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.

     

Methamphetamine induces autophagy as a pro-survival response against apoptotic endothelial cell death through the Kappa opioid receptor

Methamphetamine (METH) is a psychostimulant with high abuse potential and severe neurotoxicity. Recent studies in animal models have indicated that METH can impair the blood–brain barrier (BBB), suggesting that some of the neurotoxic effects resulting from METH abuse could be due to barrier disruption. We report here that while chronic exposure to METH disrupts barrier function of primary human brain microvascular endothelial cells (HBMECs) and human umbilical vein endothelial cells (HUVECs), an early pro-survival response is observed following acute exposure by induction of autophagic mechanisms. Acute METH exposure induces an early increase in Beclin1 and LC3 recruitment. This is mediated through inactivation of the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p70S6K pathway, and upregulation of the ERK1/2. Blockade of Kappa opioid receptor (KOR), and treatment with autophagic inhibitors accelerated METH-induced apoptosis, suggesting that the early autophagic response is a survival mechanism for endothelial cells and is mediated through the kappa opioid receptor. Our studies indicate that kappa opioid receptor can be therapeutically exploited for attenuating METH-induced BBB dysfunction.     

Proteomic analysis revealed association of aberrant ROS signaling with suberoylanilide hydroxamic acid-induced autophagy in Jurkat T-leukemia cells

Suberoylanilide hydroxamic acid (SAHA) is a newly emerging histone deacetylase inhibitor (HDACi) and has been approved in phase II clinical trials for treating patients with cutaneous T-cell lymphoma. Autophagy is a conserved self-digestion process that degrades cytoplasmic materials and recycles long-lived proteins and organelles within cells. In this study, we demonstrate that SAHA stimulates autophagy in Jurkat T-leukemia cells, which was evidenced by the appearance of autophagic vacuoles, formation of acidic vesicular organelles, recruitment of LC3-II to the autophagosomes and conversion of LC3-I to LC3-II. Moreover, SAHA treatment upregulated expression of Beclin 1 and Atg7 and promoted formation of the Atg12-Atg5 conjugate. Furthermore, inhibition of autophagy by chloroquine (CQ) enhanced SAHA-induced apoptosis. To determine the underlying mechanism of SAHA-induced autophagy, two complementary proteomic approaches (2-DE and SILAC), coupled with ESI-Q-TOF MS/MS analysis are utilized to profile differentially expressed proteins between control and SAHA-treated Jurkat T-leukemia cells. In total, 72 proteins were identified with significant alterations. Cluster analysis of the changed proteins reveal several groups of enzymes associated with energy metabolism, anti-oxidative stress and cellular redox control, which suggested an abnormal reactive oxygen species (ROS) production in SAHA-treated Jurkat T-leukemia cells. These observations were further confirmed by ROS chemiluminescence assay. Mechanistic studies revealed that SAHA-triggered autophagy was mediated by ROS production, which could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we illustrated that Akt-mTOR signaling, a major suppressive cascade of autophagy, was inactivated by SAHA treatment. Taken together, our study identifies autophagy as a reaction to counter increased ROS and is thus involved as a cellular prosurvival mechanism in response to SAHA treatment.     

Localization of Beclin1 in mouse developing tooth germs: possible implication of the interrelation between autophagy and apoptosis

Our previous study identified the appearance of autophagy in developing tooth germs, and suggested its possible association with apoptosis in odontogenesis. Beclin1 was recently indicated to play a central role in bridging autophagy and apoptosis, and occupied a key position in the process of development. This study hypothesized that Beclin1 may be involved, and act as the molecular basis of the connection between autophagy and apoptosis in odontogenesis. Immunohistochemical analysis showed the spatiotemporal expression pattern of Beclin1 in odontogenesis from embryonic (E) day 13.5 to postnatal (P) day 5.5. At E stages, Beclin1 was mainly immunolocalized in the cytoplasm of the cells in the enamel organ. Meanwhile, the nucleus localization of Beclin1 was detected in part of the stellate reticulum, outer and inner enamel epithelium, especially at E16.5 and E18.5. At P stages, Beclin1 was detected in the cytoplasm of the odontoblasts, besides the dental epithelium cells. Triple immunofluorescence analysis showed the partial colocalization of Beclin1, autophagic marker LC3, or activated caspase-3 in the E14.5 tooth germs, especially the Beclin1⁺LC3⁺Caspase-3⁺ cells in the PEK. Furthermore, western blot analysis revealed that the full-length (60 kDa) and/or cleaved (50, 37, and 35 kDa) Beclin1 in the developing tooth germs. Taken together, our findings indicate that Beclin1 is involved, and might be responsible for the crosstalk between autophagy and apoptosis in mouse odontogenesis.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

1-Methyl-4-Phenylpyridinium-Induced Cell Death via Autophagy Through a Bcl-2/Beclin 1 Complex-Dependent Pathway

Several lines of evidence suggest that the mechanism underlying drug-induced neuronal apoptosis is initiated by the increased production of reactive oxygen species (ROS). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin, has been shown to initiate an apoptotic cascade by increasing ROS in the dopaminergic neurons of the substantia nigra, leading to the morphological and physiological features associated with Parkinson’s disease. Recently, it has been reported that autophagy, a type of programmed cell death independent of the apoptotic cascade, also plays a role in neuronal damage. Although autophagy is negatively regulated by the mammalian target of rapamycin receptor (mTOR), there is some evidence showing a novel function for the anti-apoptotic protein Bcl-2. Bcl-2 is proposed to play a role in negatively regulating autophagy by blocking an essential protein in the signaling pathway, Beclin 1. Nevertheless, it is unclear whether autophagy is also correlated with apoptotic signaling in 1-methyl-4-phenylpyridinium (MPP⁺) toxicity. Therefore, we hypothesized that the MPP⁺ toxicity generally associated with initiating the apoptotic signaling cascade also increases an autophagic phenotype in neuronal cells. Using the SK-N-SH dopaminergic cell lines, we demonstrate that MPP⁺ increases the expression of microtubule-associated protein light chain 3 (LC3-II), an autophagosome membrane marker and the mTOR signaling pathway, and Beclin 1 while decreasing the Bcl-2 levels. Moreover, these expressions correlate with a decreased binding ratio between Bcl-2 and Beclin 1, in effect limiting the regulation of the downstream autophagic markers, such as LC3-II. Our results indicate that MPP⁺ can induce autophagy in SK-N-SH cells by decreasing the Bcl-2/Beclin 1 complex.     

Autophagy is a protective response to ethanol neurotoxicity

Ethanol is a neuroteratogen and neurodegeneration is the most devastating consequence of developmental exposure to ethanol. The mechanisms underlying ethanol-induced neurodegeneration are complex. Ethanol exposure produces reactive oxygen species (ROS) which cause oxidative stress in the brain. We hypothesized that ethanol would activate autophagy to alleviate oxidative stress and neurotoxicity. Our results indicated that ethanol increased the level of the autophagic marker Map1lc3-II (LC3-II) and upregulated LC3 puncta in SH-SY5Y neuroblastoma cells. It also enhanced the levels of LC3-II and BECN1 in the developing brain; meanwhile, ethanol reduced SQSTM1 (p62) levels. Bafilomycin A₁, an inhibitor of autophagosome and lysosome fusion, increased p62 levels in the presence of ethanol. Bafilomycin A₁ and rapamycin potentiated ethanol-increased LC3 lipidation, whereas wortmannin and a BECN1-specific shRNA inhibited ethanol-promoted LC3 lipidation. Ethanol increased mitophagy, which was also modulated by BECN1 shRNA and rapamycin. The evidence suggested that ethanol promoted autophagic flux. Activation of autophagy by rapamycin reduced ethanol-induced ROS generation and ameliorated ethanol-induced neuronal death in vitro and in the developing brain, whereas inhibition of autophagy by wortmannin and BECN1-specific shRNA potentiated ethanol-induced ROS production and exacerbated ethanol neurotoxicity. Furthermore, ethanol inhibited the MTOR pathway and downregulation of MTOR offered neuroprotection. Taken together, the results suggest that autophagy activation is a neuroprotective response to alleviate ethanol toxicity. Ethanol modulation of autophagic activity may be mediated by the MTOR pathway.     

Selenium Deficiency Induces Autophagy in Immune Organs of Chickens

The aim of the present study was to investigate the effects of selenium (Se) deficiency on autophagy-related genes and on ultrastructural changes in the spleen, bursa of Fabricius, and thymus of chickens. The Se deficiency group was fed a basal diet containing Se at 0.033 mg/kg and the control group was fed the same basal diet containing Se at 0.15 mg/kg. The messenger RNA (mRNA) levels of the autophagy genes microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, Beclin 1, dynein, autophagy associated gene 5 (ATG5), and target of rapamycin complex 1 (TORC1) were assessed using real-time qPCR. The protein levels of LC3-II, Beclin 1, and dynein were investigated using western blot analysis. Furthermore, the ultrastructure was observed using an electron microscope. The results indicated that spleen mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of LC3-II, Beclin 1, and dynein were increased in the Se deficiency group compared with the control group. In the bursa of Fabricius, the mRNA levels of LC3-I, LC3-II, Beclin 1, dynein, ATG5, and TORC1 and the protein levels of Beclin 1 and dynein were increased; furthermore, the protein level of LC3-II was decreased in the Se deficiency group compared to the control group. In the thymus, the mRNA levels of LC3-I, Beclin 1, and ATG5 increased; the levels of LC3-II, dynein, and TORC1 were decreased; the protein level of Beclin 1 increased; and the levels of LC3-II and dynein decreased in the Se deficiency group compared to those in the control group. Further cellular morphological changes, such as autophagy vacuoles, autolysosomes, and lysosomal degradation, were observed in the spleen, bursa of Fabricius, and thymus of the Se-deficiency group. In summary, Se deficiency caused changes in autophagy-related genes, which increased the autophagic process and also caused structural damages to the immune organs of chickens.     

Ketamine induces reactive oxygen species and enhances autophagy in SV-HUC-1 human uroepithelial cells

This study was aimed at exploring the underlying mechanisms of ketamine in the SV-40 immortalized human ureteral epithelial (SV-HUC-1) cells. The viability and apoptosis of SV-HUC-1 cells treated with 0.01, 0.1, and 1 mM ketamine were respectively detected via cell counting kit-8 (CCK-8) assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Reactive oxygen species (ROS) level was measured through ROS probe staining. Apoptosis-related proteins (B-cell lymphoma 2 [Bcl-2] and Bax) and autophagy-associated proteins (light chain 3-I [LC3-I] and LC3-II) were determined by western blot or immunofluorescent assay. Additionally, transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes. After cotreatment of 3-methyladenine (3-MA) or N-acetyl-l -cysteine (NAC), the biological functions of SV-HUC-1 cells were analyzed to determine the association of ROS with cell viability and autophagy. CCK-8 assay and TUNEL staining indicated that ketamine effectively decreased the viability of SV-HUC-1 cells and accelerated apoptosis of SV-HUC-1 cells through regulating the expression level of IKBα (phospho), nuclear factor кB (P65), Bcl-2, and Bax proteins. Enhanced ROS production was also confirmed in ketamine-treated SV-HUC-1 cells treated with ketamine. Ketamine-induced autophagosomes in SV-HUC-1 cells were observed by means of TEM, and increased levels of LC3 II/I ratio and Beclin 1 were examined through western blot and immunofluorescent assay. Furthermore, ketamine exerted effects on SV-HUC-1 cells in a dose-dependent and time-dependent manner. Additionally, cotreatment of NAC with 3-MA significantly attenuated the ROS level and suppressed the cell autophagy. Ketamine promoted SV-HUC-1 cell autophagy and impaired the cell viability of SV-HUC-1 cells by inducing ROS.     

Licarin A induces cell death by activation of autophagy and apoptosis in non-small cell lung cancer cells

Lung cancer has a relatively poor prognosis with a low survival rate and drugs that target other cell death mechanism like autophagy may help improving current therapeutic strategy. This study investigated the anti-proliferative effect of Licarin A (LCA) from Myristica fragrans in non-small cell lung cancer cell lines—A549, NCI-H23, NCI-H520 and NCI-H460. LCA inhibited proliferation of all the four cell lines in a dose and time dependent manner with minimum IC50 of 20.03?±?3.12, 22.19?±?1.37 µM in NCI-H23 and A549 cells respectively. Hence NCI-H23 and A549 cells were used to assess the ability LCA to induce autophagy and apoptosis. LCA treatment caused G1 arrest, increase in Beclin 1, LC3II levels and degradation of p62 indicating activation of autophagy in both NCI-H23 and A549 cells. In addition, LCA mediated apoptotic cell death was confirmed by MMP loss, increased ROS, cleaved PARP and decreased pro-caspase3. To understand the role of LCA induced autophagy and its association with apoptosis, cells were analysed following treatment with a late autophagy inhibitor-chloroquine and also after Beclin 1 siRNA transfection. Data indicated that inhibition of autophagy resulted in reduced anti-proliferative as well as pro-apoptotic ability of LCA. These findings confirmed that LCA brought about autophagy dependent apoptosis in non-small cell lung cancer cells and hence it may serve as a potential drug candidate for non-small cell lung cancer therapy.     

Evaluation of the effects of alpha-phenyl-N-tert-butyl nitrone pretreatment on the neurobehavioral effects of methamphetamine

A relationship between formation of reactive oxygen species (ROS) and energy depletion has been proposed to play an important role in mediating methamphetamine (METH)-induced neurotoxicity. To evaluate this relationship, we examined the effect of the spin-trap agent, alpha-phenyl-N-tert-butyl nitrone (PBN) on hyperthermia and self-injurious behavior (SIB) and striatal dopamine (DA) depletion produced by METH (4 injections of 4 mg/kg, 2 hr intervals, s.c.) in BALB/c mice. Repeated administration of METH induced hyperthermia, incidence of SIB and striatal DA depletion (84% after 3 days). Pretreatment with PBN (4 injections of 60 or 120 mg/kg, i.p.) reduced METH-induced hyperthermia, but did not significantly attenuate METH-induced SIB or the striatal DA depletion. On the other hand, pretreatment with high doses of PBN (4 injections of 180 or 240 mg/kg, i.p.) protected against METH-induced hyperthermia and SIB, and PBN (180 mg/kg) also completely protected against the acute striatal DA depletion 60 min after the last injection of the drug. However, the long-lasting striatal DA depletion was only attenuated by 52 or 56%, respectively. These results indicate that METH-induced hyperthermia contributes to, but is not solely responsible for METH-induced neurotoxicity, and supports a role for formation of ROS and other mechanisms in the generation of METH-induced striatal dopaminergic neurotoxicity. In addition, the difference in the efficacy of PBN to protect against the acute or long-lasting striatal DA depletion induced by METH may indicate that both ROS formation and other mechanisms are required for METH-induced neurotoxicity to develop.     

Aristolochic acid I induced autophagy extenuates cell apoptosis via ERK 1/2 pathway in renal tubular epithelial cells

Autophagy is a lysosomal degradation pathway that is essential for cell survival and tissue homeostasis. However, limited information is available about autophagy in aristolochic acid (AA) nephropathy. In this study, we investigated the role of autophagy and related signaling pathway during progression of AAI-induced injury to renal tubular epithelial cells (NRK52E cells). The results showed that autophagy in NRK52E cells was detected as early as 3-6 hrs after low dose of AAI (10 μM) exposure as indicated by an up-regulated expression of LC3-II and Beclin 1 proteins. The appearance of AAI-induced punctated staining of autophagosome-associated LC3-II upon GFP-LC3 transfection in NRK52E cells provided further evidence for autophagy. However, cell apoptosis was not detected until 12 hrs after AAI treatment. Blockade of autophagy with Wortmannin or 3-Methyladenine (two inhibitors of phosphoinositede 3-kinases) or small-interfering RNA knockdown of Beclin 1 or Atg7 sensitized the tubular cells to apoptosis. Treatment of NRK52E cells with AAI caused a time-dependent increase in extracellular signal-regulated kinase 1 and 2 (ERK1/2) activity, but not c-Jun N-terminal kinase (JNK) and p38. Pharmacological inhibition of ERK1/2 phosphorylation with U0126 resulted in a decreased AAI-induced autophagy that was accompanied by an increased apoptosis. Taken together, our study demonstrated for the first time that autophagy occurred earlier than apoptosis during AAI-induced tubular epithelial cell injury. Autophagy induced by AAI via ERK1/2 pathway might attenuate apoptosis, which may provide a protective mechanism for cell survival under AAI-induced pathological condition.     

Airborne fine particulate matter induces multiple cell death pathways in human lung epithelial cells

Our group was the first one reporting that autophagy could be triggered by airborne fine particulate matter (PM) with a mean diameter of less than 2.5 μm (PM_2.5) in human lung epithelial A549 cells, which could potentially lead to cell death. In the present study, we further explored the potential interactions between autophagy and apoptosis because it was well documented that PM_2.5 could induce apoptosis in A549 cells. Much to our surprise, we found that PM_2.5-exposure caused oxidative stress, resulting in activation of multiple cell death pathways in A549 cells, that is, the tumor necrosis factor-alpha (TNF-α)-induced pathway as evidenced by TNF-α secretion and activation of caspase-8 and -3, the intrinsic apoptosis pathway as evidenced by increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic protein Bcl-2, disruption of mitochondrial membrane potential, and activation of caspase-9 and -3, and autophagy as evidenced by an increased number of double-membrane vesicles, accompanied by increases of conversion and punctuation of microtubule-associated proteins light chain 3 (LC3) and expression of Beclin 1. It appears that reactive oxygen species (ROS) function as signaling molecules for all the three pathways because pretreatment with N-acetylcysteine, a scavenger of ROS, almost completely abolished TNF-α secretion and significantly reduced the number of apoptotic and autophagic cells. In another aspect, inhibiting autophagy with 3-methyladenine, a specific autophagy inhibitor, enhanced PM_2.5-induced apoptosis and cytotoxicity. Intriguingly, neutralization of TNF-α with an anti-TNF-α special antibody not only abolished activation of caspase-8, but also drastically reduced LC3-II conversion. Thus, the present study has provided novel insights into the mechanism of cytotoxicity and even pathogenesis of diseases associated with PM_2.5 exposure.     

Cadmium results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells

Environmental exposure to cadmium (Cd) links to neurodegenerative disorders. Autophagy plays an important role in controlling cell survival/death. However, how autophagy contributes to Cd's neurotoxicity remains enigmatic. Here, we show that Cd induced significant increases in autophagosomes with a concomitant elevation of LC3-II and p62 in PC12 cells and primary neurons. Using autophagy inhibitor 3-MA, we demonstrated that Cd-increased autophagosomes contributed to neuronal apoptosis. Impairment of Cd on autophagic flux was evidenced by co-localization of mCherry and GFP tandem-tagged LC3 puncta in the cells. This is further supported by the findings that administration of chloroquine (CQ) potentiated the basic and Cd-elevated LC3-II and p62 levels, autophagosome accumulation and cell apoptosis, whereas rapamycin relieved the effects in the cells in response to Cd. Subsequently, we noticed that Cd evoked the phosphorylation of Akt and BECN1. Silencing BECN1 and especially expression of mutant BECN1 (Ser295A) attenuated Cd-increased autophagosomes and cell death. Of note, inhibition of Akt with Akt inhibitor X, or ectopic expression of dominant negative Akt (dn-Akt), in the presence or absence of 3-MA, significantly alleviated Cd-triggered phosphorylation of Akt and BECN1, autophagosomes, and apoptosis. Importantly, we found that Cd activation of Akt functioned in impairing autophagic flux. Collectively, these results indicate that Cd results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells. Our findings underscore that inhibition of Akt to improve autophagic flux is a promising strategy against Cd-induced neurotoxicity and neurodegeneration.     

Down-Regulation of miRNA-30a Alleviates Cerebral Ischemic Injury Through Enhancing Beclin 1-Mediated Autophagy

The understanding of molecular mechanism underlying ischemia/reperfusion-induced neuronal death and neurological dysfunction may provide therapeutic targets for ischemic stroke. The up-regulated miRNA-30a among our previous identified 19 MicroRNAs (miRNAs) in mouse brain after 6 h middle cerebral artery occlusion (MCAO) could negatively regulate Beclin 1 messenger RNA (mRNA) resulting in decreased autophagic activity in tumor cells and cardiomyocytes, but its role in ischemic stroke is unclear. In this study, the effects of miRNA-30a on ischemic injury in N2A cells and cultured cortical neurons after oxygen glucose deprivation (OGD), and mouse brain with MCAO-induced ischemic stroke were evaluated. The results showed that miRNA-30a expression levels were up regulated in the brain of mice after 6 h MCAO without reperfusion, but significantly down regulated in the peri-infarct region of mice with 1 h MCAO/24 h reperfusion and in N2A cells after 1 h OGD/6–48 h reoxygenation. Both the conversion ratio of microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I and Beclin 1 protein level increased in N2A cells and cultured cortical neurons following 1 h OGD/24 h reoxygenation. The down-regulated miRNA-30a could attenuate 1 h OGD/24 h reoxygenation-induced ischemic injury in N2A cells and cultured cortical neurons through enhancing Beclin 1-mediated autophagy, as miRNA-30a recognized the 3′-untranslated region of beclin 1 mRNA to negatively regulate Beclin 1-protein level via promoting beclin 1 messenger RNA (mRNA) degradation, and Beclin 1 siRNA abolished anti-miR-30a-induced neuroprotection in 1 h OGD/24 h reoxygenation treated N2A cells. In addition, anti-miR-30a attenuated the neural cell loss and improved behavioral outcome of mice with ischemic stroke. These results suggested that down-regulation of miRNA-30a alleviates ischemic injury through enhancing beclin 1-mediated autophagy, providing a potential therapeutic target for ischemic stroke.     

Crocin Inhibits Apoptosis and Astrogliosis of Hippocampus Neurons Against Methamphetamine Neurotoxicity via Antioxidant and Anti-inflammatory Mechanisms

Methamphetamine (METH) is a stimulant drug, which can cause neurotoxicity and increase the risk of neurodegenerative disorders. The mechanisms of acute METH intoxication comprise intra-neuronal events including oxidative stress, dopamine oxidation, and excitotoxicity. According to recent studies, crocin protects neurons by functioning as an anti-oxidant, anti-inflammatory, and anti-apoptotic compound. Accordingly, this study aimed to determine if crocin can protect against METH-induced neurotoxicity. Seventy-two male Wistar rats that weighed 260–300 g were randomly allocated to six groups of control (n?=?12), crocin 90 mg/kg group (n?=?12), METH (n?=?12), METH?+?crocin 30 mg/kg (n?=?12), METH?+?crocin 60 mg/kg (n?=?12), and METH?+?crocin 90 mg/kg (n?=?12). METH neurotoxicity was induced by 40 mg/kg of METH in four injections (e.g., 4?×?10 mg/kg q. 2 h, IP). Crocin was intraperitoneally (IP) injected at 30 min, 24 h, and 48 h after the final injection of METH. Seven days after METH injection, the rats’ brains were removed for biochemical assessment using the ELISA technique, and immunohistochemistry staining was used for caspase-3 and glial fibrillary acidic protein (GFAP) detection. Crocin treatment could significantly increase superoxide dismutase (P?<?0.05) and glutathione (P?<?0.01) levels and reduce malondialdehyde and TNF-α in comparison with the METH group (P?<?0.05). Moreover, crocin could significantly decline the level of caspase-3 and GFAP-positive cells in the CA1 region (P?<?0.01). According to the results, crocin exerts neuroprotective effects on METH neurotoxicity via the inhibition of apoptosis and neuroinflammation.     

Parkin,as a Regulator,Participates in Arsenic Trioxide-Triggered Mitophagy in HeLa Cells

Parkin is a well-established synergistic mediator of mitophagy in dysfunctional mitochondria. Mitochondria are the main target of arsenic trioxide (ATO) cytotoxicity, and the effect of mitophagy on ATO action remains unclear. In this study, we used stable Parkin-expressing (YFP-Parkin) and Parkin loss-of-function mutant (Parkin C431S) HeLa cell models to ascertain whether Parkin-mediated mitophagy participates in ATO-induced apoptosis/cell death. Our data showed that the overexpression of Parkin significantly sensitized HeLa cells to ATO-initiated proliferation inhibition and apoptosis; however, the mutation of Parkin C431S significantly weakened this Parkin-mediated responsiveness. Our further investigation found that ATO significantly downregulated two fusion proteins (Mfn1/2) and upregulated fission-related protein (Drp1). Autophagy was also activated as evidenced by the formation of autophagic vacuoles and mitophagosomes, increased expression of PINK1, and recruitment of Parkin to impaired mitochondria followed by their degradation, accompanied by the increased transformation of LC3-I to LC3-II, increased expression of Beclin1 and decreased expression of P62 in YFP-Parkin HeLa cells. Enhanced mitochondrial fragmentation and autophagy indicated that mitophagy was activated. Furthermore, during the process of mitophagy, the overproduction of ROS implied that ROS might represent a key factor that initiates mitophagy following Parkin recruitment to mitochondria. In conclusion, our findings indicate that Parkin is critically involved in ATO-triggered mitophagy and functions as a potential antiproliferative target in cancer cells.     

Apolipoprotein L6, induced in atherosclerotic lesions, promotes apoptosis and blocks Beclin 1-dependent autophagy in atherosclerotic cells

Inflammatory cytokine-regulated apoptosis and autophagy play pivotal roles in plaque rupture and thrombosis of atherosclerotic lesions. However, the molecular interplay between apoptosis and autophagy in vascular cells has not been investigated. Our prior study showed that human apolipoprotein L6 (ApoL6), a pro-apoptotic BH3-only member of the Bcl-2 family, was one of the downstream targets of interferon-γ (INFγ), which sensitizes atherosclerotic lesion-derived cells (LDCs) to Fas-induced apoptosis. To investigate whether ApoL6 plays a causal role in atherosclerotic apoptosis and autophagy, in this study, we demonstrate that IFNγ treatment itself strongly induces ApoL6, and ApoL6 is highly expressed and partially co-localized with activated caspase 3 in activated smooth muscle cells in atherosclerotic lesions. In addition, overexpression of ApoL6 promotes reactive oxygen species (ROS) generation, caspase activation, and subsequent apoptosis, which can be blocked by pan caspase inhibitor and ROS scavenger. Knockdown of ApoL6 expression by siApoL6 suppresses INFγ- and Fas-mediated apoptosis. Further, ApoL6 binds Bcl-X(L), one of the most abundant anti-death proteins in LDCs. Interestingly, forced ApoL6 expression in LDCs induces degradation of Beclin 1, accumulation of p62, and subsequent attenuation of LC3-II formation and translocation and thus autophagy, whereas siApoL6 treatment reverts the phenotype. Taken together, our results suggest that ApoL6 regulates both apoptosis and autophagy in SMCs. IFNγ-initiated, ApoL6-induced apoptosis in vascular cells may be an important factor causing plaque instability and a potential therapeutic target for treating atherosclerosis and cardiovascular disease.     

Dihydrocapsaicin (DHC), a saturated structural analog of capsaicin, induces autophagy in human cancer cells in a catalase-regulated manner

Although capsaicin, a pungent component of red pepper, is known to induce apoptosis in several types of cancer cells, the mechanisms underlying capsaicin-induced cytotoxicity are unclear. Here, we showed that dihydrocapsaicin (DHC), an analog of capsaicin, is a potential inducer of autophagy. DHC was more cytotoxic than capsaicin in HCT116, MCF-7 and WI38 cell lines. Capsaicin and DHC did not affect the sub-G(1) apoptotic peak, but induced G(0)/G(1) arrest in HCT116 and MCF-7 cells. DHC caused the artificial autophagosome marker GFP-LC3 to redistribute and upregulated expression of autophagy-related proteins. Blocking of autophagy by 3-methyladenine (3MA) as well as siRNA Atg5 induced a high level of caspase-3 activation. Although pretreatment with zVAD completely inhibited caspase-3 activation by 3MA, it did not prevent cell death. DHC-induced autophagy was enhanced by zVAD pretreatment, as shown by increased accumulation of LC3-II protein. DHC attenuated basal ROS levels through catalase induction; this effect was enhanced by antioxidants, which increased both LC3-II expression and caspase-3 activation. The catalase inhibitor 3-amino-1,2,4-triazole (3AT) abrogated DHC-induced expression of LC3-II, overexpression of the catalase gene increased expression of LC3-II protein, and knockdown decreased it. Additionally, DHC-induced autophagy was independent of p53 status. Collectively, DHC activates autophagy in a p53-independent manner and that may contribute to cytotoxicity of DHC.