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AIM: To explore the effect of polyphenolic compound 3, 5-dihydroxy-4-methoxybenzyl alcohol （DHMBA） on hypoxia/reoxygenation（ H/R） injury of human umbilical vein endothelial cells（ EA. hy926 cells） and its potential mechanisms. METHODS: To construct an H/R model, the EA. hy926 cells were cultured in an acidic hypoxia buffer while in an anaerobic workstation. The cells were divided into control, H/R, H/R+different doses of DHMBA, H/R+ edaravone （antioxidant） and H/R+reactive oxygen species （ROS） inducer oligomycin A+DHMBA groups. Cell viability was measured by CCK-8 assay, and tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） levels in cells were measured by ELISA. Phosphorylation of endothelial nitric oxide synthase （eNOS） and nuclear factor-κB （NF-κB） p65 were measured by Western blot. Intracellular NO levels were determined by laser confocal microscopy. Glutathione（ GSH）/glutathione disulfide （GSSG） oxidation balance was determined by the dinitrobenzoic acid chromogenic method. Intracellular ROS levels were measured by flow cytometry. Lactate dehydrogenase （LDH） leakage was determined using nitro blue tetrazolium staining. Scratch assays were performed to assess cell migration. RESULTS: DHMBA exhibited no significant cytotoxicity between 125 and 1 000 μmol/L. In H/R-injured human umbilical vein endothelial cells, DHMBA improved cell survival, inhibited phosphorylation of NF-κB p65, reduced the content of TNF-α and IL-6, and increased phosphorylation of eNOS and NO levels. DHMBA also suppressed ROS overload and restored the ratio between GSH and oxidized GSH, decreased in LDH leakage and increased cell migration in H/R-injured human umbilical vein endothelial cells. CONCLUSION: DHMBA can alleviate H/R-induced oxidative stress, inflammation, cellular damage, and dysfunction, which are associated with the ability of DHMBA to inhibit ROS production in human umbilical vein endothelial cells. [ABSTRACT FROM AUTHOR]

Objective: Panax quinquefolius saponins (PQS) and Panax notoginseng saponins (PNS) are key bioactive compounds in Panax quinquefolius L. and Panax notoginseng, commonly used in the treatment of clinical ischemic heart disease. However, their potential in mitigating myocardial ischemia-reperfusion injury remains uncertain. This study aims to evaluate the protective effects of combined PQS and PNS administration in myocardial hypoxia/reoxygenation (H/R) injury and explore the underlying mechanisms. Methods: To investigate the involvement of HIF-1α/BNIP3 mitophagy pathway in the myocardial protection conferred by PNS and PQS, we employed small interfering BNIP3 (siBNIP3) to silence key proteins of the pathway. H9C2 cells were categorized into four groups: control, H/R, H/R + PQS + PNS, and H/R + PQS + PNS+siBNIP3. Cell viability was assessed by Cell Counting Kit-8, apoptosis rates determined via flow cytometry, mitochondrial membrane potential assessed with the JC-1 fluorescent probes, intracellular reactive oxygen species detected with 2′,7′-dichlorodihydrofluorescein diacetate, mitochondrial superoxide production quantified with MitoSOX Red, and autophagic flux monitored with mRFP-GFP-LC3 adenoviral vectors. Autophagosomes and their ultrastructure were visualized through transmission electron microscopy. Moreover, mRNA and protein levels were analyzed via real-time PCR and Western blotting. Results: PQS + PNS administration significantly increased cell viability, reduced apoptosis, lowered reactive oxygen species levels and mitochondrial superoxide production, mitigated mitochondrial dysfunction, and induced autophagic flux. Notably, siBNIP3 intervention did not counteract the cardioprotective effect of PQS + PNS. The PQS + PNS group showed downregulated mRNA expression of HIF-1α and BNIP3, along with reduced HIF-1α protein expression compared to the H/R group. Conclusions: PQS + PNS protects against myocardial H/R injury, potentially by downregulating mitophagy through the HIF-1α/BNIP3 pathway. [ABSTRACT FROM AUTHOR]

Abstract Acute kidney injury (AKI) is a serious disorder associated with significant morbidity and mortality. AKI and ischemia/reperfusion (hypoxia/reoxygenation, H/R) injury can be induced due to several reasons. Paeoniflorin (PF) is a traditional herbal medicine derived from Paeonia lactiflora Pall. It exerts diverse therapeutic effects, including anti-inflammatory, antioxidative, antiapoptotic, and immunomodulatory properties; thus, it is considered valuable for treating several diseases. However, the effects of PF on H/R injury-induced AKI remain unknown. In this study, we established an in vitro H/R model using COCL2 and investigated the functions and underlying mechanisms of PF on H/R injury in HK-2 cells. The cell vitality was evaluated using the cell count kit-8 assay. The DCFH-DA fluorescence probe was used to measure the levels of reactive oxygen species (ROS). Oxidative damage was detected using superoxide dismutase (SOD) and malondialdehyde (MDA) assay kits. Apoptotic relative protein and Keap1/Nrf2/HO-1 signaling were evaluated by Western blotting. Our results indicated that PF increased cell viability and SOD activity and decreased the ROS and MDA levels in HK-2 cells with H/R injury. PF inhibits apoptosis by increasing Bcl-2 and decreasing Bax. Furthermore, PF significantly upregulated the expression of HO-1 and Nrf2, but downregulated the expression of HIF-1α and Keap1. PF considerably increased Nrf2 nuclear translocation and unregulated the HO-1 expression. The Nrf2 inhibitor (ML385) could reverse the abovementioned protective effects of PF, suggesting that Nrf2 can be a critical target of PF. To conclude, we found that PF attenuates H/R injury-induced AKI by decreasing the oxidative damage via the Nrf2/HO-1 pathway and inhibiting apoptosis.

Acute kidney injury (AKI) is a serious disorder associated with significant morbidity and mortality. AKI and ischemia/reperfusion (hypoxia/reoxygenation, H/R) injury can be induced due to several reasons. Paeoniflorin (PF) is a traditional herbal medicine derived from Paeonia lactiflora Pall. It exerts diverse therapeutic effects, including anti-inflammatory, antioxidative, antiapoptotic, and immunomodulatory properties; thus, it is considered valuable for treating several diseases. However, the effects of PF on H/R injury-induced AKI remain unknown. In this study, we established an in vitro H/R model using COCL2 and investigated the functions and underlying mechanisms of PF on H/R injury in HK-2 cells. The cell vitality was evaluated using the cell count kit-8 assay. The DCFH-DA fluorescence probe was used to measure the levels of reactive oxygen species (ROS). Oxidative damage was detected using superoxide dismutase (SOD) and malondialdehyde (MDA) assay kits. Apoptotic relative protein and Keap1/Nrf2/HO-1 signaling were evaluated by Western blotting. Our results indicated that PF increased cell viability and SOD activity and decreased the ROS and MDA levels in HK-2 cells with H/R injury. PF inhibits apoptosis by increasing Bcl-2 and decreasing Bax. Furthermore, PF significantly upregulated the expression of HO-1 and Nrf2, but downregulated the expression of HIF-1α and Keap1. PF considerably increased Nrf2 nuclear translocation and unregulated the HO-1 expression. The Nrf2 inhibitor (ML385) could reverse the abovementioned protective effects of PF, suggesting that Nrf2 can be a critical target of PF. To conclude, we found that PF attenuates H/R injury-induced AKI by decreasing the oxidative damage via the Nrf2/HO-1 pathway and inhibiting apoptosis. [ABSTRACT FROM AUTHOR]

Background: Ischemic stroke, caused neurological dysfunction due to inadequate blood supply to brain, has a high morbidity and mortality. Ethyl pyruvate (EP), a simple aliphatic ester derived from pyruvic acid, has the advantages of safety and stability. Studies have confirmed that EP has anti-oxidative, anti-inflammation, anti-tumor, and other pharmacological effects, and it demonstrates significant therapeutic effects on multiple diseases. GAS6 and its high affinity Axl receptor play an important role in cell adhesion, anti-apoptosis, proliferation and migration by activating downstream signal transduction pathways. Previous studies have demonstrated the neuroprotective effects of the GAS6/Axl axis. Methods: A series of experimental methods were employed to confirm the effect of EP against cerebral hypoxia/reoxygenation (HR) injury. Results: In this study, the protective effect and mechanism of EP on HR injury in N2a cells was explored. The results found that treatment with EP could increase HR-injured neuronal viability, improve cell morphology, and reduce LDH release and ROS accumulation, thereby exhibiting a neuroprotective effect. Furthermore, EP treatment restored the down-regulated expression of GAS6, Axl, NQO1, PGC-1α, NRF1, and UCP2 caused by HR injury. Specifically, it was observed that the neuroprotective effect of EP was partially inhibited by GAS6 siRNA. Conclusion: In conclusion, these results suggest that EP treatment attenuates HR-induced oxidative stress injury in neuroblastoma cells via activating GAS6/Axl signaling.

There are limited therapeutic options for patient with traumatic spinal cord injury (SCI). Phosphoinositide 3-kinase family (PI3Ks) are the key molecules for regulating cell autophagy, which is a possible way of treating SCI. As we know, PI3K family are composed of eight isoforms, which are distributed into three classes. While the role of PI3Ks in regulating autophagy is controversial and the effects may be in a cell-specific manner. Different isoforms do not distribute in neural cells consistently and it is not clear how the PI3K isoforms regulate and interact with autophagy. Therefore, we explored the distributions and expression of different PI3K isoforms in two key neural cells (PC12 cells and astrocytes). The results showed that the expression of LC3II/I and p62, which are the markers of autophagy, changed in different patterns in PC12 cells and astrocytes after hypoxia/reoxygenation injury (H/R). Furthermore, the mRNA level of eight PI3K isoforms did not change in the same way, and even for the same isoform the mRNA activities are different between PC12 cells and astrocytes. What is more, the results of western blot of PI3K isoforms after H/R were inconsistent with the relevant mRNA. Based on this study, the therapeutic effects of regulating autophagy on SCI are not confirmed definitely, and its molecular mechanisms may be related with different temporal and spatial patterns of activation and distributions of PI3K isoforms. [ABSTRACT FROM AUTHOR]

Purpose: Endoplasmic reticulum stress (ERS) plays a crucial role in myocardial ischemia-reperfusion injury (MIRI). Cellular FLICE-inhibitory protein (cFLIP) is an essential regulator of apoptosis and plays a major role in regulating ERS. The present study aimed to investigate the effects of long isoform cFLIP (cFLIPL) on endogenous apoptosis and the mechanism of ERS in MIRI. Methods: The cFLIPL recombinant adenovirus vector was used to infect H9c2 cells and Sprague–Dawley (SD) rats. After infection for 72 h, ischemia was induced for 30 min, and reperfusion was then performed for 2 h to establish the MIRI model in SD rats. H9c2 cells were hypoxic for 4 h and then reoxygenated for 12 h to simulate ischemia/reperfusion (I/R) injury. Model parameters were evaluated by assessing cardiomyocyte viability, cell death (apoptosis), and ERS-related protein expression. In addition, tunicamycin (TM), an ERS agonist, was also added to the medium for pretreatment. Coimmunoprecipitation (Co-IP) of cFLIPL and p38 MAPK protein was performed. Results: cFLIPL expression was decreased in I/R injury and hypoxia/reoxygenation (H/R) injury, and cFLIPL overexpression reduced myocardial infarction in vivo and increased the viability of H9c2 cells in vitro. I/R and H/R upregulated the protein expression of GRP78, IRE-1, and PERK to induce ERS and apoptosis. Interestingly, overexpression of cFLIPL significantly inhibited ERS and subsequent apoptosis, which was reversed by an agonist of ERS. Moreover, Co-IP showed that cFLIPL attenuated ERS and was associated with inhibiting the activation of p38 protein. Conclusion: The expression of cFLIPL is significantly downregulated in MIRI, and it is accompanied by excessive ERS and apoptosis. Upregulated cFLIPL suppresses ERS to reduce myocardial apoptosis, which is associated with inhibiting the activity of p38 MAPK. Therefore, cFLIPL may be a potential intervention target for MIRI. [ABSTRACT FROM AUTHOR]

Autophagy plays various roles at different stages of ischemia reperfusion (I/R) injury in cardiomyocytes. It has been reported that tissue factor pathway inhibitor (TFPI) has a protective effect on I/R injury. This study aimed to determine the roles of TFPI in autophagy during the I/R injury process in cardiomyocytes and the possible mechanisms. An isolated hypoxia/reoxygenation (H/R) pattern of cardiomyocytes was established by the MIC101 system. The cell viability and oxidative stress of cardiomyocytes were detected by an MTT assay and ROS assay, respectively. The autophagy level was measured by Ad-mCherry-GFP-LC3B and MDC. We detected the expression levels of autophagy-related proteins by western blotting. After 2 h of hypoxia and 12 h of reoxygenation, the cardiomyocyte viability in the H/R group was significantly lower than that in the control group (p < 0.05) than in the H/R group. According to intracellular ROS production, the fluorescence intensity in the H/R group was enhanced compared with that in the negative control group, and it was weaker in the H/R + rTFPI group compared with the H/R group. The level of autophagy and the expression levels of autophagy-related proteins (LC3-II/LC3-I, Beclin-1 and PI3K) were markedly increased in the H/R group compared to the control group (p < 0.05) whereas the levels were markedly decreased in the H/R + rTFPI group compared to the H/R group (p < 0.05). TFPI could relieve cardiomyocyte injury by inhibiting the Class III PI3K/Beclin-1 pathway and oxidative stress; thus, TFPI decreased autophagy and protected cardiomyocytes induced by H/R injury. In conclusion, TFPI may be a new direction for the prevention of myocardial I/R injury. [ABSTRACT FROM AUTHOR]

Background: H2S (hydrogen sulfide) protects cerebral vasodilatation and endothelial cells against oxygen-glucose deprivation/reoxygenation injury via the inhibition of the RhoA-ROCK pathway and ROCK2 expression. However, the inhibitory mechanism of H2S on ROCK2 expression is still unclear. The study aimed to investigate the target and mechanism of H2S in inhibition of ROCK2. Methods: His-ROCK2wild protein was constructed, expressed, and was used for phosphorylation assay in vitro. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) was used to determine the potential phosphorylation sites of ROCK2. Recombinant ROCK2wild-pEGFP-N1, ROCK2T436A-pEGFP-N1, and ROCK2S575F-pEGFP-N1 plasmids were constructed and transfected into rat hippocampal neurons (RHNs). ROCK2 expression, cell viability, the release of lactate dehydrogenase (LDH), nerve-specific enolase (NSE), and Ca2+ were detected to evaluate the neuroprotective mechanism of H2S. Results: Phosphorylation at Thr436 and Ser575 of ROCK2 was observed by mass spectrometry when Polo-like kinase 1 (PLK1) and protein kinase A (PKA) were added in vitro, and NaHS significantly inhibited phosphorylation at Thr436 and Ser575. Additionally, NaHS significantly inhibited the expression of ROCK2 and recombinant proteins GFP-ROCK2, GFP-ROCK2T436A, and GFP-ROCK2S575F in transfected RHNs. Compared with empty plasmid, GFP-ROCK2T436A, and GFP-ROCK2S575F groups, NaHS significantly inhibited the release of LDH, NSE, and Ca2+ and promoted ROCK2 activity in the GFP-ROCK2wild group. Thr436 and Ser575 may be dominant sites that mediate NaHS inhibition of ROCK2 protein activity in RHNs. Compared with the empty plasmid, GFP-ROCK2T436A, and the GFP-ROCK2S575F group, NaHS had more significant inhibitory effects on hypoxia/reoxygenation (H/R) injury-induced cell viability reduction and increased LDH and NSE release in the GFP-ROCK2wild group. Conclusion: Exogenous H2S protected the RHNs against H/R injury via Thr436 and Ser575 of ROCK2. These findings suggested that Thr436 and Ser575 may be the dominant sites that mediated the effect of NaHS on protecting RHNs against H/R injury. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the mechanism through which N-acetylneuraminic acid (Neu5Ac) exacerbates hypoxia/reoxygenation (H/R) injury in rat cardiomyocytes (H9C2 cells)., Methods: H9C2 cells were cultured in hypoxia and glucose deprivation for 8 h followed by reoxygenation for different durations to determine the optimal reoxygenation time. Under the optimal H/R protocol, the cells were treated with 0, 5, 10, 20, 30, 40, 50, and 60 mmol/L Neu5Ac during reoxygenation to explore the optimal drug concentration. The cells were then subjected to H/R injury followed by treatment with Neu5Ac, Fer-1 (a ferroptosis inhibitor), or both. The changes in SOD activity, intracellular Fe 2+ and lipid ROS levels in the cells were evaluated, and the cellular expressions of Nrf2, GPX4, HO-1, FSP1, and xCT proteins were detected using Western blotting., Results: Following hypoxia and glucose deprivation for 8 h, the cells with reoxygenation for 6 h, as compared with other time lengths of reoxygenation except for 9 h, showed the lowest expression levels of Nrf2, GPX4, HO-1, and FSP1 proteins ( P <0.001). Neu5Ac treatment of dose-dependently decreased the viability of the cells with H/R injury with an IC 50 of 30.07 mmol/L. Reoxygenation for 3 h with normal glucose supplementation and a Neu5Ac concentration of 30 mmol/L were selected as the optimal conditions in the subsequent experiments. The results showed that Neu5Ac could significantly increase SOD activity, Fe 2+ and lipid ROS levels and reduce Nrf2, GPX4, HO-1, and FSP1 protein expressions in H9C2 cells with H/R injury, but its effects were significantly attenuated by treatment with Fer-1., Conclusions: Neu5Ac exacerbates ferroptosis of myocardial cells with H/R injury by inhibiting the Nrf2 axis to promote the production of ROS and lipid ROS.

Background: The volatile anaesthetic sevoflurane protects cardiac tissue from reoxygenation/reperfusion. Mitochondria play an essential role in conditioning. We aimed to investigate how sevoflurane and its primary metabolite hexafluoroisopropanol (HFIP) affect necrosis, apoptosis, and reactive oxygen species formation in cardiomyocytes upon hypoxia/reoxygenation injury. Moreover, we aimed to describe the similarities in the mode of action in a mitochondrial bioenergetics analysis. Methods: Murine cardiomyocytes were exposed to hypoxia (0.2% O2 for 6 h), followed by reoxygenation (air with 5% CO2 for 2 h) in the presence or absence sevoflurane 2.2% or HFIP 4 mM. Lactate dehydrogenase (LDH) release (necrosis), caspase activation (apoptosis), reactive oxygen species, mitochondrial membrane potential, and mitochondrial function (Seahorse XF analyser) were measured. Results: Hypoxia/reoxygenation increased cell death by 44% (+31 to +55%, P

Significant pancreatic islet dysfunction and loss shortly after transplantation to the liver limit the widespread implementation of this procedure in the clinic. Nonimmune factors such as reactive oxygen species and inflammation have been considered as the primary driving force for graft failure. The adipokine adiponectin plays potent roles against inflammation and oxidative stress. Previous studies have demonstrated that systemic administration of adiponectin significantly prevented islet loss and enhanced islet function at post‐transplantation period. In vitro studies indicate that adiponectin protects islets from hypoxia/reoxygenation injury, oxidative stress as well as TNF‐α‐induced injury. By applying adenovirus mediated transfection, we now engineered islet cells to express exogenous adiponectin gene prior to islet transplantation. Adenovirus‐mediated adiponectin transfer to a syngeneic suboptimal islet graft transplanted under kidney capsule markedly prevented inflammation, preserved islet graft mass and improved islet transplant outcomes. These results suggest that adenovirus‐mediated adiponectin gene therapy would be a beneficial clinical engineering approach for islet preservation in islet transplantation. [ABSTRACT FROM AUTHOR]

Psoralidin (PSO) is a natural phenolic coumarin extracted from the seeds of Psoralea corylifolia L. Growing preclinical evidence indicates that PSO has anti‐inflammatory, anti‐vitiligo, anti‐bacterial, and anti‐viral effects. Growth arrest‐specific gene 6 (GAS6) and its receptor, Axl, modulate cellular oxidative stress, apoptosis, survival, proliferation, migration, and mitogenesis. Notably, the neuroprotective role of the GAS6/Axl axis has been identified in previous studies. We hypothesize that PSO ameliorates cerebral hypoxia/reoxygenation (HR) injury via activating the GAS6/Axl signaling. We first confirmed that PSO was not toxic to the cells and upregulated GAS6 and Axl expression after HR injury. Moreover, PSO exerted a marked neuroprotective effect against HR injury, represented by restored cell viability and cell morphology, decreased lactate dehydrogenase (LDH) release, and reactive oxygen species (ROS) generation. Furthermore, PSO pretreatment also elevated the levels of nuclear factor‐related factor 2 (Nrf‐2), NAD(P)H dehydrogenase quinone‐1 (NQO1), heme oxygenase‐1 (HO‐1), silent information regulator 1 (SIRT1), peroxisome proliferator‐activated receptor coactivator 1α (PGC‐1α), nuclear respiratory factor 1 (NRF1), uncoupling protein 2 (UCP2), and B‐cell lymphoma 2 (BCl2) both in the condition of baseline and HR injury. However, GAS6 siRNA or Axl siRNA inhibited the neuroprotective effects of PSO. Our findings suggest that PSO pretreatment attenuated HR‐induced oxidative stress, apoptosis, and mitochondrial dysfunction in neuroblastoma cells through the activation of GAS6/Axl signaling. [ABSTRACT FROM AUTHOR]

Zhipeng Zhu,1,* Xiaoyan Ling,2,* Hongmei Zhou,1 Caijun Zhang,1 Weiwei Yan1 1Department of Anesthesiology, The Second Affiliated Hospital of Jiaxing University, Jiaxing City, Zhejiang Province 314000, People’s Republic of China; 2The Outpatient Nursing Department of the Second Affiliated Hospital of Jiaxing University, Jiaxing City, Zhejiang Province 314000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhipeng ZhuDepartment of Anesthesiology and Pain, The Second Affiliated Hospital of Jiaxing University, Jiaxing City, Zhejiang Province, People’s Republic of ChinaEmail xiaozhu781126@163.comBackground: Myocardial ischaemia-reperfusion injury (IRI) has been confirmed to induce endoplasmic reticulum stress (ERS) when myocardial cell function continues to deteriorate to a certain degree. The clinical applications of effective tested strategies are sometimes inconsistent with the applications evaluated in experiments, although reasonable mechanisms and diverse signalling pathways have been broadly explored. Dexmedetomidine (DEX) has been shown to attenuate IRI of the heart in animal studies. This study aimed to determine whether DEX can protect injured cardiomyocytes under hypoxia/reoxygenation (H/R) at the cellular level and whether the mechanism is related to ERS and the p38 MAPK pathway.Methods: H9c2 cells were subjected to H/R or thapsigargin (TG) to build a model. DEX or 4-PBA was added to the medium either 1 h or 24 h before modelling, respectively. Model parameters were determined by assessing cell viability and injury, which were measured by assessing cell counting kit-8 (CCK8), lactate dehydrogenase (LDH) release and flow cytometry results, and the expression of GRP78, CHOP and caspase-12. In addition, the protein expression of p38MAPK and p-p38MAPK was examined, and SB202190, a negative regulator, was also preincubated in medium.Results: Compared to that of cells in the control group, the activity of cells in the H/R and TG groups was decreased dramatically, and the LDH concentration and proportion of apoptotic cells were increased. DEX could correspondingly reverse the changes induced by H/R or TG. Additionally, DEX effectively attenuated ERS defined as increased expression of GRP78, CHOP and caspase-12. Additionally, DEX could obviously depress the P38 MAPK phosphorylation and high p-p38 MAPK expression in the TG group, indicating DEX has a function similar to that of SB202190.Conclusion: H/R injury in H9c2 cells can lead to abnormal ERS and apoptosis, as well as activation of the p38MAPK signalling pathway. DEX can protect cardiomyocytes by intervening in ERS, regulating p38MAPK and the downstream apoptotic signalling pathway.Keywords: dexmedetomidine, hypoxia/reoxygenation injury, endoplasmic reticulum stress signalling pathway, p38 mitogen-activated protein kinase

This study investigated the mechanism by which ginsenoside Rg&#95;(1 )attenuates hypoxia/reoxygenation(H/R) injury in HL-1 cardiomyocytes by inhibiting the acetylation of ATP synthase subunit alpha(ATP5A1) through silent information regulator 3(SIRT3). In this study, an H/R injury model was constructed by hypoxia for 6 h and reoxygenation for 2 h in HL-1 cardiomyocytes. First, the optimal effective concentration of ginsenoside Rg&#95;1 was determined using a cell viability assay kit. Then, lactate dehydrogenase(LDH) leakage was measured using a microplate method to evaluate the protective effect of ginsenoside Rg&#95;1 against H/R injury in HL-1 cardiomyocytes. Western blot was further used to detect SIRT3 expression and the acetylation level of mitochondrial proteins in cardiomyocytes. ATP content was measured using a luciferase assay. Immunoprecipitation was used to detect the acetylation level of ATP5A1. The oxygen consumption rate(OCR) was measured using the Seahorse XFp analyzer. Flow cytometry was used to assess cell apoptosis to explore the specific mechanism. The results showed that compared with the control group, the model group had a significant increase in LDH leakage, a decrease in SIRT3 expression, a significant increase in the acetylation levels of mitochondrial proteins and ATP5A1, a decrease in OCR and ATP content, and an increase in cell apoptosis rate. Compared with the model group, the ginsenoside Rg&#95;1 group showed a significant decrease in LDH leakage, an increase in SIRT3 expression, a significant decrease in the acetylation levels of mitochondrial proteins and ATP5A1, an increase in OCR and ATP content, and a decrease in cell apoptosis rate. Compared with the ginsenoside Rg&#95;1 group, the ginsenoside Rg&#95;1 + si SIRT3 group showed a significant increase in LDH leakage, a decrease in SIRT3 expression, a significant increase in the acetylation levels of mitochondrial proteins and ATP5A1, a decrease in OCR and ATP content, and an increase in cell apoptosis rate. In conclusion, ginsenoside Rg&#95;1 alleviates H/R injury in HL-1 cells by improving mitochondrial respiratory function through SIRT3-mediated inhibition of ATP5A1 acetylation.

Objective To investigate the effect of microRNA-21（miR-21）on renal tubular epithelial cells（NRK- 52E）injured by hypoxia/reoxygenation（H/R）in rats. Methods NRK-52E cells were cultured in vitro and randomly divided into 4 groups：pre-miR-21+H/R group，antagomiR-21+H/R group，model group，and normal group. The H/R injury models were established in the model group，and the cells in the normal group were routinely cultured. The cells in the pre-miR-21+H/R group and the antagomiR-21+H/R group were pre-treated with pre-miR-21 and antagomiR-21 at a final concentration of 30 nmol/L 24 h before hypoxia，respectively. Cell viability was detected by CCK-8，the apoptosis rate was measured by flow cytometry，miR-21 and PDCD4 mRNA expression levels were detected by RT-PCR，and Caspase-3 protein was detected by Western blotting. The activity of antioxidant enzymes and total antioxidant capacity of cells were detected by chemical colorimetry and endpoint colorimetry，and inflammatory cytokines in supernatant of cell culture were detected by ELISA. Results Compared with the normal group，the model group had lower cell viability，higher apoptosis rate，higher relative expression level of miR-21，lower relative expression level of PDCD4 mRNA，higher relative expression level of Caspase-3 protein P17，lower antioxidant enzyme activity and total antioxidant capacity，and higher level of inflammatory cytokines（all P<0. 05）. Compared with the model group，the pre-miR-21 +H/R group had higher cell viability， lower apoptosis rate，lower relative expression level of miR-21，higher relative expression level of PDCD4 mRNA， lower relative expression level of Caspase-3 protein P17，higher antioxidant enzyme activity and total antioxidant capacity， and lower level of inflammatory cytokines（all P<0. 05）. There was no significant difference between antagomiR-21+H/R group and model group in the indicators except the relative expression of miR-21（all P>0. 05）. Conclusion MiR-21 can inhibit the apoptosis of NRK-52E cells damaged by H/R，increase the antioxidant capacity of NRK-52E cells，and alleviate inflammation. [ABSTRACT FROM AUTHOR]

Prokineticin 2 (PK2) was reported to be decreased in the hearts of end-state heart failure patients. Our study aimed to explore the effects of PK2 on hypoxia/reoxygenation (H/R) injury and the underlying mechanism. H9c2 cardiomyocytes were treated with 5 nM PK2 in the presence or absence of 5 mM dual phosphatidylinositol 3-kinase (PI3K)/the mammalian target of rapamycin (mTOR) inhibitor (BEZ235) for 24 h and then subjected to H/R treatment. Cell viability and lactate dehydrogenase (LDH) release were evaluated by CCK-8 and LDH release assays, respectively. Apoptosis was determined by flow cytometry analysis. Oxidative stress was assessed by measuring superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content. Results showed that H/R treatment decreased PK2 expression and inactivated the Akt/mTOR pathway in H9c2 cardiomyocytes. PK2 treatment activated the Akt/mTOR pathway in H/R-exposed H9c2 cardiomyocytes. H/R stimulation suppressed cell viability, increased LDH release, induced apoptosis and oxidative stress in H9c2 cardiomyocytes, while these effects were neutralised by treatment with PK2. However, the inhibitory effects of PK2 on H/R-induced injury in H9c2 cardiomyocytes were abolished by the addition of BEZ235. In conclusion, PK2 relieved H/R-induced injury in H9c2 cardiomyocytes by activation of the Akt/mTOR pathway.

Background and Aims: Myocardial ischemia/reperfusion injury (MI/RI) is a result of coronary revascularization, and often increases cell apoptosis and autophagy. Downregulated cellular FADD-like-IL-1β-converting enzyme-inhibitory protein (cFLIP) was associated with development of several myocardial diseases, whether overexpression of cFLIP can attenuate MI/RI remains unclear. This study aimed to determine the effects of cFLIP on apoptosis and autophagy in MI/RI.Methods and Results: Ischemia/reperfusion (I/R) rat model and hypoxia/reoxygenation (H/R) cardiomyocytes model were established. Both I/R injury and H/R injury down-regulated expression of two cFLIP isoforms (cFLIPL and cFLIPS), and instigated apoptosis and autophagy simultaneously. Overexpression of cFLIPL and/or cFLIPS led to a significant increase in cardiomyocytes viability in vitro, and also reduced the myocardial infarct volume in vivo, these changes were associated with suppressed apoptosis and autophagy. Mechanistically, overexpression of cFLIP significantly downregulated pro-apoptotic molecules (Caspase-3, -8, -9), and pro-autophagic molecules (Beclin-1 and LC3-II). Moreover, cFLIP significantly suppressed activity of NF-κB pathway to upregulate the expression of Bcl-2, which is the molecular of interplay of apoptosis and autophagy.Conclusion: Overexpression of cFLIP significantly attenuated MI/RI both in vivo and vitro via suppression of apoptosis and lethal autophagy. cFLIP can suppress activity of NF-κB pathway, and further upregulated expression of Bcl-2. [ABSTRACT FROM AUTHOR]

Objective To study the protective effect of Shengxian decoction and the single herb decoction against myocardial injury induced by hypoxia/reoxygenation. Methods The H9c2 cells were cultured to establish hypoxia/reoxygenation model. Rats were divided into 8 groups: normal control group, hypoxia/reoxygenation group (model group) and treated groups (Shengxian decoction and the single herb decoction). The apoptotic rate of cardiomyocytes, the activity of reactive oxygen species (ROS) and intracellular calcium concentration (Ca2+) were measured. Results Compared with hypoxia/reoxygenation group, the apoptosis rate, ROS activity and intracellular Ca2+ concentration were significantly lower in all treated groups (P<0.05). The ROS activity and intracellular Ca2+ concentration was decreased by 41.37% and 15.20% in Shengxian decoction group compared to the model group. Conclusion Shengxian decoction and the single herb decoction had protective effect on myocardial injury induced by hypoxia/reoxygenation. [ABSTRACT FROM AUTHOR]

Human umbilical cord mesenchymal stromal cell-derived extracellular vesicles (HuMSC-EVs) can repair damaged tissues. The expression profile of circular RNAs (circRNAs) provides valuable insights into the regulation of the repair process and the exploration of the repair mechanism. AC16 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) injury and subsequently cultured with or without HuMSC-EVs (Group T and Group C, respectively). High-throughput RNA sequencing was implemented for the two groups. On the basis of the transcriptome data, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and network analyses were carried out to determine the differential gene expression profiles between the two groups. After screening the circRNA database, the results were proved by quantitative real-time polymerase chain reaction. The survival rate of cardiomyocytes exposed to H/R was increased by treatment with HuMSC-EVs. RNA-seq analysis showed that 66 circRNAs were differentially expressed in cardiomyocytes in the co-cultured group. The cellular responses to hypoxia and to decreased oxygen levels were at the top of the GO upregulated list for the two groups, while the vascular endothelial growth factor signaling pathway, long-term potentiation, and the glucagon signaling pathway were at the top of the KEGG pathway upregulated list for the two groups. In the same samples, the 10 most aberrantly upregulated circRNAs were chosen for further verification of their RNA sequences. Seven of the 10 most aberrant circRNAs were significantly upregulated in the co-cultured group and in the HuMSC-EVs. Our results revealed that upregulated circRNAs were abundant during the repair of damaged cardiomyocytes by HuMSC-EVs, which provides a new perspective for the repair of H/R by HuMSC-EVs.

Subject: Cardiovascular disease, as a very common and serious coexisting disease in diabetic patients, and is one of the risk factors that seriously affect the prognosis and complications of surgical patients. Previous studies have shown that sevoflurane post-conditioning (SPostC) exerts a protective effect against myocardial ischemia/reperfusion injury by HIF-1α, but the protective effect is weakened or even disappeared under hyperglycemia. This study aims to explore whether regulating the HIF-1α/MIF/AMPK signaling pathway can restore the protective effect and reveal the mechanism of SPostC on cardiomyocyte hypoxia/reoxygenation injury under high glucose conditions.Methods: H9c2 cardiomyocytes were cultured in normal and high-concentration glucose medium to establish a hypoxia/reoxygenation (H/R) injury model of cardiomyocytes. SPostC was performed with 2.4% sevoflurane for 15 min before reoxygenation. Cell damage was determined by measuring cell viability, lactate dehydrogenase activity, and apoptosis; Testing cell energy metabolism by detecting reactive oxygen species (ROS) generation, ATP content and mitochondrial membrane potential; Analysis of the change of HIF-1α, MIF and AMPKα mRNA expression by RT-PCR. Western blotting was used to examine the expression of HIF-1α, MIF, AMPKα and p-AMPKα proteins. HIF-1α and MIF inhibitors and agonists were administered 40 min before hypoxia.Results: 1) SPostC exerts a protective effect by increasing cell viability, reducing LDH levels and cell apoptosis under low glucose (5 μM) after undergoing H/R injury; 2) High glucose concentration (35 μM) eliminated the cardioprotective effect of SPostC, which is manifested by a significantly decrease in the protein and mRNA expression level of the HIF-1α/MIF/AMPK signaling pathway, accompanied by decreased cell viability, increased LDH levels and apoptosis, increased ROS production, decreased ATP synthesis, and decreased mitochondrial membrane potential; 3. Under high glucose (35 μM), the expression levels of HIF-1α and MIF were up-regulated by using agonists, which can significantly increase the level of p-AMPKα protein, and the cardioprotective effect of SPostC was restored.Conclusion: The signal pathway of HIF-1α/MIF/AMPK of H9c2 cardiomyocytes may be the key point of SPostC against H/R injure. The cardioprotective of SPostC could be restored by upregulating the protein expression of HIF-1α and MIF under hyperglycemia.

BackgroundExploring the effective therapy for neonatal hypoxic-ischemic brain injury is an important goal. This study was designed to investigate how dexmedetomidine (DEX) contribute to hypoxic brain injury.MethodsDeveloping Sprague-Dawley rat models of hypoxia/reoxygenation (H/R) injury were constructed to simulate neonatal hypoxic brain injury for DEX treatment. Immunohistochemistry and western blot were performed to measure neuroglobin (Ngb) protein expression in hippocampal tissues. Hippocampal neuron injury and apoptosis were detected by Nissl staining and TUNEL assay, respectively. A Morris water maze (MWM) test was performed to evaluate the long-term learning and memory function.ResultsThe expression of Ngb was increased following H/R model establishment and up-regulated by medium and high doses of DEX, but not up-regulated by low doses of DEX. Medium and high doses of DEX alleviated the H/R injury as well as induced the reduction of Nissl bodies and apoptosis. Besides, medium and high doses of DEX down-regulated cytosolic Cyt-c, Apaf-1, and caspase-3 in H/R injury model. MWM test showed that medium and high doses of DEX significantly shortened the escape latency and enhanced the number of platform crossings. However, low doses of DEX have no effect on Nissl bodies, mitochondrial apoptosis, expression of apoptosis-related proteins and long-term learning functions.ConclusionsDEX induced Ngb expression in H/R rat models. The neuroprotection of DEX-mediated Ngb up-regulation may be achieved by inhibiting neuronal apoptosis through the mitochondrial pathway. Findings indicated that DEX may be useful as an effective therapy for neonatal hypoxic brain injury.

Previous studies have shown an attenuating effect of ginsenoside Re on myocardial injury induced by hypoxia/reoxygenation (H/R). However, the underlying mechanism remains unclear. This study was designed to determine the underlying mechanism by which ginsenoside Re protects from myocardial injury induced by H/R. HL-1 cells derived from AT-1 mouse atrial cardiomyocyte tumor line were divided into control, H/R, and H/R + ginsenoside Re groups. Cell viability was measured by CCK-8 assay. ATP levels were quantified by enzymatic assays. Signaling pathway was predicted by network pharmacology analyses and verified by luciferase assay and gene-silencing experiment. The relationship between ginsenoside Re and its target genes and proteins was analyzed by docking experiments, allosteric site analysis, real-time PCR, and ubiquitination and immunoprecipitation assays. Our results showed that ginsenoside Re treatment consistently increased HL-1 cell viability and significantly up-regulated ATP levels after H/R-induced injury. Network pharmacology analysis suggested that the effect of ginsenoside Re was associated with the regulation of the Hypoxia-inducing factor 1 (HIF-1) signaling pathway. Silencing of HIF-1α abrogated the effect of ginsenoside Re on HL-1 cell viability, which was restored by transfection with an HIF-1α-expressing plasmid. Results of the bioinformatics analysis suggested that ginsenoside Re docked at the binding interface between HIF-1α and the von Hippel-Lindau (VHL) E3 ubiquitin ligase, preventing VHL from binding HIF-1α, thereby inhibiting the ubiquitination of HIF-1α. To validate the results of the bioinformatics analysis, real-time PCR, ubiquitination and immunoprecipitation assays were performed. Compared with the mRNA expression levels of the H/R group, ginsenoside Re did not change expression of HIF-1α mRNA, while protein level of HIF-1α increased and that of HIF-1α[Ub]n decreased following ginsenoside Re treatment. Immunoprecipitation results showed that the amount of HIF-1α bound to VHL substantially decreased following ginsenoside Re treatment. In addition, ginsenoside Re treatment increased the expression of GLUT1 (glucose transporter 1) and REDD1 (regulated in development and DNA damage response 1), which are targets of HIF-1α and are critical for cell metabolism and viability. These results suggested that Ginsenoside Re treatment attenuated the myocardial injury induced by H/R, and the possible mechanism was associated with the inhibition of HIF-1α ubiquitination.

Myocardial ischemia-reperfusion injury (IRI) is a severe trauma which is characterized by inflammatory reaction, oxidative stress and cardiomyocyte apoptosis. Anesthetics such as sevoflurane have been proved to exhibit cardioprotective effect on IRI and the present study aimed to explore the underlying mechanism. In this study, H9C2 cells were randomly divided into the following groups: Control group; hypoxia/reoxygenation (H/R) group; 2.5% sevoflurane (Sev) 1 h group (H9C2 cells were exposed to 1 h of 2.5% sevoflurane 24 h before H/R); 2.5% Sev 2 h group (H9C2 cells were exposed to 2 h of 2.5% sevoflurane 24 h before H/R); 2.5% sevoflurane (Sev) 2 h + LY294002 group (H9C2 cells were pretreated with 10 μL LY294002 for 24 h before sevoflurane treatment). Cell proliferation and apoptosis were examined by CCK8 assay and Flow cytometry. Then, the expression levels of key proteins, including Bcl-2, Mcl-1, iNOS, p-AKT, t-AKT, PIM1, P-Bad, p-GSK3β, t-GSK3β and cyclinD1, were examined by western blot. Furthermore, nitric oxide (NO) concentration was detected with an ELISA kit, and TNF-α, IL-1β, IL-6 and IL-10 levels were examined by western blot. The CCK8 assay and flow cytometry results indicated that sevoflurane pretreatment reduced the apoptosis of H9C2 cells with H/R injury. In addition, sevoflurane pretreatment significantly inhibited the inflammatory injury induced by H/R. Furthermore, sevoflurane activated AKT/Pim-1 and AKT/GSK3β signaling pathway. These beneficial effects of sevoflurane were canceled by phosphoinositide-3-kinase inhibitor LY294002. In conclusion, these results verified that sevoflurane attenuates H/R-induced cardiomyocyte injury via AKT/Pim-1 and AKT/GSK3β signaling pathways.

Background/Aims: The present study aimed to detect the expression of miR-449a and investigate the effect of miR-449a on cell injury in cardiomyocytes subjected to hypoxia/ reoxygenation (H/R) and its underlying mechanisms. Methods: The expression of miR-449a was determined using reverse transcription–polymerase chain reaction in both neonatal rat ventricular myocytes and H9C2 cells. For gain-of-function and loss-of-function studies, H9C2 cells were transfected with either miR-449a mimics or miR-449a inhibitor. The target gene of miR-449a was confirmed by a dual-luciferase reporter assay. Apoptosis was analyzed by both flow cytometry using Annexin V and propidium iodide and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL). Necrosis was confirmed by the detection of lactate dehydrogenase release. The cell viability was measured using the methylthiotetrazole method. The protein levels of Notch-1, Notch-1 intracellular domain, hairy and enhancer of split-1 (Hes-1), and apoptosis-related genes were measured by Western blot analysis. Results: MiR-449a was significantly upregulated in both neonatal rat ventricular myocytes and H9C2 cells subjected to H/R. However, H/R-induced cell apoptosis and necrosis were markedly reduced by miR-449a inhibition. By targeting Notch-1, miR-449a regulated the Notch-1/ Hes-1 signaling pathway. The blockade of the Notch signaling pathway partly abolished the protective effect of miR-449a suppression against H/R injury, whereas the overexpression of Notch-1 intracellular domain partly reversed the effect of miR-449a overexpression on H/R-induced cell injury. Conclusions: The present study suggested that miR-449a inhibition protected H9C2 cells against H/R-induced cell injury by targeting the Notch-1 signaling pathway, providing a novel insight into the molecular basis of myocardial ischemia–reperfusion injury and a potential therapeutic target.

Our current research aimed to decipher the role and underlying mechanism with regard to miR-29b-3p involving in myocardial ischemia/reperfusion (I/R) injury. In the present study, cardiomyocyte H9c2 cell was used, and hypoxia/reoxygenation (H/R) model was established to mimic the myocardial I/R injury. The expressions of miR-29b-3p and pentraxin 3 (PTX3) were quantified deploying qRT-PCR and Western blot, respectively. The levels of LDH, TNF-α, IL-1β and IL-6 were detected to evaluate cardiomyocyte apoptosis and inflammatory response. Cardiomyocyte viability and apoptosis were examined employing CCK-8 assay and flow cytometry, respectively. Verification of the targeting relationship between miR-29b-3p and PTX3 was conducted using a dual-luciferase reporter gene assay. It was found that miR-29b-3p expression in H9c2 cells was up-regulated by H/R, and a remarkable down-regulation of PTX3 expression was demonstrated. MiR-29b-3p significantly promoted of release of inflammatory cytokines of H9c2 cells, and it also constrained the proliferation and promoted the apoptosis of H9c2 cells. Additionally, PTX3 was inhibited by miR-29b-3p at both mRNA and protein levels, and it was identified as a direct target of miR-29b-3p. PTX3 overexpression could reduce the inflammatory response, increase the viability of H9c2 cells, and inhibit apoptosis. Additionally, PTX3 counteracted the function of miR-29b-3p during the injury of H9c2 cells induced by H/R. In summary, miR-29b-3p was capable of aggravating the H/R injury of H9c2 cells by repressing the expression of PTX3. [ABSTRACT FROM AUTHOR]

Background. Sevoflurane preconditioning (SPC) can provide myocardial protective effects similar to ischemic preconditioning (IPC). However, the underlying molecular mechanism of SPC remains unclear. Studies confirm that hypoxia-inducible factor-1 (HIF-1) can transform cells from aerobic oxidation to anaerobic glycolysis by activating the switch protein pyruvate dehydrogenase kinase-1 (PDK-1), thus providing energy for the normal life activities of cells under hypoxic conditions. The purpose of this study was to investigate whether the cardioprotective effects of SPC are associated with activation of the HIF-1a/PDK-1 signal pathway. Methods. The H9c2 cardiomyocytes hypoxia/reoxygenation model was established and treated with 2.4% sevoflurane at the end of equilibration. Lactate dehydrogenase (LDH) level, cell viability, cell apoptosis, mitochondrial membrane potential, key enzymes of glycolysis, ATP concentration of glycolysis were assessed after the intervention. Apoptosis related protein(Bcl-2, Bax), HIF-1a protein, and PDK-1 protein were assessed by western blot. Results. Compared with the H/R group, SPC significantly increased the expression of HIF-1a, PDK-1, and Bcl-2 and reduced the protein expression of Bax, which markedly decreased the apoptosis ratio and Lactate dehydrogenase (LDH) level, increasing the cell viability, content of key enzymes of glycolysis, ATP concentration of glycolysis and stabilizing the mitochondrial membrane potential. However, the cardioprotective effects of SPC were disappeared by treatment with a HIF-1a selective inhibitor. Conclusion. This study demonstrates that the cardioprotective effects of SPC are associated with the activation of the HIF-1a/PDK-1 signaling pathway. The mechanism may be related to increasing the content of key enzymes and ATP of glycolysis in the early stage of hypoxia. [ABSTRACT FROM AUTHOR]

Prokineticin 2 (PK2) was reported to be decreased in the hearts of end-state heart failure patients. Our study aimed to explore the effects of PK2 on hypoxia/reoxygenation (H/R) injury and the underlying mechanism. H9c2 cardiomyocytes were treated with 5 nM PK2 in the presence or absence of 5 mM dual phosphatidylinositol 3-kinase (PI3K)/the mammalian target of rapamycin (mTOR) inhibitor (BEZ235) for 24 h and then subjected to H/R treatment. Cell viability and lactate dehydrogenase (LDH) release were evaluated by CCK-8 and LDH release assays, respectively. Apoptosis was determined by flow cytometry analysis. Oxidative stress was assessed by measuring superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content. Results showed that H/R treatment decreased PK2 expression and inactivated the Akt/mTOR pathway in H9c2 cardiomyocytes. PK2 treatment activated the Akt/mTOR pathway in H/R-exposed H9c2 cardiomyocytes. H/R stimulation suppressed cell viability, increased LDH release, induced apoptosis and oxidative stress in H9c2 cardiomyocytes, while these effects were neutralised by treatment with PK2. However, the inhibitory effects of PK2 on H/R-induced injury in H9c2 cardiomyocytes were abolished by the addition of BEZ235. In conclusion, PK2 relieved H/R-induced injury in H9c2 cardiomyocytes by activation of the Akt/mTOR pathway. [ABSTRACT FROM AUTHOR]

Background: Exploring the effective therapy for neonatal hypoxic-ischemic brain injury is an important goal. This study was designed to investigate how dexmedetomidine (DEX) contribute to hypoxic brain injury. Methods: Developing Sprague-Dawley rat models of hypoxia/reoxygenation (H/R) injury were constructed to simulate neonatal hypoxic brain injury for DEX treatment. Immunohistochemistry and western blot were performed to measure neuroglobin (Ngb) protein expression in hippocampal tissues. Hippocampal neuron injury and apoptosis were detected by Nissl staining and TUNEL assay, respectively. A Morris water maze (MWM) test was performed to evaluate the long-term learning and memory function. Results: The expression of Ngb was increased following H/R model establishment and up-regulated by medium and high doses of DEX, but not up-regulated by low doses of DEX. Medium and high doses of DEX alleviated the H/R injury as well as induced the reduction of Nissl bodies and apoptosis. Besides, medium and high doses of DEX down-regulated cytosolic Cyt-c, Apaf-1, and caspase-3 in H/R injury model. MWM test showed that medium and high doses of DEX significantly shortened the escape latency and enhanced the number of platform crossings. However, low doses of DEX have no effect on Nissl bodies, mitochondrial apoptosis, expression of apoptosis-related proteins and long-term learning functions. Conclusions: DEX induced Ngb expression in H/R rat models. The neuroprotection of DEX-mediated Ngb up-regulation may be achieved by inhibiting neuronal apoptosis through the mitochondrial pathway. Findings indicated that DEX may be useful as an effective therapy for neonatal hypoxic brain injury. [ABSTRACT FROM AUTHOR]

[Objectives] To explore the protection mechanism of crocin against ischemia-reperfusion injury of myocardial cells. [Methods] Newborn male SD rats were selected, left ventricular cardiomyocytes (CMs) were isolated, and a hypoxia/reoxygenation model of CMs was established to simulate the process of ischemia reperfusion injury. The cells were randomly divided into four groups: normal cell group (control group), crocin group), hypoxia/reoxygenation group (H/R group), hypoxia/reoxygenation o crocin group (H/R + crocin group). H/R + crocin group selected the concentration of crocin 1, 10, and 100 µmol/L, and determined the optimal concentration of crocin by detecting the cell proliferation ability. After the cells were pretreated using the optimal concentration of crocin, the levels of superoxide anion, cell proliferation, apoptosis and Nox2 levels in each group of cells were detected. [Results] Compared with the control group, the proliferation ability of CMs after hypoxia-reoxygenation injury was reduced (P < 0.05), while cell apoptosis and intracellular superoxide anion levels were significantly in creased (P < 0.01); the CMs pretreated with crocin can reduce the level of Nox2 (P < 0.01), increase the cell proliferation ability of CMs, reduce cell apoptosis, and accordingly reduce the level of superoxide anion in the cell (P < 0.05). [Conclusions] Crocin protects CMs from hypoxia/reoxygenation injury through down-regulating the level of Nox2 and reducing oxidative stress injury. [ABSTRACT FROM AUTHOR]

Previous studies have shown an attenuating effect of ginsenoside Re on myocardial injury induced by hypoxia/reoxygenation (H/R). However, the underlying mechanism remains unclear. This study was designed to determine the underlying mechanism by which ginsenoside Re protects from myocardial injury induced by H/R. HL-1 cells derived from AT-1 mouse atrial cardiomyocyte tumor line were divided into control, H/R, and H/R + ginsenoside Re groups. Cell viability was measured by CCK-8 assay. ATP levels were quantified by enzymatic assays. Signaling pathway was predicted by network pharmacology analyses and verified by luciferase assay and gene-silencing experiment. The relationship between ginsenoside Re and its target genes and proteins was analyzed by docking experiments, allosteric site analysis, real-time PCR, and ubiquitination and immunoprecipitation assays. Our results showed that ginsenoside Re treatment consistently increased HL-1 cell viability and significantly up-regulated ATP levels after H/R-induced injury. Network pharmacology analysis suggested that the effect of ginsenoside Re was associated with the regulation of the Hypoxia-inducing factor 1 (HIF-1) signaling pathway. Silencing of HIF-1α abrogated the effect of ginsenoside Re on HL-1 cell viability, which was restored by transfection with an HIF-1α-expressing plasmid. Results of the bioinformatics analysis suggested that ginsenoside Re docked at the binding interface between HIF-1α and the von Hippel-Lindau (VHL) E3 ubiquitin ligase, preventing VHL from binding HIF-1α, thereby inhibiting the ubiquitination of HIF-1α. To validate the results of the bioinformatics analysis, real-time PCR, ubiquitination and immunoprecipitation assays were performed. Compared with the mRNA expression levels of the H/R group, ginsenoside Re did not change expression of HIF-1α mRNA, while protein level of HIF-1α increased and that of HIF-1α[Ub]n decreased following ginsenoside Re treatment. Immunoprecipitation results showed that the amount of HIF-1α bound to VHL substantially decreased following ginsenoside Re treatment. In addition, ginsenoside Re treatment increased the expression of GLUT1 (glucose transporter 1) and REDD1 (regulated in development and DNA damage response 1), which are targets of HIF-1α and are critical for cell metabolism and viability. These results suggested that Ginsenoside Re treatment attenuated the myocardial injury induced by H/R, and the possible mechanism was associated with the inhibition of HIF-1α ubiquitination. [ABSTRACT FROM AUTHOR]

Lycopene (lyc) has an effect on preventing cancer, yet its effects on hypoxia/reoxygenation (H/R) injury remained obscure. The study aimed at discovering its role in preventing hepatic cells against H/R injury. Hepatic cells were incubated in hypoxia incubator to simulate ischemia/reperfusion injury in vitro. Cell viability was detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after Lycopene treatment with or without ML385 (nuclear factor erythroid 2-related factor 2 [Nrf2] inhibitor). Lactate dehydrogenase (LDH) and malondialdehyde (MDA) content were detected. Cellular cytokine (tumor necrosis factor-α, TNF-α; interleukin-6, IL-6) levels were measured using enzyme-linked immunosorbent assay (ELISA). Hepatic cell apoptosis and cellular reactive oxygen species (ROS) content was detected by flow cytometry. Nrf2 transfer was observed using immunofluorescence staining. Nrf2 and heme oxygenase-1 (HO-1) expressions were detected with quantitative real-time polymerase chain reaction and western blot as needed. In hepatic cells, after H/R, the viability was dropped, TNF-α and IL-6 levels and LDH and MDA content were increased, with high apoptosis rate and ROS content. Lycopene led to a reversed effect, with promotion on Nrf2 transfer from cytoplasm into nucleus and Nrf2/HO-1 pathway activation. Further experiments showed that ML385 could reverse the effects of Lycopene. Lycopene could activate Nrf2/HO-1 pathway to protect hepatic cells against H/R injury. [ABSTRACT FROM AUTHOR]

Astragaloside IV (AS/IV) is one of the extracted components from the traditional Chinese medicine Astragalus which has been demonstrated to have potential capacity for anti-inflammation activity and for treating cardiovascular disease. Our purpose was to determine the function and underlying molecular mechanism of AS/IV in hypoxia/reoxygenation (H/R) injured in cardiomyocytes. Differentially expressed genes (DEGs) were screened using bioinformatic analysis, and the molecular targeting relationship was verified by the dual-luciferase report system. H/R injured cardiomyocytes were employed to explore the effect of AS/IV. QRT-PCR and Western blot analysis were applied to detect the expression of mRNA and proteins, respectively. Additionally, superoxide dismutase (SOD), lactic dehydrogenase (LDH) and MDA (malondialdehyde) levels were detected to determine the oxidative damage. Cell viability was assessed by CCK-8, and flow cytometry was used to evaluate cell apoptosis ratio. TGFBR1 and TLR2 were selected as DEGs. Additionally, AS/IV could enhance cell proliferation and upregulated miR-101a expression, which suppressed TGFBR1 and TLR2 expression in H/R injured cardiomyocytes. Moreover, the results of Western blot exhibited that the downstream genes (p-ERK and p-p38) in the MAPK signaling pathway were suppressed, which meant AS/IV could inhibit this pathway in H/R injured cardiomyocytes. Overall, this study demonstrated AS/IV could attenuate H/R injury in human cardiomyocytes via the miR-101a/TGFBR1/TLR2/MAPK signaling pathway axis, which means that it could serve as a possible alternate for H/R treatment. [ABSTRACT FROM AUTHOR]

• Hypothermia protected hippocampal neurons against from H/R induced pyroptosis. • The PTEN and PI3K/Akt/GSK-3β signaling participated in activating inflammasome and promoting pyroptosis. • Methylated level of PTEN mRNA was elevated in hippocampal neurons upon H/R. Cerebral ischemia/reperfusion (I/R) injury often leads to irreversible neuronal injury and even death, and hypothermia is the only therapeutic method that has been proven to be effective. However, the molecular mechanisms underlying the effect of hypothermia treatment on I/R injury have not been fully elucidated. In the present study, we aimed to evaluate the neuroprotective effects and mechanisms of hypothermia against hypoxia/reoxygenation (H/R)-induced neuronal damage. Primary hippocampal neurons were exposed to H/R and were then treated with hypothermia. We observed that hypothermia significantly increased cellular viability, downregulated the expression of pyroptosis-related proteins—including NLR pyrin domain containing 3 (NLRP3), apoptotic speck-like protein containing CARD (ASC), cleaved Caspase-1, and Gasdermin-D (GsdmD) p30—and reduced secretion of the pro-inflammatory cytokines, IL-1β and IL-18. Additionally, pretreatment with MCC950, a specific small-molecule inhibitor of the NLRP3 inflammasome, yielded a protective effect on cellular viability that was comparable to that of hypothermia treatment. Furthermore, hypothermia also significantly elevated the expression level of phosphatase and tensin homologous protein (PTEN) and activated the phosphorylation levels of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β). These protective effects of hypothermia on pyroptosis-related proteins and pro-inflammatory cytokines were partially reversed by the specific PI3K/Akt inhibitor, LY294002. Moreover, the methylated level of PTEN mRNA was elevated in hippocampal neurons upon H/R, whereas this level remained stable in the hypothermia group. Therefore, our findings suggest that hypothermia protects neurons against neuronal H/R-induced pyroptosis, and that m6A-mediated activation of PTEN and the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/GSK-3β signaling pathway may play crucial roles during this process. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRNAs) are involved in myocardial ischemia-reperfusion injury. miRNA-421 (miR-421) plays a significant role in the initiation of apoptosis and myocardial infarction. However, the molecular regulation of miR-421 in myocardial ischemia-reperfusion injury requires further elucidation. Methods: An in vitro hypoxia/reoxygenation model was established, and the expression levels of miR-421 and Sirtuin-3 (Sirt3) in H9c2 cells were quantified using quantitative real-time polymerase chain reaction. Flow cytometry was employed to measure the effects of miR-421 on myocardial apoptosis induced by hypoxia/reoxygenation. The activity of lactate dehydrogenase and superoxide dismutase and levels of malondialdehyde were measured. The binding sites of miR-421 on Sirt3 were predicted using TargetScan software. A luciferase reporter assay was used to validate the direct targeting of Sirt3 with miR-421. Protein expression levels of Sirt3 and its downstream proteins were evaluated using Western blot analysis. Results: Exposure of H9c2 cells to hypoxia/reoxygenation led to increased apoptosis, levels of malondialdehyde and lactate dehydrogenase, and decreased levels of superoxide dismutase. miR-421 knockdown resulted in decreased apoptosis, levels of lactate dehydrogenase and malondialdehyde, and increased superoxide dismutase levels in H9c2 cells. Hypoxia/reoxygenation significantly decreased the relative expression levels of Sirt3. Down-regulation of Sirt3 resulted from overexpression of miR-421, which directly targeted Sirt3. Knockdown of miR-421 up-regulated Sirt3 expression, inhibited activation of the Jun N-terminal kinase/activator protein 1 pathway and caspase 9/3-dependent cell death. Conclusion: The miR-421-Sirt3-Jun N-terminal kinase/activator protein 1 axis is a novel molecular mechanism that accommodates hypoxia/reoxygenation-induced oxidative stress and apoptosis and provides a new direction for the study and treatment of hypoxia/reoxygenation. [ABSTRACT FROM AUTHOR]

Renal ischemia-reperfusion injury is a major cause of acute kidney injury. In the present study, we investigated the effects of pioglitazone on hypoxia/reoxygenation (H/R) injury in rat renal tubular epithelial cells (RTECs) under normal- (NG) or high-glucose (HG) culture conditions via evaluating oxidative stress and endoplasmic reticulum stress (ERS). The RTECs (NRK-52E cells) were divided into six groups as follows: NG group, HG group, NG + H/R group, HG + H/R group, NG + Pio + H/R group, and HG + Pio + H/R group, among which cells in H/R groups were subjected to 4 h of hypoxia followed by 12 h of reoxygenation. After that, the cells were evaluated using the Cell Counting Kit-8 assay for the determination of their viability and flow cytometry assay for the detection of apoptosis. The levels of superoxide dismutase (SOD), glutathione reductase (GSH), catalase (CAT), and malondialdehyde (MDA) were determined via colorimetric chemical assays. In addition, the expression of ERS-associated proteins, i.e. ATF4, ATF6, GRP78, and CHOP, was determined via western blotting. A HG environment could reduce the viability and increase the apoptotic rate of NRK-52E cells with increased MDA levels and decreased SOD, CAT, and GSH levels, and upregulate the expression of ERS-associated proteins, i.e. ATF4, ATF6, and GRP78. H/R injury could further aggravate changes in the above indicators, but pioglitazone could significantly reverse such changes and alleviate cell injury. Thus, Pioglitazone exhibits a cytoprotective effect on RTECs against H/R injury under NG or HG culture conditions by inhibiting oxidative stress and ERS.

Docosahexaenoic acid (DHA) can alleviate cerebral ischemia/reperfusion injury by reducing blood–brain barrier permeability and maintaining its integrity, accompanied by an increased Ang-1/Ang-2 ratio; however, the underlying mechanisms of these effects remain unclear. Src-suppressed C kinase substrates (SSeCKS), a substrate of protein kinase C, plays an important role in maintaining cell junctions and cell morphology and regulating cell permeability. However, whether DHA can increase SSeCKS expression and then mediate the Ang-1/Ang-2 ratio still needs to be studied. Human cerebrovascular pericytes (HBVPs) cultured in vitro were divided into groups, treated with or without DHA along with SSeCKS siRNA to knockdown SSeCKS expression, and then subjected to 24 h of hypoxia followed by 6 h of reoxygenation. Cell viability; lactate dehydrogenase (LDH) release; and Ang-1, Ang-2 and VEGF activity were detected by using ELISA kits. The apoptosis rate was assessed by TUNEL flow cytometry. Expression of the SSeCKS, Ang-1, Ang-2 and VEGF proteins was evaluated by western blotting. Pretreatment with 10 μM or 40 μM DHA efficiently attenuated hypoxia/reoxygenation (H/R) injury by activating SSeCKS to increase the Ang-1/Ang-2 ratio and downregulate VEGF expression in HBVPs, as evidenced by decreased LDH release and apoptotic rates and increased HBVPs viability. Meanwhile, after we used SSeCKS siRNA to knock down SSeCKS protein expression, the protective effect of DHA on HBVPs following H/R injury was reversed. In conclusion, DHA can activate SSeCKS to increase the Ang-1/Ang-2 ratio and downregulate VEGF expression in HBVPs, thus reducing H/R injury. [ABSTRACT FROM AUTHOR]

Renal ischemia-reperfusion injury is a major cause of acute kidney injury. In the present study, we investigated the effects of pioglitazone on hypoxia/reoxygenation (H/R) injury in rat renal tubular epithelial cells (RTECs) under normal- (NG) or high-glucose (HG) culture conditions via evaluating oxidative stress and endoplasmic reticulum stress (ERS). The RTECs (NRK-52E cells) were divided into six groups as follows: NG group, HG group, NG + H/R group, HG + H/R group, NG + Pio + H/R group, and HG + Pio + H/R group, among which cells in H/R groups were subjected to 4 h of hypoxia followed by 12 h of reoxygenation. After that, the cells were evaluated using the Cell Counting Kit-8 assay for the determination of their viability and flow cytometry assay for the detection of apoptosis. The levels of superoxide dismutase (SOD), glutathione reductase (GSH), catalase (CAT), and malondialdehyde (MDA) were determined via colorimetric chemical assays. In addition, the expression of ERS-associated proteins, i.e. ATF4, ATF6, GRP78, and CHOP, was determined via western blotting. A HG environment could reduce the viability and increase the apoptotic rate of NRK-52E cells with increased MDA levels and decreased SOD, CAT, and GSH levels, and upregulate the expression of ERS-associated proteins, i.e. ATF4, ATF6, and GRP78. H/R injury could further aggravate changes in the above indicators, but pioglitazone could significantly reverse such changes and alleviate cell injury. Thus, Pioglitazone exhibits a cytoprotective effect on RTECs against H/R injury under NG or HG culture conditions by inhibiting oxidative stress and ERS. [ABSTRACT FROM AUTHOR]

Hypoxia/reoxygenation (H/R) is used as an in vivo model of ischemia/reperfusion injury, and myocardial ischemia can lead to heart disease. Therefore, it is necessary to prevent myocardial H/R injury to avoid the risk of heart disease. The aim of the present study was to investigate whether inhibiting microRNA (miR)-101a-3p attenuated H9C2 cell H/R injury, apoptosis mechanisms and key target proteins. Cell viability and apoptosis were determined by Cell Counting Kit-8 assays and flow cytometry using a cell apoptosis kit, respectively. The contents of creatine kinase (CK) and lactate dehydrogenase (LDH) were detected using colorimetric assays. Dual luciferase assays were carried out to determine if miR-101a-3p inhibited Janus kinase (JAK)2. Western blot analysis and reverse transcription-quantitative PCR were used to determine proteins levels and mRNAs expression. It was found that the inhibition of miR-101a-3p increased the growth of H9C2 cells and decreased H9C2 cell apoptosis during H/R injury. The inhibition of miR-101a-3p reduced the amounts of CK and LDH in H/R model H9C2 cells. The inhibition of miR-101a-3p lowered the levels of Bax, interleukin-6 and tumor necrosis factor-α, but raised the levels of phosphorylated (p)-STAT3 and p-JAK2 in H9C2 cells subjected to H/R injury treatment. miR-101a-3p mimic was found to inhibit H9C2 cell viability, raise p-JAK2 level and slightly increase p-STAT3 during H/R injury. AG490 induced H9C2 cell apoptosis, and decreased the levels of p-JAK2 and p-STAT3 during H/R injury. The data indicated that inhibiting miR-101a-3p reduced H/R damage in H9C2 cells and decreased apoptosis via Bax/Bcl-2 signaling during H/R injury. In addition, it was suggested that the inhibition of miR-101a-3p decreased H/R injury in H9C2 cell by regulating the JAK2/STAT3 signaling pathway. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress is implicated in the pathogenesis of many diseases, including myocardial ischemia/reperfusion injury. We hypothesized that human umbilical cord mesenchymal stromal cells derived extracellular vesicles (HuMSC-EVs) could protect cardiac cells against hyperactive ER stress induced by hypoxia/reoxygenation (H/R) injury. The H/R model was generated using the H9c2 cultured cardiac cell line. HuMSC-EVs were extracted using a commercially available exosome isolation reagent. Levels of apoptosis-related signaling molecules and the degree of ER stress were assessed by western blot. The role of the PI3K/Akt pathway was investigated using signaling inhibitors. Lactate dehydrogenase leakage and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) analysis were used for evaluating the therapeutic effects of HuMSC-EVs in vitro. The results showed that ER stress and the rate of apoptosis were increased in the context of H/R injury. Treatment with HuMSC-EVs inhibited ER stress and increased survival in H9c2 cells exposed to H/R. Mechanistically, the PI3K/Akt pathway was activated by treatment with HuMSC-EVs after H/R. Inhibition of the PI3K/Akt pathway by a specific inhibitor, LY294002, partially reduced the protective effect of HuMSC-EVs. Our findings suggest that HuMSC-EVs could alleviate ER stress–induced apoptosis during H/R via activation of the PI3K/Akt pathway. [ABSTRACT FROM AUTHOR]

Podocyte injury was reported to be involved in the major pathogenesis of ischemia/reperfusion (I/R)-induced ischemic acute renal failure. Our purpose was to study the mechanism of miR-187 improving I/R-induced podocytes injury. The miR-187 mimics and inhibitor were transfected into the immortalized mouse podocyte (MPC-5) cells, and then transfected cells were subjected to hypoxia/reoxygenation (H/R, 3/3 h) to establish an H/R cell model. To investigate the effects of miR-187 on H/R-induced cell injury, cell viability and apoptosis were measured by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Dual-luciferase report system was used to verify whether miR-187 could directly target acetylcholinesterase (ACHE). The animal ischemia/reperfusion model was established and injected with miR-187 agomir. Kidney tissue sections were subjected to histological examination by hematoxylin and eosin staining to assess the renal injury. Real-time quantitative PCR and western blot were performed to determine gene expressions. The transfection of miR-187 mimics contributed to MPC-cells resistance to H/R-induced cell injury, which was reflected by enhanced cell viability and reduced apoptosis (from 20.05% to 9.43%) in H/R + negative control group. ACHE was confirmed as a target of miR-187, and ACHE siRNA had a similar efficiency to miR-187 mimic. The injection of miR-187 agomir not only effectively protected the kidney from I/R-induced injury, but also reduced the concentrations of serum creatinine. Moreover, nephrin was noticeably increased and desmin was decreased under the effects of agomir. Our findings indicated that miR-187 improved I/R-induced ischemic acute renal failure through protecting glomerular filtration barrier by blocking the expression of ACHE. [ABSTRACT FROM AUTHOR]

Curcumin (Cur) has been reported to function as an antioxidant and anti-inflammatory agent and to play a role in anti-atherosclerosis. The present study aimed to explore the protective effect of Cur on hypoxia/reoxygenation (H/R) injury. The morphological changes in H9c2 cardiomyocytes were observed under an inverted microscope. Cell viability was determined by Cell Counting Kit-8 (CCK-8). Lactate dehydrogenase (LDH) level, malondialdehyde (MDA) level and the antioxidant superoxide dismutase (SOD) activity were determined by corresponding kits. Apoptosis and reactive oxygen species (ROS) levels were determined by flow cytometry. Endoplasmic reticulum (ER) stress-related factors, which were examined by quantitative real-time polymerase chain reaction (qPCR) and western blot analysis, included 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP). Extracellular signal regulating kinase 1/2 (ERK1/2), p38, c-Jun NH2-terminal kinase (JNK) and the phosphorylation levels of key proteins in the mitogen-activated protein kinase (MAPK) signaling pathway were all determined by western blot analysis. Compared to the control group, the cell morphology of the H9c2 cells was obviously altered upon H/R. Cell viability was significantly decreased, while apoptosis was significantly increased by H/R. We also observed that the levels of LDH and MDA were elevated and the activity of SOD was decreased in the H/R group. Notably, LDH, MDA and SOD levels were reversed following treatment with Cur; while apoptosis and ROS levels in the H/R injury group were decreased by Cur. H/R injury-triggered ER stress and the MAPK signaling pathway were suppressed by Cur. These results demonstrated that Cur has a protective effect on cardiomyocytes via suppression of ER stress and the MAPK pathway. [ABSTRACT FROM AUTHOR]

Myocardial ischemia-reperfusion injury (IRI) is a severe trauma which is characterized by inflammatory reaction, oxidative stress and cardiomyocyte apoptosis. Anesthetics such as sevoflurane have been proved to exhibit cardioprotective effect on IRI and the present study aimed to explore the underlying mechanism. In this study, H9C2 cells were randomly divided into the following groups: Control group; hypoxia/reoxygenation (H/R) group; 2.5% sevoflurane (Sev) 1 h group (H9C2 cells were exposed to 1 h of 2.5% sevoflurane 24 h before H/R); 2.5% Sev 2 h group (H9C2 cells were exposed to 2 h of 2.5% sevoflurane 24 h before H/R); 2.5% sevoflurane (Sev) 2 h + LY294002 group (H9C2 cells were pretreated with 10 μL LY294002 for 24 h before sevoflurane treatment). Cell proliferation and apoptosis were examined by CCK8 assay and Flow cytometry. Then, the expression levels of key proteins, including Bcl-2, Mcl-1, iNOS, p-AKT, t-AKT, PIM1, P-Bad, p-GSK3β, t-GSK3β and cyclinD1, were examined by western blot. Furthermore, nitric oxide (NO) concentration was detected with an ELISA kit, and TNF-α, IL-1β, IL-6 and IL-10 levels were examined by western blot. The CCK8 assay and flow cytometry results indicated that sevoflurane pretreatment reduced the apoptosis of H9C2 cells with H/R injury. In addition, sevoflurane pretreatment significantly inhibited the inflammatory injury induced by H/R. Furthermore, sevoflurane activated AKT/Pim-1 and AKT/GSK3β signaling pathway. These beneficial effects of sevoflurane were canceled by phosphoinositide-3-kinase inhibitor LY294002. In conclusion, these results verified that sevoflurane attenuates H/R-induced cardiomyocyte injury via AKT/Pim-1 and AKT/GSK3β signaling pathways. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress and autophagy are involved in myocardial ischemia-reperfusion (I/R) injury; however, their roles in this type of injury remain unclear. The present study investigated the roles of ER stress and autophagy, and their underlying mechanisms, in H9c2 cells during hypoxia/reoxygenation (H/R) injury. Cell viability was detected by CCK-8 assay. The autophagy flux was monitored with mCherry-GFP-LC3-adenovirus transfection. The expression levels of autophagy-related proteins and ER stress-related proteins were measured by western blotting. Apoptosis was detected by flow cytometry and western blotting. The results indicated that autophagy was induced, ER stress was activated and apoptosis was promoted in H9c2 cells during H/R injury. The inhibition of ER stress by 4-phenylbutyrate or C/EBP homologous protein (CHOP)-targeting small interfering RNA (siRNA) decreased autophagy and ameliorated cell apoptosis during H/R injury. Activation of autophagy by rapamycin attenuated ER stress and ameliorated cell apoptosis. Inhibition of autophagy by 3-methyladenine or Beclin1-targeting siRNA aggravated ER stress and exacerbated cell apoptosis, and activation of ER stress by thapsigargin decreased autophagy and induced cell apoptosis. Collectively, the findings of the present study demonstrated that H/R induced apoptosis and autophagy via ER stress in H9c2 cells, and that CHOP may serve an important role in ER stress-induced autophagy and apoptosis. Autophagy, as an adaptive response, was activated by ER stress and alleviated ER stress-induced cell apoptosis during H/R injury. [ABSTRACT FROM AUTHOR]

Resveratrol, a multi-functional phytoalexin, has been well indicated to exert cardioprotective effects by weakening ischemia/reperfusion (I/R) injury, and cell apoptosis is a vital way in I/R injury. SIRT1-p53 pathway has strong significance in regulating cell apoptosis. DJ-1 can directly bind to SIRT1 and stimulate the activity of SIRT1-p53. Therefore, the current study was determined whether Resveratrol attenuates hypoxia/reoxygenation (H/R)-induced cell apoptosis, and whether DJ-1-mediated SIRT1 activation involves in the cardioprotective effects of Resveratrol. The results showed that remarkable decrease in the number of apoptotic cells along with reduction of lactate dehydrogenase release and restoration of cell viability emerged when Resveratrol was applied in the H9c2 cells exposed to H/R. Moreover, Resveratrol increased DJ-1 expression and promoted the interaction of DJ-1 with SIRT1, which further contributed to subsequent restoration of SIRT1 activity and decrease of acetylation level of p53. However, above cardioprotective effects of Resveratrol were abrogated by DJ-1 siRNA and SIRT1 specific inhibitor Sirtinol. In conclusion, the current study demonstrated that Resveratrol suppressed H/R-induced cell apoptosis, which may be conducted by up-regulating DJ-1, and later activating SIRT1 activity and subsequently inhibiting p53 acetylation level in the H9c2 cells. • Resveratrol increased the DJ-1 expression and promoted the interaction of DJ-1 with SIRT1 in the H9c2 cells exposed to H/R. • Resveratrol restored SIRT1 activity in a way of DJ-1-dependence in the H9c2 cells exposed to H/R. • Resveratrol decreased H/R-induced p53 acetylation level by DJ-1-mediated SIRT1 activation in the H9c2 cells. • Resveratrol attenuated H/R-induced cell apoptosis by DJ-1-mediated SIRT1 activation in the H9c2 cells. [ABSTRACT FROM AUTHOR]

Increasing evidence has indicated that opening of the mitochondrial permeability transition pore (mPTP) has a vital role in myocardial ischemia/reperfusion (I/R) injury. Shenmai injection (SMI) plus Danshen injection (DSI) combination, termed Yiqi Yangyin Huoxue (YYH) therapy is used in the clinic to treat cardiovascular diseases, including myocardial I/R injury. Previous studies by our group have demonstrated the protective effect of pretreatment with YYH against myocardial I/R injury in isolated rat hearts. The present study aimed to examine the protective effect of YYH against hypoxia/reoxygenation (H/R)- and H2O2-induced cardiomyocyte injury, and to determine whether this effect is produced by inhibition of mPTP opening. Primary cardiomyocytes isolated from neonatal rats were cultured and randomly grouped into a control group, injury group and pretreatment group, with six duplicated wells in each group during each assay. Cardiomyocytes in the injury group were subjected to H/R to simulate I/R or exposed to H2O2 for 2 h to induce oxidative injury. Cellular injury was assessed via release of creatine kinase (CK) and lactate dehydrogenase (LDH), and cell viability was measured by an MTT assay. The mitochondrial membrane potential (ΔΨm) and cytosolic reactive oxygen species (ROS) were detected using the fluorescent probes rhodamine123 (Rh123) and chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), respectively. Intracellular Ca2+, mitochondrial Ca2+ and mPTP opening were measured using fluo-4 acetoxymethyl (Fluo-4/AM), rhodamine-2 acetoxymethyl (Rhod-2/AM) and calcein acetoxymethyl (Calcein/AM) probes, respectively. The results indicated that pretreatment with YYH enhanced cell viability, increased ΔΨm, reduced CK and LDH release, and decreased intracellular ROS and Ca2+, thus reducing cardiomyocyte injury induced by H/R or H2O2. LY294002, a specific phosphoinositide 3-kinase (PI3K) inhibitor, and PD98059, a specific inhibitor of the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway, eliminated the protective effects of the combination therapy on cell viability and the change in the ΔΨm in cardiomyocytes. In conclusion, pre-treatment with YYH has cardioprotective effects against H/R injury and oxidative stress via activation of the PI3K/Akt and Erk1/2 signaling pathways, which reduces mPTP opening, overproduction of ROS and calcium overload. [ABSTRACT FROM AUTHOR]