injury and the underling mechanisms in vitro were investigated. Four-hour oxygen and glucose deprivation (OGD) followed by 20 h reperfusion (adding back oxygen and glucose, OGD-R) was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. HSYA (1, 10, and 100 μmol/l) was added to the cultures 30 min prior to the ischemic insult and was present during OGD and reoxygenation phases. The survival rate of PC12 cells was detected by MTT assay. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) activity were elevated by biochemical method. Hoechst 33258 staining and flow cytometric analysis were used to detect apoptosis; western blotting was used to detect the expression of Bcl-2, Bax, and Cytochrome C protein. The activity of caspase-3 was assessed by colorimetry. HSYA concentration-dependently attenuated neuronal damage with characteristics of increasing injured neuronal absorbance of MTT, decreasing cell apoptosis, and antagonizing decreases in SOD activity and increase in MDA level induced by OGD-R. Moreover, the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol and the consequent activation of caspase-3 were reversed by HSYA in a dose-dependent manner. These results suggest that apoptosis is an important characteristic of OGD-R-induced PC 12 death and that treatment of PC12 cells with HSYA can block OGD-R-induced apoptosis through suppression of intracellular oxidative stress and mitochondria dependent caspase cascade.     

Neuroprotective Effects of Theaflavins Against Oxidative Stress-Induced Apoptosis in PC12 Cells

Oxidative stress can induce neuronal apoptosis via the production of superoxide and hydroxyl radicals. This process is as a major pathogenic mechanism in neurodegenerative disorders. In this study, we aimed to clarify whether theaflavins protect PC12 cells from oxidative stress damage induced by H₂O₂. A cell model of PC12 cells undergoing oxidative stress was created by exposing cells to 200 μM H₂O₂ in the presence or absence of varying concentrations of theaflavins (5, 10, and 20 μM). Cell viability was monitored using the MTT assay and Hoechst 33258 staining, showing that 10 μM theaflavins enhanced cell survival following 200 μM H₂O₂ induced toxicity and increased cell viability by approximately 40?%. Additionally, we measured levels of intracellular reactive oxygen species (ROS) and antioxidant enzyme activity. This suggested that the neuroprotective effect of theaflavins against oxidative stress in PC12 cells is derived from suppression of oxidant enzyme activity. Furthermore, Western blot analyses indicated that theaflavins downregulated the ratio of pro-apoptosis/anti-apoptosis proteins Bax/Bcl-2. Theaflavins also downregulated the expression of caspase-3 compared with a H₂O₂-treated group that had not been treated with theaflavins. Interestingly, this is the first study to report that the four main components of theaflavins found in black tea can protect neural cells (PC12) from apoptosis induced by H₂O₂. These findings provide the foundations for a new field of using theaflavins or its source, black tea, in the treatment of neurodegenerative diseases caused by oxidative stress.     

Protective Effects of Walnut Extract Against Amyloid Beta Peptide-Induced Cell Death and Oxidative Stress in PC12 Cells

Amyloid beta-protein (Aβ) is the major component of senile plaques and cerebrovascular amyloid deposits in individuals with Alzheimer’s disease. Aβ is known to increase free radical production in neuronal cells, leading to oxidative stress and cell death. Recently, considerable attention has been focused on dietary antioxidants that are able to scavenge reactive oxygen species (ROS), thereby offering protection against oxidative stress. Walnuts are rich in components that have anti-oxidant and anti-inflammatory properties. The inhibition of in vitro fibrillization of synthetic Aβ, and solubilization of preformed fibrillar Aβ by walnut extract was previously reported. The present study was designed to investigate whether walnut extract can protect against Aβ-induced oxidative damage and cytotoxicity. The effect of walnut extract on Aβ-induced cellular damage, ROS generation and apoptosis in PC12 pheochromocytoma cells was studied. Walnut extract reduced Aβ-mediated cell death assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, and release of lactate dehydrogenase (membrane damage), DNA damage (apoptosis) and generation of ROS in a concentration-dependent manner. These results suggest that walnut extract can counteract Aβ-induced oxidative stress and associated cell death.     

Neuroprotective Effects of n-3 Polyunsaturated Fatty Acid-Enriched Phosphatidylserine Against Oxidative Damage in PC12 Cells

Neurodegenerative diseases are defined by progressive loss of specific neuronal cell populations and are associated with protein aggregates. Oxidative stress has been implicated in their pathological processes. Previous studies revealed that docosahexaenoic acid (DHA) is beneficial in neurodegenerative diseases. Phospholipids (PLs) derived from marine products are rich in DHA and eicosapentaenoic acid (EPA). In the present study, we investigated the neuroprotective effects of DHA-enriched and unenriched phosphatidylcholine (PC) and phosphatidylserine (PS) on oxidative stress induced by hydrogen peroxide (H₂O₂) and tert-butylhydroperoxide in PC12 cells. Cell viability and leakage of lactate dehydrogenase results showed that the neuroprotective effect of PS was superior to that of PC. DHA- and EPA-enriched PC and PS were superior to that without DHA or EPA; in addition, the improvement with n-3 polyunsaturated fatty acid-enriched PS (n-3 PS) was dose dependent. Acridine orange/ethidium bromide staining showed that DHA- and EPA-enriched PS (DHA/EPA-PS) could significantly inhibit apoptosis. Mechanistic studies revealed that EPA-PS and DHA-PS were effective to increase superoxide dismutase (SOD) levels by 48.4 and 58.2 % and total antioxidant capacity (T-AOC) level by 51 and 94 %, respectively, in the H₂O₂ model. Similar results for SOD and T-AOC levels were shown in the t-BHP model. EPA/DHA-PS could downregulate the messenger RNA level of Caspase-3, Caspase-9, and Bax, upregulate Bcl-2, inhibit Bax, and increase Bcl-2 at protein level. In conclusion, EPA/DHA-PS could protect PC12 cells from oxidative stress and prevent mitochondrial-mediated apoptosis. Our findings indicate that the neuroprotective effects of DHA/EPA-PLs depend on the molecular form. Further studies are necessary to reveal detailed mechanisms and structure–effect relationships.     

l-DOPA Up-Regulates Glutathione and Protects Mesencephalic Cultures Against Oxidative Stress

Abstract: Incubation with l -DOPA induced a rise in GSH level in cultures of fetal rat mesencephalon, mouse neuroblastoma (Neuro-2A), human neuroblastoma (SK-N-MC), pig kidney epithelial cells (LLC-PK₁), and glia from newborn rat brain, but not C6 glioma cells or neuronal cultures (no glia) from the mesencephalon. The pure neuronal cultures were destroyed by incubation with l -DOPA; added ascorbic acid or superoxide dismutase protected the cells. Washout of l -DOPA after 48 h amplified the rise in GSH content in mixed cultures (neurons plus glia). Examination of structure-activity relationships for elevating GSH levels in responsive cell types revealed that autooxidizable compounds (α-methyl-DOPA, dopamine, apomorphine, catechol, and hydroquinone) behaved similarly to l -DOPA, whereas structural analogues that cannot undergo autooxidation (3- O -methyl-DOPA, tyrosine, 2,4-dihydroxyphenylalanine, and resorcinol) failed to elevate GSH levels. Therefore, up-regulation of GSH appears to be a response to a mild oxidative stress. When mixed mesencephalic cultures were exposed to a strong oxidant stress by incubation with tert -butyl hydroperoxide, a loss in viability was seen. Cultures pretreated with l -DOPA or hydroquinone were protected from loss of viability. However, when cultures were pretreated with both l -DOPA and ascorbate, which prevents the rise in GSH level, protection was lost, in accord with the failure to up-regulate GSH. These results show that the up-regulation of cellular GSH evoked by autooxidizable agents is associated with significant protection of cells. Glia play an essential role in the response of mesencephalic cell cultures. An ability to up-regulate GSH may serve a protective role in vivo.     

Nerve Growth Factor Protects PC12 Cells Against Peroxynitrite-Induced Apoptosis via a Mechanism Dependent on Phosphatidylinositol 3-Kinase

Abstract: Nerve growth factor (NGF) prevents apoptosis induced by the oxidant peroxynitrite in undifferentiated PC12 rat pheochromocytoma cells. Previous studies have shown that activation of phosphatidylinositol 3-kinase (PI 3-kinase) by NGF via the TrkA receptor tyrosine kinase protects PC12 cells from serum deprivation-induced apoptosis. We found that two PI 3-kinase inhibitors, wortmannin and LY294002, eliminated the protection NGF provided against peroxynitrite-induced apoptosis at concentrations consistent with their effectiveness as PI 3-kinase inhibitors. When the activity of PI 3-kinase was assayed in phosphotyrosine immunoprecipitates after treatment of PC12 cells with peroxynitrite, PI 3-kinase activity was reduced by 50% of that detected in control cells, whereas PI 3-kinase activity in NGF-treated cells was unaffected by peroxynitrite. If an antibody against PI 3-kinase was used to immunoprecipitate the enzyme, treatment with peroxynitrite had no effect on activity. Therefore, peroxynitrite appeared to disrupt interactions between PI 3-kinase and phosphotyrosine proteins, rather than directly inhibiting the enzyme. NGF also activates p21^Ras-dependent pathways, but this did not appear to be required for NGF to exert its protective effect against peroxynitrite. PC12 cells expressing a dominant inhibitory mutant of p21^Ras were equally susceptible to peroxynitrite-induced apoptosis, which was prevented by NGF. Wortmannin was also able to block the protective effect of NGF in the p21^Ras mutant cell line. Although many signaling pathways are activated by NGF, these results suggest that a PI 3-kinase-dependent pathway is important for inhibiting peroxynitrite-induced apoptosis.     

Role of Peroxiredoxin 2 in the Protection Against Ferrous Sulfate-Induced Oxidative and Inflammatory Injury in PC12 Cells

Peroxiredoxin 2 (Prdx2) is a ubiquitous antioxidant enzyme in mammalian brain. Although a protective role of Prdx2 has been established in cerebral ischemia and several neurodegenerative diseases, its contribution against iron-induced neurocytotoxicity still remains to be determined. Accordingly, in this study, we aimed to investigate the effects of Prdx2 on iron-induced cytotoxicity using an in vitro model in which PC12 cells are exposed to ferrous sulfate (FS). The FS treatment increased Prdx2 expression, and promoted lactate dehydrogenase (LDH) release and cell apoptosis in PC12 cells, accompanied by the increase in the Bax/Bcl2 ratio, cytochrome c release, and caspase-3 cleavage. FS exposure also increased the malondialdehyde content (lipid peroxidation), 3′-nitrotyrosine expression (protein nitration), γ-H2A.X formation (DNA oxidation), and promoted nuclear factor kappa B nuclear translocation, cyclooxygenase-2 expression, and release of tumor necrosis factor-α and interleukin-1β. Lentivirus-mediated Prdx2 knockdown intensified the FS-induced LDH release and cell apoptosis by aggravating the oxidative and inflammatory damage. In conclusion, our findings demonstrated that Prdx2 played a vital role in the protection against iron-induced cytotoxicity in PC12 cells.     

L-Ascorbate Protects Against Methamphetamine-Induced Neurotoxicity of Cortical Cells via Inhibiting Oxidative Stress,Autophagy, and Apoptosis

Methamphetamine (METH)-induced cell death contributes to the pathogenesis of neurotoxicity; however, the relative roles of oxidative stress, apoptosis, and autophagy remain unclear. L-Ascorbate, also called vitamin (Vit.) C, confers partial protection against METH neurotoxicity via induction of heme oxygenase-1. We further investigated the role of Vit. C in METH-induced oxidative stress, apoptosis, and autophagy in cortical cells. Exposure to lower concentrations (0.1, 0.5, 1 mM) of METH had insignificant effects on ROS production, whereas cells exposed to 5 mM METH exhibited ROS production in a time-dependent manner. We confirmed METH-induced apoptosis (by nuclear morphology revealed by Hoechst 33258 staining and Western blot showing the protein levels of pro-caspase 3 and cleaved caspase 3) and autophagy (by Western blot showing the protein levels of Belin-1 and conversion of microtubule-associated light chain (LC)3-I to LC3-II and autophagosome staining by monodansylcadaverine). The apoptosis as revealed by cleaved caspase-3 expression marked an increase at 18 h after METH exposure while both autophagic markers, Beclin 1 and LC3-II, marked an increase in cells exposed to METH for 6 and 24 h, respectively. Treating cells with Vit. C 30 min before METH exposure time-dependently attenuated the production of ROS. Vitamin C also attenuated METH-induced Beclin 1 and LC3-II expression and METH toxicity. Treatment of cells with Vit. C before METH exposure attenuated the expression of cleaved caspase-3 and reduced the number of METH-induced apoptotic cells. We suggest that the protective effect of Vit. C against METH toxicity might be through attenuation of ROS production, autophagy, and apoptosis.