miR-30a-5p attenuates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by regulating PTEN protein expression and activating PI3K/Akt signaling pathway.

BACKGROUND: This study was designed to investigate the mechanism by which miR-30a-5p mediates cardiomyocyte apoptosis after acute myocardial infarction (AMI) induced by hypoxia/reoxygenation (H/R). METHODS: Differentially expressed miRNAs were analyzed by RNA high-throughput sequencing in acute myocardial infarction (ST-elevation myocardial infarction) patients versus healthy individuals (controls). The H/R model was used to assess the regulatory mechanism of miRNAs in AMI. Lentivirus-associated vectors were used to overexpress or knock down miR-30a-5p in cellular models. The pathological mechanisms of miR-30a-5p regulating the development of acute myocardial infarction were serially explored by qPCR, bioinformatics, target gene prediction, dual luciferase, enzyme-linked immunosorbent assays (ELISAs) and Western blotting. RESULTS: The results showed that the expression of miR-30a-5p was significantly increased in AMI patients and H9C2 cells. Hypoxia decreased cardiomyocyte survival over time, and reoxygenation further reduced cell survival. Bax and Phosphatase and tensin homolog (PTEN)were suppressed, while Bcl-2 was upregulated. Additionally, miR-30a-5p specifically targeted the PTEN gene. According to the GO and KEGG analyses, miR-30a-5p may participate in apoptosis by interacting with PTEN. The miR-30a-5p mimic decreased the expression of apoptosis-related proteins and the levels of the proinflammatory markers IL-1ß, IL-6, and TNF-α by activating the PTEN/PI3K/Akt signaling pathway. Conversely, anti-miR-30a-5p treatment attenuated these effects. Additionally, silencing PTEN and anti-miR-30a-5p had opposite effects on H/R-induced cell apoptosis. CONCLUSIONS: miR-30a-5p plays a crucial role in cardiomyocyte apoptosis after hypoxia-induced acute myocardial infarction. Our findings provide translational evidence that miR-30a-5p is a novel potential therapeutic target for AMI.

Autophagy decreases alveolar epithelial cell injury by regulating the release of inflammatory mediators.

To research the impact of autophagy on alveolar epithelial cell inflammation and its possible mechanism in the early stages of hypoxia, we established a cell hypoxia-reoxygenation model and orthotopic left lung ischemia-reperfusion model. Rat alveolar epithelial cells stably expressing GFP-LC3 were treated with an autophagy inhibitor (3-MA) or an autophagy promoter (rapamycin), followed by hypoxia-reoxygenation treatment for 2, 4, and 6 hr in vitro. In vivo, 20 male Sprague Dawley rats were randomly divided into four groups (model group: No blocking of the hilum in the left lung; control group: Blocking of the hilum in the left lung for 1 hr with dimethyl sulfoxide lavage; 3-MA group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of 3-MA (5 µmol/L) solution lavage; and rapamycin group: Blocking of the hilum in the left lung for 1 hr with 100 ml/kg of rapamycin (250 nmol/L) solution lavage) to establish an orthotopic left lung ischemia model. This study demonstrated that rapamycin significantly suppressed the nuclear factor kappa B signaling pathway and limited the expression of proinflammatory factors. A contrary result was found after the 3-MA pretreatment. These findings indicate that autophagy reduces ischemia-reperfusion injury by repressing inflammatory signaling pathways in the early stages of hypoxia in vitro and in vivo. Autophagy could be a new protective method for application in lung ischemia-reperfusion injury.

Astragaloside IV enhances GATA-4 mediated myocardial protection effect in hypoxia/reoxygenation injured H9c2 cells.

BACKGROUND AND AIM: The transcription factor GATA-4 plays an important role in myocardial protection. Astragaloside IV (Ast-IV) was reported with the effects on improving cardiac function after ischemia. In this study, we explored how Ast-IV interacts with GATA-4 to protect myocardial cells H9c2 against Hypoxia/Reoxygenation (H/R) stress. METHODS AND RESULTS: H9c2 cells were cultured under the H/R condition. Various cell activity and morphology assays were used to assess the rates of apoptosis and autophagy. In these H/R injured H9c2 cells, increased apoptosis (P < 0.01) and autophagosome number (P < 0.01) were observed, and the addition of Ast-IV ameliorated this tendency. Mechanistically, we used the RT-qPCR and Western blot to evaluate the expressions of various molecules. The results showed that Ast-IV treatment upregulated gene expression of GATA-4 (P < 0.01) and the survival factors (Bcl-2, P < 0.05; p62, P < 0.01), but suppressed apoptosis and autophagy related genes (PARP, Caspase-3, Beclin-1, and LC3-II; All P < 0.01). Furthermore, overexpressing of GATA-4 by its agonist phenylephrine can also protect H/R injured H9c2 cells, and the addition of Ast-IV further enhanced this protection of GATA-4. In contrast, silencing GATA-4 expression abolished the H/R protection of Ast-IV, which demonstrated that the myocardial protection of Ast-IV is mediated by GATA-4. Lastly, along with GATA overexpression, enhanced interactions between Bcl-2 and Beclin-1 were detected by Chromatin immunoprecipitation (P < 0.01). CONCLUSION: Ast-IV rescued the H/R injury induced apoptosis and autophagy in H9c2 cells. Ast-IV treatment can stimulate the overexpression of GATA-4, and further enhanced the myocardial protection effect of GATA-4.

MiR-134-5p Regulates Myocardial Apoptosis and Angiogenesis by Directly Targeting KDM2A After Myocardial Infarction.

MiR-134-5p was found to have potential diagnostic value for myocardial infarction (MI), but its biological role in MI has not been reported. In this study, MI mouse model was established. Quantitative real-time PCR (qRT-PCR) and western blot were used to measure the expression of miR-134-5p, lysine demethylase 2A (KDM2A), and vascular endothelial growth factor (VEGF). Dual-Luciferase reporter (DLR) assay was used to explore the relationship between miR-134-5p and KDM2A. The influence of miR-134-5p on cardiomyocytes apoptosis was detected using methyl thiazolyl tetrazolium (MTT) assay. The results revealed that miR-134-5p was highly expressed in infarction tissues of MI mice. Knockdown of miR-134-5p inhibited hypoxia/reoxygenation (H/R) -induced cardiomyocyte apoptosis. In addition, KDM2A was the target gene of miR-134-5p and negatively regulated by miR-134-5p. The promotion effect on the protein level of KDM2A and VEGF induced by miR-134-5p inhibitor can be reversed by shKDM2A in cardiomyocytes. Further, silencing of miR-134-5p promoted myocardial angiogenesis and inhibited myocardial apoptosis via upregulating KDM2A in MI mice. Taken together, our research revealed that knockdown of miR-134-5p increased KDM2A expression, thereby suppressing myocardial apoptosis and promoting myocardial angiogenesis.

Inhibition of Frizzled-2 by small interfering RNA protects rat hepatic BRL-3A cells against cytotoxicity and apoptosis induced by Hypoxia/Reoxygenation.

Frizzled-2 plays an important role in maintaining normal hepatic cell functionality. This study aimed to investigate the role of inhibition of Frizzled-2 in protecting rat liver BRL-3A cells from Hypoxia/Reoxygenation (H/R). In vitro H/R hepatic cell model was established by culturing BRL-3A cells under H/R condition. Frizzled-2 siRNA was transfected into BRL-3A cells to inhibit Frizzled-2 signaling. Wnt5a and Frizzled-2 were significantly increased in BRL-3A cells upon H/R treatment. H/R treatment induced cell cytotoxicity, the early apoptosis rate and the intracellular Ca2+ level in BRL-3A cells while silencing frizzled-2 gene decreased the H/R induced cell cytotoxicity, apoptosis and intracellular Ca2+ level. In vivo mice study further showed the up-regulation of Frizzled-2/Wnt 5 pathway and cleaved Caspase-3 expression in liver tissues under ischemia and reperfusion injury (IRI). In summary, inhibition of Frizzled-2 by its siRNA may protects BRL-3A cells by attenuating the H/R induced cell cytotoxicity and apoptosis.

Oxygen partial pressure plays a crucial role in B16 melanoma cell survival by regulating autophagy and mitochondrial functions.

The oxygen partial pressure generally increases when cancerous cells become part of the blood vessels. The study was to investigate the influence of oxygen partial pressure on the apoptosis of B16 melanoma cells. Our results demonstrated that both short-term and long-term hypoxia/reoxygenation (H/R) treatment increased stress-induced intracellular reactive oxygen species (ROS). H/R treatment also increased apoptosis and autophagy in B16 cells. N-acetylcysteine (NAC), a ROS scavenger, can reduce ROS and aid survival. However, Bafilomycin A1, an autophagy inhibitor, can accelerate cell death. Thus, our work revealed that ROS and autophagy play critical roles in cellular H/R.

Salidroside protects renal tubular epithelial cells from hypoxia/reoxygenation injury in vitro.

Oxidative stress, inflammation and cell apoptosis are important mechanisms of renal ischemia/reperfusion (I/R) injury. Salidroside, a natural phenylpropanoid glycoside, possesses anti-inflammatory, anti-oxidative, and anti-apoptotic effects. However, the effect of salidroside on renal I/R injury has not been fully elucidated. The present study aimed to investigate the effect of salidroside on renal I/R injury in vitro. Our results showed that salidroside improved the viability of human renal tubular epithelial cells (HK-2) in response to hypoxia/reoxygenation (H/R). Salidroside caused apparent decrease in the levels of reactive oxygen species (ROS) and malondiaidehyde (MDA), and significant increase in superoxide dismutase (SOD) activity in HK-2 cells. Pretreatment with salidroside markedly inhibited the production levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 in a dose-dependent manner. Salidroside treatment exhibited significant increase in Bcl-2 expressions, and decrease in Bax expressions and caspase-3 activity when compared with the H/R group. Salidroside decreased the levels of toll-like receptor 4 (TLR4) and p-p65 in HK-2 cells. Overexpression of TLR4 significantly attenuated the effects of salidroside on cell viability, oxidative stress, cytokine production and cell apoptosis in HK-2 cells. These findings indicated that salidroside protected HK-2 cells from H/R stimulation, which was mediated by the TLR4/NF-κB pathway.

Knockdown of KLF11 attenuates hypoxia/reoxygenation injury via JAK2/STAT3 signaling in H9c2.

KLF11 is a Krüppel-like factor (KLF) family member, which plays a central role in cardiac hypertrophy and cerebrovascular protection during ischemic insults. However, the roles of KLF11 in hypoxia/reoxygenation (H/R) injury of rat cardiomyocytes H9c2 have not been elucidated. The aim of this study was to evaluate the effects of KLF11 on H/R injury and investigate the molecular mechanisms involved. Here, we found that KLF11 was increased following H/R and reached the highest level with 24 h hypoxia followed by 12 h reoxygenation. Moreover, we found that inhibition of KLF11 by small RNA suppressed cell apoptosis, the activity of caspase3, the expression of cleaved-caspase3 and cytochrome C in the cytoplasm and the damage of mitochondrial membrane induced by H/R in H9c2, suggesting that KLF11 silencing protects against H/R injury. In addition, we observed that knockdown of KLF11 elevated the expression of p-JAK2 and p-STAT3 in H9c2, and AG490, a selective inhibitor of JAK2/STAT3 abrogated the potential roles of KLF11 in cell apoptosis and mitochondrial damage. In aggregates, our results showed that depletion of KLF11 protected H9c2 against H/R injury through activating the JAK2/STAT3 signaling pathway, suggesting that KLF11 may be provide therapeutic targets for H/R or other heart diseases.

Sulfated Polysaccharide Isolated from the Sea Cucumber Stichopus japonicus Against PC12 Hypoxia/Reoxygenation Injury by Inhibition of the MAPK Signaling Pathway.

In this report, the sulfated polysaccharide (SJP) isolated from the sea cucumber Stichopus japonicus can protect PC12 from Na2S2O4-induced hypoxia/reoxygenation (H/R) injury. SJP effectively improves cell viability and reduces extracellular LDH release in PC12 cells after H/R. Moreover, SJP significantly increases SOD activity but decreases MDA levels. Our experiments showed that SJP could significantly reduce cell apoptosis caused by H/R. Our current results demonstrate that SJP suppressed the activation of MAPKs, resulting in a significant decrease in Bax/Bcl-2 ratio, cleaved caspase-3/caspase-3, p53 phosphorylation, and cytochrome c release in a concentration-dependent manner. MAPK is closely related to H/R injury. SJP inhibited JNK1/2 and p38 MAPK activation but did not affect the increased ERK1/2 expression. These results suggested that JNK1/2 and p38 MAPK pathways could be involved in SJP-mediated attenuation of PC12 H/R injury. SJP prevented PC12 H/R injury in a dose-dependent manner, indicating that SJP may be developed as a candidate drug to prevent or treat cerebral ischemia-reperfusion injury.

Downregulation of connexin 32 attenuates hypoxia/reoxygenation injury in liver cells.

Gap junction intercellular communication is involved in ischemia-reperfusion (IR) injury of organs. Connexins are proteins that are critical to the function of gap junctions. To clarify the role of gap junctions in IR injury in liver cells, the function of gap junctions was modulated in an in vitro hypoxia/reoxygenation (H/R) model. BRL-3A rat liver cells, endogenously expressing connexins Cx32 and Cx43, were used to model the process of hepatic IR injury. Suppression of gap junction activity was achieved genetically, using Cx32-specific small interfering RNA (siRNA), or chemically, with pharmacological inhibitors, oleamide, and 18-α-GA. BRL-3A cells subjected to H/R exhibited reduced cell survival and pathologies indicative of IR injury. Cx32-specific siRNA, oleamide, and 18-α-GA, respectively, decreased gap junction permeability, as assessed by the parachute assay. Pretreatment with Cx32-specific siRNA increased cell survival. Pretreatment with oleamide or 18-α-GA did not improve cell survival. Modulating gap junction by Cx32 gene silencing protected BRL-3A liver cells from H/R.

Lycium barbarum polysaccharides (LBP) suppresses hypoxia/reoxygenation (H/R)-induced rat H9C2 cardiomyocytes pyroptosis via Nrf2/HO-1 signaling pathway.

This study aimed to explore the mechanism that Lycium barbarum polysaccharides (LBP) suppress hypoxia/reoxygenation (H/R)-caused pyroptosis in cardiomyocytes (H9C2) via the Nrf2/HO-1 pathway. Initially, we established the cell model of H/R (6 h hypoxia plus with 24 h reoxygenation), and found that 90 µg/mL LBP was the optimal concentration. Subsequently, we confirmed that LBP reduced the pyroptosis rate of cells after H/R, the activity of LDH, the inflammatory factors IL-1ß and IL-18, and the levels of pyroptosis-specific markers ASC, NLRP3, and Caspase-1 (mRNAs and proteins). It increased the cell survival rate and the mRNA levels of the Nrf2/HO-1 pathway markers Nrf2 and HO-1, and allowed cytoplasmic Nrf2 protein to enter the nucleus to activate HO-1 protein. The Nrf2 siRNA2 caused the following events in H/R model: (1) the increases of the apoptosis rate, LDH activity, the levels of inflammatory factors (IL-1ß and IL-18), the levels of ACS, NLRP3, and Caspase-1 (mRNAs and proteins); and (2) the decreases of the cell survival rate, the mRNA levels of Nrf2 and HO-1, and the protein levels of cytoplasm-Nrf2, nucleus-Nrf2, and HO-1. Therefore we concluded that 90 µg/mL LBP suppressed H/R-induced H9C2 cardiomyocyte pyroptosis via the Nrf2/HO-1 pathway.

KLF15 alleviates oxidative stress and apoptosis of H/R-induced trophoblast cells to improve invasion and migration capacity via the activation of IGF1R.

BACKGROUND: Krüppel-like factor 15 (KLF15) has been reported to be involved in ischemia injury of multiple types of diseases. Nevertheless, the roles and underlying mechanisms of KLF15 in preeclampsia (PE) are still unclear. METHODS: In this study, the expression of KLF15 in placenta tissues and hypoxia/reoxygenation (H/R)-induced HTR8/SVneo cells was evaluated by GSE66273 database, qRT-PCR and western blot assay. CCK-8 assay was employed to detect cell proliferation. Wound healing assay and transwell assay were used to detect cell migration and invasion. Cell oxidative stress was measured by DCFH-DA staining and kits. Cell apoptosis was evaluated by TUNEL assay and western blot assay. The JASPAR database was used to analyze the binding site of KLF15 and insulin-like growth factor-1 receptor (IGF1R) promoter region. The luciferase reporter assay was used to detect IGF1R promoter activity and ChIP assay was used to verify the combination of KLF15 and IGF1R promoter. Moreover, western blot was employed to measure the expressions of PI3K/Akt-related proteins. RESULTS: The data showed that the expression of KLF15 was significantly downregulated in GSE66273 database, tissues and HTR8/SVneo cells. KLF15 overexpression increased H/R-induced HTR8/SVneo cell proliferation, invasion and migration, and inhibited oxidative stress and cell apoptosis. In addition, IGF1R was highly expressed in H/R-induced HTR8/SVneo cells after KLF15 overexpression, and the binding of KLF15 and IGF1R promoter was verified. Silencing of IGF1R reversed the effects of KLF15 overexpression on H/R-induced HTR8/SVneo cell proliferation, migration, invasion, oxidative stress and cell apoptosis. Moreover, KLF15 overexpression and IGF1R silencing regulated the expressions of PI3K/Akt-related proteins in H/R-induced HTR8/SVneo cells. CONCLUSION: In conclusion, KLF15 overexpression promoted the proliferation and metastasis, and suppressed oxidative stress and cell apoptosis of H/R-induced HTR8/SVneo cells through mediating the PI3K/Akt pathway, which may provide a promising target for the treatment of preeclampsia.

Stachydrine ameliorates hypoxia reoxygenation injury of cardiomyocyte via enhancing SIRT1-Nrf2 pathway.

BACKGROUND: Hypoxia/reoxygenation (H/R)-induced cardiomyocyte cell apoptosis is critical in developing myocardial infarction. Stachydrine (STA), an active constituent of Leonurus heterophyllus sweet, could have a protective effect on myocardial H/R injury, which remains unexplored. Therefore, the study aimed to investigate the protective effects and mechanisms of STA on H/R injury of cardiomyocytes. METHODS: Rat cardiomyocyte H9c2 cells underwent H/R (hypoxia for 4 h and reoxygenation for 12 h). Cells were pretreated with STA (50 µM) 2 h before H/R. Cardiomyocyte injury was evaluated by CCK-8 assay and lactate dehydrogenase (LDH) release. Apoptosis was assessed by TUNEL staining and caspase-3 activity. Oxidative stress was assessed by lipid oxidation product MDA and a ROS-scavenging enzyme SOD in culture media. Western blot was performed to measure the protein expressions of SIRT1, Nrf2, and heme oxygenase-1 (HO-1). RESULTS: STA reversed the decrease in cell viability and increased LDH release in H9c2 cells with the H/R insult. STA significantly suppressed oxidative stress, reduced MDA content, and increased SOD activity in H9c2 cells exposed to H/R. STA reduced apoptosis in H9c2 cells exposed to H/R, as evidenced by the reduced TUNEL positive cells and caspase-3 activity. In addition, STA enhanced SIRT1, Nrf2, and HO-1 protein expression in H/R-stimulated H9c2 cells. SIRT1 and Nrf2 involved the protective effect of STA in H/R-exposed H9c2 cells, as the changes in cell viability and caspase-3 activity by STA can be reversed by SIRT1 inhibitor EX-527 or Nrf2 siRNA. CONCLUSIONS: Our data speculated that STA protects H/R injury and inhibits oxidative stress and apoptosis in cardiomyocytes by activation of the SIRT1-Nrf2 pathway.

MiR-183-5p overexpression in bone mesenchymal stem cell-derived exosomes protects against myocardial ischemia/reperfusion injury by targeting FOXO1.

OBJECTIVE: Exosomes have been suggested to serve as possible drug delivery vehicles due to their nanometer-size range and capability of transferring biological materials to recipient cells. Thus, whether miR-183-5p-overexpressing bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) could protect against myocardial ischemia/reperfusion (MI/R) injury by targeting FOXO1 was investigated. METHODS: Exosomes were isolated from rat BMSCs, and ischemia/reperfusion (I/R) rat models were established. I/R rats were treated with Exo/NC-Exo/miR-183-5p-Exo/anti-miR-183-5p-Exo. Cardiac function, serum biochemical indices, apoptosis, myocardial infarction size, and the expression of miR-183-5p, FOXO1 and cleaved caspase 3 were assessed. Primary cardiomyocytes were isolated to establish hypoxia/reoxygenation (H/R) models to observe the function of miR-183-5p-Exo in vitro. RESULTS: Rats in the I/R group exhibited a decreased left ventricular ejection fraction (LVEF), left ventricular fraction shortening (LVFS) and left ventricular systolic pressure (LVSP) but an increased left ventricular end-diastolic pressure (LVEDP), myocardial infarct size and apoptosis index (AI). In addition, in I/R rats, miR-183-5p expression was decreased, but FOXO1 and cleaved caspase 3 expression was increased. Both Exo and miR-183-5p-Exo improved the above indices in I/R rats, but miR-183-5p-Exo showed better effects. However, anti-miR-183-5p-Exo reversed the protective effect of Exo. FOXO1 was a target gene of miR-183-5p. Experiments in vitro revealed that Exo and miR-183-5p-Exo suppressed apoptosis and oxidative stress injury in H/R-induced cardiomyocytes, whereas overexpressed FOXO1 reversed the protective role of miR-183-5p-Exo. CONCLUSION: BMSC-derived exosomal miR-183-5p could target FOXO1 to reduce apoptosis and oxidative stress in I/R cardiomyocytes and improve cardiac function, thereby protecting against MI/R injury.

MiR-181c protects cardiomyocyte injury by preventing cell apoptosis through PI3K/Akt signaling pathway.

BACKGROUND: Cardiomyocyte apoptosis plays an important role in the development of heart failure, which leads to high mortality in patients with cardiovascular diseases. In this study, we are focused to identify the role of miRNA-181c in the regulating of myocardial tissue apoptosis in the doxorubicin (DOX) or hypoxia/reoxygenation (H/R) induced H9C2 cardiomyocyte injury. METHODS: DOX-induced heart failure animal model was established using mice. Total RNA was extracted from tissue and cell using Trizol. RT-PCR was conducted for real-time RNA quantification. H9c2 cells were collected and labeled using an Annexin V-PI apoptosis kit. Flow cytometry was conducted to identify the cell apoptosis. Rat cardiomyocyte H9c2 cell was treated by 16 hours' hypoxia and 2 hours' reoxygenation to induce cell apoptosis. TUNEL assay was employed for myocardial tissue apoptosis analysis. RESULTS: It was revealed that miR-181c was suppressed on the heart tissue of DOX-induced heart failure animal model. We observed miR-181c overexpression reduced apoptosis through TUNEL assay, which suggested the inhibitory effect of miR-181c on myocardial tissue apoptosis. Transfection of miR-181c mimic could decrease cell apoptosis in H/R treated H9C2 cells in vitro. Under the stimulation of H/R or DOX, miR-181c could downregulate protein expression of Fas, IL-6 and TNF-α, and upregulated Bcl2 and the phosphorylation of Akt. CONCLUSIONS: Our study revealed that miR-181c protected heart failure by impeding cardiomyocyte apoptosis through PI3K/Akt pathway, implying the therapeutic role of miR-181c during the exacerbation of the cardiovascular disease.

MiR-137-3p exacerbates the ischemia-reperfusion injured cardiomyocyte apoptosis by targeting KLF15.

Ischemia-reperfusion (I/R) injury is a kind of the tissue damage caused by an abrupt re-supplying tissue with blood after a period of ischemia or hypoxia. It contributes to a wide range of pathological processes including kidney injury, circulatory arrest, and especially cardiovascular disease. However, the underlying pathological mechanism is not fully elucidated. Previously, extensive studies demonstrated that miRNAs participate in the pathogenesis of I/R injury, such as I/R-induced cardiomyocyte apoptosis. Here, we found that miR-137-3p, a mature form of miR-137, was up-regulated in I/R-injured cardiomyocytes of myocardial infarction patients. Deficiency of miR-137-3p partly alleviated the cardiomyocyte apoptosis and oxidative stress induced by hypoxia-reoxygenation (H/R) treatment in H9c2 cells. Also, we provided evidences that miR-137-3p directly targeted the 3' UTR of KLF15 mRNA to down-regulate its expression, and loss function of KLF15 significantly abolished the deleterious effects of ectopic miR-137-3p on cardiomyocytes both in vitro and in vivo. Collectively, these observations highlight a molecular perturbation in the pathogenesis of I/R injury in cardiomyocytes.

Nobiletin suppresses oxidative stress and apoptosis in H9c2 cardiomyocytes following hypoxia/reoxygenation injury.

Nobiletin (3',4',5,6,7,8-hexamethoxyflavone), a dietary polymethoxylated flavonoid found in Citrus fruits, was reported to exhibit protective activity against ischemia/reperfusion (I/R). However, the role of nobiletin in myocardial I/R injury remains unclear. This study was designed to examine the cardioprotective effect of nobiletin from myocardial hypoxia/reoxygenation (H/R) injury in vitro, and to explore the potential molecular mechanisms. Our results showed that nobiletin improved cell viability in H9c2 cells after H/R treatment. In addition, nobiletin significantly inhibited the production of reactive oxygen species and malondialdehyde (MDA), cell apoptosis, as well as suppressed the levels of pro-inflammatory factors in H/R-stimulated H9c2 cells. Furthermore, we observed that pretreatment with nobiletin significantly activated the Akt/GSK-3ß signaling pathway in H/R-stimulated H9c2 cells. Taken together, these findings demonstrated that nobiletin attenuates myocardial I/R injury via the activation of Akt/GSK-3ß pathway in H9c2 cardiomyocytes. Thus, nobiletin may be regarded as a promising drug for the prevention of myocardial I/R injury and ischemic heart disease.

Rutin alleviates hypoxia/reoxygenation-induced injury in myocardial cells by up-regulating SIRT1 expression.

Rutin possesses multiple pharmacological activities, including the cardioprotective effect. The present study aimed to evaluate the protective effects of rutin on hypoxia/reoxygenation (H/R)-induced myocardial injury and its underlying mechanism involved. H9c2 cells were pretreated with 50 µM rutin or combined with 1 µM silent information regulator 1 (SIRT1) inhibitor (EX-527) for 1 h, and subjected to hypoxia for 6 h, followed by reoxygenation for 24 h. SIRT1 expression was detected by qRT-PCR and western blot. The effects of rutin or combined with EX-527 on cell viability, myocardial injury, apoptotic rate, and oxidative stress in H/R-stimulated H9c2 cells were assayed. The results showed that rutin elevated SIRT1 expression in H9c2 cells, as well as H/R-stimulated H9c2 cells. Rutin increased cell viability in H9c2 cells exposed to H/R. H/R stimulation induced myocardial injury, as evidenced by the increased levels of lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB) and aspartate transaminase (AST), which were abolished in the presence of rutin. Rutin attenuated H/R-induced increase of apoptotic rate and caspase-3 activity in H/R-treated cells. Moreover, H/R-induced decrease in the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and increase in malondialdehyde (MDA) content were reversed by rutin treatment. The presence of EX-527 abolished these protective effects of rutin. In conclusion, rutin protected H9c2 cells against H/R injury through increasing SIRT1 expression. Our findings suggested that rutin might be a potential therapeutic agent for the treatment of myocardial H/R injury.

Teneligliptin protects against hypoxia/reoxygenation-induced endothelial cell injury.

Cardiovascular complications are the main causes of mortality in diabetic patients. Teneligliptin is a newly developed anti-diabetic agent. It has been reported that teneligliptin has a vascular protective capacity in preclinical studies and diabetes patients. In this study, we investigated the effect of teneligliptin on hypoxia/reoxygenation (H/R)-induced endothelial cell injury in rat cardiac microvascular endothelial cells (CMECs). We showed that teneligliptin pretreatment suppressed H/R-induced production of reactive oxygen species (ROS), NADPH oxidase 4 (NOX4) expression and promoted glutathione production. Teneligliptin pretreatment reduced H/R-induced LDH release and improved cell viability. Teneligliptin significantly relieved the reduction in mitochondrial membrane potential (MMP) induced by H/R. Moreover, teneligliptin suppressed H/R-induced cytokine production and production of vascular adhesion molecules such as IL-1ß, TNF-α and ICAM-1. Mechanistically, we showed that teneligliptin inhibited the expression of transcriptional factor Egr-1, which regulates cytokine production and vascular adhesion. Collectively, our data support the notion that teneligliptin is a protective agent in CMECs and has the potential for therapeutic use in the treatments of vascular complications in diabetes patients.

Hesperetin post-treatment prevents rat cardiomyocytes from hypoxia/reoxygenation injury in vitro via activating PI3K/Akt signaling pathway.

Hesperidin (HES), a citrus fruit extract, has beneficial effects on various ischemia/reperfusion (I/R) models. Here, we investigated the possible positive effect of hesperetin (HPT), an active metabolite of HES, and identified the potential molecular mechanisms involved in cardiomyocytes H/R-induced injury. To construct the cardiomyocyte model of hypoxia/reoxygenation (H/R) injury, cultured neonatal rat cardiomyocytes were subjected to 3h of hypoxia followed by 3h of reoxygenation. Cell viability and apoptosis were detected. The levels of Apoptosis-related proteins and PI3K/Akt proteins were detected by western blot. Our results showed that HPT post-treatment significantly inhibited apoptosis by elevating the expression of Bcl-2, decreasing the expression of Bax and cleaved caspase-3, and diminished the apoptotic cardiomyocytes ratio. Mechanism studies demonstrated that HPT post-treatment up-regulated the expression levels of p-PI3K, and p-Akt. Co-treatment of the cardiomyocytes with the PI3K/Akt-specific inhibitor LY294002 blocked the HPT-induced cardioprotective effects. Taken together, these data suggested that HPT post-treatment prevented cardiomyocytes from H/R injury in vitro most likely through the activation of PI3K/Akt signaling pathway.