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Purpose To investigate the effect of prolonged sevoflurane (SEV) exposure on differentiation potential and hypoxia tolerance of neural stem cells (NSCs). Materials and methods NSCs were extracted from 15-day fetal mice. After sub-culture, Sevoflurane exposure treatment was performed. Cell cycle were detected by flow cytometry. Western Blot and immunofluorescence assay were used to detect the expression and spatial distribution of Nestin, NSE, GFAP, Oct4, and SOX2; CCK-8 detected cell viability. Cell growth morphology was observed under a microscope. Tunel detected cell apoptosis; the concentration of extracel-lular lactate dehydrogenase (LDH) was determined by ELISA. Results Compared with the control group, the proportion of NSCs in the G2/M phase increased in the Sevoflurane exposure group; our results also suggested the sphere- formation rate decreased significantly, Increased apoptosis and decreased cell viability; Besides, the level of LDH release increased. Conclusion Long-term exposure to sevoflurane (> 8 h) promoted the premature differentiation of NSCs and reduced their pluripotency, reserves, and hypoxia tolerance. This study reveals the reasons underlying damage to the nervous system of young children induced by long-term exposure to sevoflurane from the perspective of CNS reserve cells.

The purpose of this study was to determine whether the epidermal growth factor receptor (EGFR), which is a classical receptor tyrosine kinase, is involved in the protective effect of morphine against ischemia/reperfusion (I/R)-induced myocardial mitochondrial damage. Isolated rats hearts were subjected to global ischemia followed by reperfusion. Cardiac H9c2 cells were exposed to a simulated ischemia solution followed by Tyrode’s solution to induce hypoxia/reoxygenation (H/R) injury. Triphenyltetrazolium chloride (TTC) was used to measure infarct size. The mitochondrial morphological and functional changes were determined using transmission election microscopy (TEM), mitochondrial stress assay, and mitochondrial swelling, respectively. Mitochondrial fluorescence indicator JC-1, DCFH-DA, and Mitosox Red were used to determine mitochondrial membrane potential (△Ψm), intracellular reactive oxygen species (ROS) and mitochondrial superoxide. A TUNUL assay kit was used to detect the level of apoptosis. Western blotting analysis was used to measure the expression of proteins. Treatment of isolated rat hearts with morphine prevented I/R-induced myocardial mitochondrial injury, which was inhibited by the selective EGFR inhibitor AG1478, suggesting that EGFR is involved in the mitochondrial protective effect of morphine under I/R conditions. In support of this hypothesis, the selective EGFR agonist epidermal growth factor (EGF) reduced mitochondrial morphological and functional damage similarly to morphine. Further study demonstrated that morphine may alleviate I/R-induced cardiac damage by inhibiting autophagy but not apoptosis. Morphine increased protein kinase B (Akt), extracellular regulated protein kinases (ERK) and signal transducer and activator of transcription-3 (STAT-3) phosphorylation, which was inhibited by AG1478, and EGF had similar effects, indicating that morphine may activate Akt, ERK, and STAT-3 via EGFR. Morphine and EGF increased intracellular reactive oxygen species (ROS) generation. This effect of morphine was inhibited by AG1478, indicating that morphine promotes intracellular ROS generation by activating EGFR. However, morphine did not increase ROS generation when cells were transfected with siRNA against EGFR. In addition, EGFR activity was markedly increased by morphine, but the effect of morphine was reversed by naltrindole. These results suggest that morphine may activate EGFR via δ-opioid receptor activation. Morphine may prevent I/R-induced myocardial mitochondrial damage by activating EGFR through δ-opioid receptors, in turn increasing RISK and SAFE pathway activity via intracellular ROS. Moreover, morphine may reduce myocardial injury by regulating autophagy but not apoptosis.

Preeclampsia (PE) is a major cause of adverse maternal and infant outcomes. Accurate screening of PE is currently the focus of clinical attention. This study aimed to develop a model for predicting PE.A retrospective case-control study was conducted with 916 pregnant women who received care at the Second Hospital of Tianjin Medical University (October 2018 to July 2020). Women were randomly divided into the training (n=680) and testing (n=236) sets based on a ratio of 3:1. Demographic and clinical data of women were collected. In training set, logistic regression (LR), classification tree (CT) model, and random forest (RF) algorithm were used to develop prediction models for PE. Using the testing set was to validate these prediction models. The predictive performance of three models were assessed by the area under the curve (AUC), sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV).Of the total 916 women, 237 had PE. The family history of hypertension, pre-pregnancy body mass index (pBMI), blood pressure (BP) ≥130/80 mmHg in early pregnancy, age, chronic hypertension, and duration of hypertension were the predictors of PE. The AUCs for the LR, CT, and RF models were 0.778, 0.850, and 0.871, respectively (all P0.05 for all pair-wise comparisons). The RF had the best predictive efficiency with sensitivity, specificity, PPV, and NPV of 79.6%, 94.7%, 79.6%, and 94.7%, respectively.The RF model could be a practical screening approach for predicting PE, which is helpful for clinicians to identify high-risk individuals and prevent the occurrence of adverse pregnancy outcomes.

Moderate hypothermia plays a major role in myocardial cell death as a result of hypoxia/reoxygenation (H/R) injury. However, few studies have investigated the molecular mechanisms of hypothermic cardioprotection. Several responses to stress and other cell functions are regulated by post‑translational protein modifications controlled by small ubiquitin‑like modifier (SUMO). Previous studies have established that high SUMOylation of proteins potentiates the ability of cells to withstand hypoxic‑ischemic stress. The level to which moderate hypothermia affects SUMOylation is not fully understood, as the functions of SUMOylation in the heart have not been studied in depth. The aim of the present study was to investigate the effect of moderate hypothermia (33˚C) on the protective functions of SUMOylation on myocardial cells. HL‑1 and H9c2 cells were treated with the hypoxia‑mimetic chemical CoCl2 and complete medium to simulate H/R injury. Hypothermia intervention was then administered. A Cell Counting kit‑8 assay was used to analyze cell viability. Mitochondrial membrane potential and the generation of reactive oxygen species (ROS) were used as functional indexes of mitochondria dysfunction. Bcl‑2 and caspase‑3 expression levels were analyzed by western blotting. The present results suggested that moderate hypothermia significantly increased SUMO1 and Bcl‑2 expression levels, as well as the mitochondrial membrane potential, but significantly decreased the expression levels of caspase‑3 and mitochondrial ROS. Thus, moderate hypothermia may enhance SUMOylation and attenuate myocardial H/R injury. Moreover, a combination of SUMOylation and moderate hypothermia may be a potential cardiovascular intervention.

It has been controversial whether patients with hepatocellular carcinoma (HCC) should receive glucocorticoid therapy during chemotherapy. Recent studies have demonstrated that glucocorticoids increase the therapeutic sensitivity of tumors to some chemotherapeutic drugs, but the specific mechanism remains unclear. In the present study, dexamethasone (Dex) was used to treat HCC stem cells. The results demonstrated that Dex reduced stemness maintenance and self‑renewal of HCC stem cells, promoted epithelial‑to‑mesenchymal transition, inhibited migration and angiogenesis and, more importantly, increased cell sensitivity to the herpes simplex virus thymidine kinase/ganciclovir drug system in vitro and in vivo. Further mechanistic analyses demonstrated that Dex inhibited small ubiquitin‑like modifier (SUMO) modification of several proteins in HCC stem cells, including hypoxia‑inducible factor (HIF)‑1α, an important hypoxia tolerance protein, and octamer‑binding transcription factor 4 (Oct4), a crucial stemness maintenance protein. Inducing deSUMOylation of HIF‑1α and Oct4 reduced their accumulation in the nucleus, thereby inhibiting tumor angiogenesis and stemness maintenance. These findings provide a new perspective to the study of the mechanism underlying the anti‑hepatocarcinogenesis effects of Dex. Due to the few side effects of glucocorticoids at low doses and their anti‑inflammatory effects, the appropriate combination of glucocorticoids and chemotherapeutic drugs is expected to improve the survival of HCC patients and their prognosis.

Cerebral infarct penumbra due to hypoxia and toxin accumulation is not conducive to the transplantation of neural stem cells (NSCs), although mild hypothermia can improve the local microenvironment of the ischemic penumbra and exert neuroprotective effects. However, insufficient understanding of the molecular mechanism by which mild hypothermia protects the brain limits widespread clinical application. This study evaluated the molecular mechanism of mild hypothermia-induced brain protection from the perspective of global protein small ubiquitin-like modifier (SUMO) modification, with the aim of improving NSC transplant survival rates in the penumbra to enhance neurological function. NSCs from neonatal rats were extracted to detect the effects of hypoxia and mild hypothermia on SUMOylation modification levels, cell stemness, and hypoxia-induced injury. Overexpression and knockdown of UBC9 in NSCs were used to evaluate their ability to maintain stemness and withstand hypoxic injury. Finally, a rat middle cerebral artery occlusion (MCAO) model was used to verify the effect of mild hypothermia treatment and UBC9 overexpression on neural function of NSCs following penumbra transplantation in rats. Results showed that hypoxia and mild hypothermia promoted both the SUMOylation modification and maintenance of NSC stemness. Overexpression of UBC9 enhanced the abilities of NSCs to maintain stemness and resist hypoxic injury, while UBC9 knockdown had the opposite effect. Following transplantation into the ischemic penumbra of MCAO model rats, mild hypothermia and Ubc9-overexpressing NSCs significantly reduced cerebral infarct areas and improved neurological function. In conclusion, this study demonstrated that global protein SUMOylation is an important molecular mechanism for NSCs to tolerate hypoxia, and mild hypothermia can further increase the degree of global SUMOylation to enhance the hypoxia tolerance of NSCs, which increases their survival during transplantation in situ and ability to perform nerve repair in the penumbra of cerebral infarction.

Idiopathic pulmonary fibrosis (IPF) is a refractory chronic respiratory disease with progressively exacerbating symptoms and a high mortality rate. There are currently only two effective drugs for IPF; thus, there is an urgent need to develop new therapeutics. Previous experiments have shown that ginkgolic acid (GA), as a SUMO-1 inhibitor, exerted an inhibitory effect on cardiac fibrosis induced by myocardial infarction. Regarding the pathogenesis of PF, previous studies have concluded that small ubiquitin-like modifier (SUMO) polypeptides bind multiple target proteins and participate in fibrosis of multiple organs, including PF. In this study, we found altered expression of SUMO family members in lung tissues from IPF patients. GA mediated the reduced expression of SUMO1/2/3 and the overexpression of SENP1 in a PF mouse model, which improved PF phenotypes. At the same time, the protective effect of GA on PF was also confirmed in the SENP1-KO transgenic mice model. Subsequent experiments showed that SUMOylation of SMAD4 was involved in PF. It was inhibited by TGF-β1, but GA could reverse the effects of TGF-β1. SENP1 also inhibited the SUMOylation of SMAD4 and then participated in epithelial-mesenchymal transition (EMT) downstream of TGF-β1. We also found that SENP1 regulation of SMAD4 SUMOylation affected reactive oxygen species (ROS) production during TGF-β1-induced EMT and that GA prevented this oxidative stress through SENP1. Therefore, GA may inhibit the SUMOylation of SMAD4 through SENP1 and participate in TGF-β1-mediated pulmonary EMT, all of which reduce the degree of PF. This study provided potential novel targets and a new alternative for the future clinical testing in PF.

Zinc homeostasis has been known to play a role in myocardial ischemia/reperfusion (I/R) injury, but the precise molecular mechanisms regulating the expression of ZIP transporters during reperfusion are still unclear. The aim of this study was to determine whether ER Stress/CaMKII/STAT3 pathway plays a role in the regulation of cellular zinc homeostasis. Zinc deficiency increased mRNA and protein expressions of the ER stress relevant markers Chop and Bip, and STAT3 phosphorylation in H9c2 or HL-1 cells, an effect that was abolished by ZnCl2. ER calcium concentration [(Ca2+)ER] was decreased and cytosolic calcium concentration [(Ca2+)I] was increased at the condition of normoxia or ischemia/reperfusion, indicating that zinc deficiency triggers ER stress and Ca2+ leak. Further studies showed that upregulation of STAT3 phosphorylation was reversed by Ca2+ chelator, indicating that intracellular Ca2+ is important for zinc deficiency-induced STAT3 activation. In support, zinc deficiency enhanced ryanodine receptors (RyR), a channel in the ER that mediate Ca2+ release, and Ca2+-calmodulin-dependent protein kinase (CaMKII) phosphorylation, implying that zinc deficiency provoked Ca2+ leak from ER via RyR and p-CaMKII is involved in STAT3 activation. Moreover, inhibition of STAT3 activation blocked zinc deficiency induced ZIP9 expression, and resulted in increased Zn2+ loss in cardiomyocytes, further confirming that STAT3 activation during reperfusion promotes the expression of ZIP9 zinc transporter to correct the imbalance in zinc homeostasis. In addition, suppressed STAT3 activation aggravated reperfusion injury. These data suggest that the ER Stress/CaMKII/STAT3 axis may be an endogenous protective mechanism, which increases the resistance of the heart to I/R.

Background Although it is well known that selective intra-arterial cooling (SI-AC) elicits cerebral protection against ischemia/reperfusion (I/R) injury, the underlying mechanism remains unclear. This study aimed to determine whether SI-AC can protect against cerebral I/R injury by inhibiting oxidative stress and mitochondrial dysfunction through regulation of Sirt3 deSUMOylation via SENP1. Methods All mice were subjected to 2 h of cerebral ischemia followed by 24 h of reperfusion. SI-AC treatment was performed by infusion with cold saline (10 °C, 20 mL/kg) for 15 min through a microcatheter placed in the internal carotid artery immediately before reperfusion. The infarct volume, survival rate, neurological deficit scores, behavioral parameters, histopathology findings, and apoptosis were assessed. HT22 cells were subjected to 2 h of oxygen and sugar deprivation (OGD) and 22 h of reoxygenation. HA-SUMO1, Flag-Sirt3, a Sirt3 mutation plasmid (Flag-Sirt3 K288R), His-SENP1, and SENP1 small interfering RNA were transfected into HT22 cells 48 h before OGD. Apoptosis-related proteins were analyzed by western blotting. SUMOylation of Sirt3, acetylation of cyclooxygenase 1 (COX1), superoxide dismutase 2 (SOD2), and isocitrate dehydrogenase 2 (IDH2), the activities of COX1, SOD2, and IDH2, oxidative stress, and mitochondrial dysfunction were evaluated. Results Compared with the I/R group, SI-AC decreased cerebral infarct volume and neurological deficit scores and increased motor coordination, exploratory behavior, and memory. Hematoxylin and eosin and Nissl staining showed that SI-CA decreased karyopyknosis, nuclear fragmentation, and nucleolysis, increased neuron density, and decreased the cell apoptosis rate. In addition, Sirt3 was revealed as a target protein of SUMO1. SI-AC attenuated cerebral I/R injury through Sirt3 deSUMOylation via SENP1. Conclusions SENP1-mediated deSUMOylation of Sirt3 plays an essential role in SI-AC-induced cerebral protection against I/R injury. Our findings provide a promising therapeutic approach for treatment of acute cerebral I/R injury.

Large numbers of elderly people have aging-associated osteoporosis, but efficient approaches to ameliorate bone loss are limited due to our poor understanding of the underlying mechanisms. In this study, we found that melatonin levels in bone marrow decreased with age, and melatonin primarily enhanced the osteogenic potential of mesenchymal stem cells (MSCs) derived from elderly donors compared with fetal- or young adult-derived MSCs. Mechanistic studies indicated melatonin treatment alleviated the senescence-related hypermethylation of the MMSET promoter, leading to elevated expression of the histone methyltransferase NSD2, and promoted the histone H3 dimethylation modification at lysine 36 of the osteogenic genes RUNX2 and SP7/OSTERIX as a consequence. MMSET depletion partially abolished the effects of melatonin on osteogenesis in senescent MSCs in vitro. Moreover, melatonin treatment promoted bone formation and alleviated the progression of osteoporosis in a mouse model of aging. Clinically, severity of senile osteoporosis (SOP) in patients was associated with melatonin levels in bone marrow plasma and the MMSET expression in MSCs, and melatonin treatment enhanced osteoblastogenesis from MSCs derived from SOP patients. Our study discovered a previously unreported epigenetic regulatory role for melatonin in alleviating MSC senescence and suggests that melatonin may be a potent agent for preventing aging-associated osteoporosis.

SUMOylation of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) has been shown to play a critical role in the abnormal Ca2+ cycle of heart failure. Ginsenoside Rg3 (Rg3), the main active constituent of Panax ginseng, exerts a wide range of pharmacological effects in cardiovascular diseases. However, the effect of Rg3 on abnormal Ca2+ homeostasis in heart failure has not been reported. In this study, we showed a novel role of Rg3 in the abnormal Ca2+ cycle in cardiomyocytes of mice with heart failure. Among mice undergoing transverse aortic constriction, animals that received Rg3 showed improvements in cardiac function and Ca2+ homeostasis, accompanied by increases in the SUMOylation level and SERCA2a activity. In an isoproterenol (ISO)-induced cell hypertrophy model, Rg3 reduced the ISO-induced Ca2+ overload in HL-1 cells. Gene knockout of SUMO1 in mice inhibited the cardioprotective effect of Rg3, and SUMO1 knockout mice that received Rg3 did not exhibit improved Ca2+ homeostasis in cardiomyocytes. Additionally, mutation of the SUMOylation sites of SERCA2a blocked the positive effect of Rg3 on the ISO-induced abnormal Ca2+ cycle in HL-1 cells, and was accompanied by an abnormal endoplasmic reticulum stress response and generation of ROS. Our data demonstrated that Rg3 has a positive effect on the abnormal Ca2+ cycle in the cardiomyocytes of mice with heart failure. SUMO1 is an important factor that mediates the protective effect of Rg3. Our findings suggest that drug intervention by regulating the SUMOylation of SERCA2a can provide a novel therapeutic strategy for the treatment of heart failure.

While it is well known that adenosine receptor activation protects the heart from ischemia/reperfusion injury, the precise mitochondrial mechanism responsible for the action remains unknown. This study probed the mitochondrial events associated with the cardioprotective effect of 5'-(N-ethylcarboxamido) adenosine (NECA), an adenosine A2 receptor agonist. Isolated rat hearts were subjected to 30min ischemia followed by 10min of reperfusion, whereas H9c2 cells experienced 20min ischemia and 10min reperfusion. NECA prevented mitochondrial structural damage, decreases in respiratory control ratio (RCR), and collapse of mitochondrial membrane potential (ΔΨm). Both the adenosine A2A receptor antagonist SCH58261 and A2B receptor antagonist MRS1706 inhibited the action of NECA. NECA reduced mitochondrial proteins carbonylation, H2O2, and superoxide generation at reperfusion, but did not change superoxide dismutase (SOD) activity. In support, the protective effects of NECA and Peg-SOD on ΔΨm upon reperfusion were additive, implying that NECA's protection is attributable to the reduced superoxide generation but not to the enhancement of the superoxide-scavenging capacity. NECA increased the mitochondrial Src tyrosine kinase activity and suppressed complex I activity at reperfusion in a Src-dependent manner. NECA also reduced mitochondrial superoxide through Src tyrosine kinase. Studies with liquid chromatography-mass spectrometer (LC-MS) identified Tyr118 of the NDUFV2 subunit of complex 1 as a likely site of the tyrosine phosphorylation. Furthermore, the complex I activity of cells transfected with the Y118F mutant was increased, suggesting that this site might be a negative regulator of complex I activity. In support, NECA failed to suppress complex I activity at reperfusion in cells transfected with the Y118F mutant of NDUFV2. In conclusion, NECA prevents mitochondrial oxidative stress by decreasing mitochondrial superoxide generation through inhibition of complex I via the mitochondrial Src tyrosine kinase. Phosphorylation of Tyr118 residue in NDUFV2 subunit may account for the inhibitory effect of NECA on complex I.

Background. Zinc plays a role in mitophagy and protects cardiomyocytes from ischemia/reperfusion injury. This study is aimed at investigating whether SUMOylation of Drp1 is involved in the protection of zinc ion on cardiac I/R injury. Methods. Mouse hearts were subjected to 30 minutes of regional ischemia followed by 2 hours of reperfusion (ischemia/reoxygenation (I/R)). Infarct size and apoptosis were assessed. HL-1 cells were subjected to 24 hours of hypoxia and 6 hours of reoxygenation (hypoxia/reoxygenation (H/R)). Zinc was given 5 min before reperfusion for 30 min. SENP2 overexpression plasmid (Flag-SENP2), Drp1 mutation plasmid (Myc-Drp1 4KR), and SUMO1 siRNA were transfected into HL-1 cells for 48 h before hypoxia. Effects of zinc on SUMO family members were analyzed by Western blotting. SUMOylation of Drp1, apoptosis and the collapse of mitochondrial membrane potential (ΔΨm), and mitophagy were evaluated. Results. Compared with the control, SUMO1 modification level of proteins in the H/R decreased, while this effect was reversed by zinc. In the setting of H/R, zinc attenuated myocardial apoptosis, which was reversed by SUMO1 siRNA. Similar effects were observed in SUMO1 KO mice exposed to H/R. In addition, the dynamin-related protein 1 (Drp1) is a target protein of SUMO1. The SUMOylation of Drp1 induced by zinc regulated mitophagy and contributed to the protective effect of zinc on H/R injury. Conclusions. SUMOylation of Drp1 played an essential role in zinc-induced cardio protection against I/R injury. Our findings provide a promising therapeutic approach for acute myocardial I/R injury.

Osteosarcoma stem cells are able to escape treatment with conventional chemotherapeutic drugs, as the majority of them are in a quiescent state. Recent reports have suggested that small ubiquitin‑like modifiers (SUMOs) serve important roles in the maintenance of cancer stem cell stemness. Therefore, a potential strategy to increase the effectiveness of chemotherapeutic agents is to interfere with SUMO modification of proteins associated with the maintenance of stemness in osteosarcoma stem cells. The present study revealed a significant decrease in the expression of SUMO1 specific peptidase 1 (SENP1) in osteosarcoma tissues and osteosarcoma cell lines, and SENP1 expression was much lower in osteosarcoma stem cells than in non‑cancer stem cells. Further experiments indicated that the low levels of SENP1 were essential for maintenance of stemness in osteosarcoma stem cells. Overexpression of SENP1 resulted in a marked decrease in the maintenance of stemness, but only slightly induced apoptosis of osteosarcoma cells, which is crucial to reduce the side effects of drugs on normal precursor cells. Finally, SENP1 overexpression led to a significant increase in the sensitivity of osteosarcoma stem cells to the herpes simplex virus 1 thymidine kinase gene in combination with ganciclovir in vitro and in vivo. In conclusion, the present study described a novel method to increase the sensitivity of osteosarcoma stem cells to chemotherapeutic drugs. Notably, this approach may significantly reduce the required dose of conventional chemotherapeutic drugs and reduce side effects.

The ability of herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) systems to kill tumor cells is partially dependent on the integrity of gap junction intercellular communication (GJIC) of targeted tumor cells. Recent studies have suggested that connexin 43 (Cx43), which serves a role in gap junction-mediated intercellular communication, is regulated by small ubiquitin-like modifiers (SUMOs). However, the roles of these post-translational modifications remain to be elucidated. The present study demonstrated overexpression of SUMO‑conjugating enzyme Ubc9 (Ubc9) protein in osteosarcoma. Silencing Ubc9 by siRNA inhibited osteosarcoma cell proliferation and migration, and significantly increased the sensitivity of cells to HSV-TK/GCV systems both in vitro and in vivo. Further experimentation demonstrated that silencing Ubc9 induced decoupling of SUMO1 from Cx43, generating increased free Cx43 levels, which is important for reconstructing GJIC and recovering cellular functions. In conclusion, the present study revealed a novel method for the effective restoration of GJIC in osteosarcoma cells, which may increase their sensitivity to conventional chemotherapy.

Zinc plays a role in autophagy and protects cardiac cells from ischemia/reperfusion injury. This study aimed to test if zinc can induce mitophagy leading to attenuation of mitochondrial superoxide generation in the setting of hypoxia/reoxygenation (H/R) in cardiac cells. H9c2 cells were subjected to 4 h hypoxia followed by 2 h reoxygenation. Under normoxic conditions, treatments of cells with ZnCl2 increased both the LC3-II/LC3-I ratio and GFP-LC3 puncta, implying that zinc induces autophagy. Further experiments showed that endogenous zinc is required for the autophagy induced by starvation and rapamycin. Zinc down-regulated TOM20, TIM23, and COX4 both in normoxic cells and the cells subjected to H/R, indicating that zinc can trigger mitophagy. Zinc increased ERK activity and Beclin1 expression, and zinc-induced mitophagy was inhibited by PD98059 and Beclin1 siRNA during reoxygenation. Zinc-induced Beclin1 expression was reversed by PD98059, implying that zinc promotes Beclin1 expression via ERK. In addition, zinc failed to induce mitophagy in cells transfected with PINK1 siRNA and stabilized PINK1 in mitochondria. Moreover, zinc-induced PINK1 stabilization was inhibited by PD98059. Finally, zinc prevented mitochondrial superoxide generation and dissipation of mitochondrial membrane potential (ΔΨm) at reoxygenation, which was blocked by both the Beclin1 and PINK1 siRNAs, suggesting that zinc prevents mitochondrial oxidative stress through mitophagy. In summary, zinc induces mitophagy through PINK1 and Beclin1 via ERK leading to the prevention of mitochondrial superoxide generation in the setting of H/R. Clearance of damaged mitochondria may account for the cardioprotective effect of zinc on H/R injury.