Apoptosis In H9c2 Cells Research Articles

The mechanism of miR-525-5p derived from hypoxia and reoxygenation in H9c2 Cardiomyocytes.

Ischemia-reperfusion injury (IRI) is associated with ischemic heart disease (IHD) which leads to patients a poor progression. According to Pubmed Datasets, we analyzed different gene and mRNA expressions in IHD patients with IRI. The relevant mRNA expression detected in H9C2 cells undergo hypoxia and reoxygenation, we selected and structured miR-525-5p gene mutation H9C2 cells, the results performed miR-525-5p mutated restored H9C2 metabolism of mitochondria which detected by relevant genes and proteins. At the same time, miR-525-5p silence resisted hypoxia and reoxygenation induced H9C2 cells apoptosis. All the results indicated miR-525-5p maybe protect H9C2 cells without hypoxia and reoxygenation induced injury through regulating the mitochondria metabolism.

ITGB1 promotes apoptosis in H9C2 cells

Myocardial infarction is a heart disease with high mortality rate. The research on the cardiomyocyte apoptosis is helpful to the prevention and treatment of myocardial infarction. As a cell surface receptor, the function of ITGB1 gene in cardiomyocytes is not clear. This study aimed to analyze the function and molecular mechanism of cardiomyocyte derived ITGB1 in myocardial apoptosis. To analyze cardiomyocyte apoptosis, TUNEL staining was performed with ITGB1 overexpression and knockdown in H9C2 cells. Apoptosis-related markers were also detected using Western blot. In addition, apoptosis-related signaling pathways were detected to elucidate the molecular mechanism regulated by ITGB1. Our results indicated that ITGB1 promoted cardiomyocyte apoptosis by inhibiting the Notch1 and Smad2/3 signal pathways. These data highlight the potential usefulness of ITGB1 as a potential target for preventing myocardial infarction.

MicroRNA-18-5p inhibits the oxidative stress and apoptosis of myocardium induced by hypoxia by targeting RUNX1.

Acute myocardial infarction (AMI) is the main cause of sudden death in the world. The aim of this paper was to explore the role of microRNA-18-5p (miR-18-5p) in myocardial infarction (MI) and its potential mechanism. The expression of miRNA and protein was detected using quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis and Western blot. The level of oxidative stress of cardiomyocytes was evaluated by detecting the contents of Superoxide dismutase (SOD), Reactive oxygen species (ROS) and Malondialdehyde (MDA). Caspase-3 activity assay, flow cytometry and TUNEL staining were employed to evaluate apoptosis of myocardium. Dual-Luciferase reporter gene assay was used to prove whether miR-18-5p targeted RUNX1. MiR-18-5p was down-regulated in hypoxia-treated H9c2 cells. Hypoxia treatment induced oxidative stress and apoptosis of H9c2 cells. The oxidative stress of H9c2 was manifested by the decrease of SOD activity, the increase of ROS and MDA levels, and the apoptosis of H9c2 was shown by the increase of caspase-3 activity and apoptosis rate. MiR-18-5p mimic was transfected into H9c2 cells and successfully up-regulated miR-18-5p. And overexpression of miR-18-5p markedly inhibited the oxidative stress and apoptosis caused by hypoxia in H9c2 cells. Through bioinformatics analysis and Dual-Luciferase reporter gene assay, RUNX1 was proved to have binding sites for miR-18-5p. Furthermore, knocking down RUNX1 using small interfering RNA-RUNX1 (siR-RUNX1) significantly protected H9c2 cells from oxidative stress and apoptosis. MiR-18-5p expression was decreased in hypoxia-treated H9c2 cells. Overexpression of miR-18-5p alleviated hypoxia-induced oxidative stress and apoptosis in H9c2 cells via targeting RUNX1.

RETRACTED ARTICLE: Propofol inhibits oxidative stress injury through the glycogen synthase kinase 3 beta/nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway

Statement of Retraction Ziyin Zhan, BaoFeng Yan, Yuguo Li, Shuo Yang, and Jinfeng Li- 2022, Propofol inhibits oxidative stress injury through the glycogen synthase kinase 3 beta/nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signaling pathway, Bioengineered 13(1), doi: 10.1080/21655979.2021.2021062 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

LncRNA FGD5-AS1 acts as a ceRNA to regulate lipopolysaccharide-induced injury via the miR-223-3p-3p/GAS5 axis in cardiomyocytes.

Long noncoding RNAs (lncRNAs) are abnormally expressed in numerous diseases, and they are closely associated with cardiac diseases. However, the role of lncRNAs in lipopolysaccharide (LPS)-induced cardiotoxicity as well as the potential mechanism remain largely unclear. In the present study, IncRNA microarray assays were performed to analyze differential lncRNA expression in LPS-treated cardiomyocytes, and lncRNA FGD5-AS1 was one of the downregulated lncRNAs. H9C2 cells were treated with LPS, and the expression of lncRNA FGD5-AS1 was markedly downregulated. LncRNA FGD5 overexpression decreased the LPS-induced cardiomyocyte apoptosis and inflammation. Bioinformatics analysis and a luciferase reporter assay indicated that lncRNA FGD5-AS1 directly binds to miR-223-3p. A miR-222-3p mimic partially reversed the inhibitory effect of lncRNA FGD5-AS1 on the LPS-induced H9C2 cell apoptosis and inflammatory response. Moreover, miR-223-3p directly targeted growth arrest-specific transcript 5 (GAS5). LncRNA FGD5-AS1 regulated LPS-induced H9C2 cell inflammation and apoptosis via the miR-223-3p/GAS5 axis.

Hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen–glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy

In this study, we investigated the protective effect of hyperbaric oxygen (HBO) on PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion and its possible mechanism. PC12 and H9C2 cell oxygen-glucose deprivation/reperfusion model were established. Cells were divided into a control group, model group, hyperbaric air (HBA) group and HBO group. The cell viability was detected by the CCK8 assay. Hoechst 33342 and PI staining assays and mitochondrial membrane potential (MMP) assays were used to detect cell apoptosis. The ultrastructure of cells, including autophagosomes, lysosomes, and apoptosis, were examined using a transmission electron microscope. The expression of autophagy-related proteins was detected by cellular immunofluorescence and immunocytochemistry. Our results showed that HBO can significantly improve the vitality of damaged PC12 and H9C2 cells caused by oxygen–glucose deprivation/reperfusion. HBO can significantly inhibit apoptosis of PC12 and H9C2 cells caused by oxygen-glucose deprivation/reperfusion. Importantly, we found that the protective mechanism of PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion may be related to the inhibition of the autophagy pathway. In this study, the results of cellular immunofluorescence and immunocytochemistry experiments showed that the 4E-BP1, p-AKt and mTOR levels of PC12 and H9C2 cells in the model group decreased, while the levels of LC3B, Atg5 and p53 increased. However, after HBO treatment, these autophagy-related indexes were reversed. In addition, observation of the cell ultrastructure with transmission electron microscopy found that in the model group, a significant increase in the number of autophagic vesicles was observed. In the HBO group, a decrease in autophagic vesicles was observed. The study demonstrated that hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy.

Knockdown of hypoxia-inducible factor 1-alpha (HIF1α) interferes with angiopoietin-like protein 2 (ANGPTL2) to attenuate high glucose-triggered hypoxia/reoxygenation injury in cardiomyocytes

ABSTRACT To investigate the role of hypoxia-inducible factor 1-alpha (HIF1A) in hypoxia/reoxygenation (H/R) injury of cardiomyocytes induced by high glucose (HG). The in vitro model of coronary heart disease with diabetes was that H9c2 cells were stimulated by H/R and HG. Quantitative reverse transcription PCR (RT-qPCR) and Western blot analysis were used to detect the expression of HIF1A and angiopoietin-like protein 2 (ANGPTL2) in H9c2 cells. Cell viability and apoptosis were, respectively, estimated by Cell Counting Kit 8 (CCK-8) and TUNEL assays. Lactate dehydrogenase (LDH) activity, inflammation and oxidative stress were in turn detected by their commercial assay kits. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to confirm the association between HIF1A and ANGPTL2 promoter. The expression of nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway-related proteins and apoptosis-related proteins were also detected by Western blot analysis. As a result, ANGPTL2 expression was upregulated in H9c2 cells induced by HG or/and H/R. ANGPTL2 positively modulated HIF1A expression in H9c2 cells. HG or/and H/R suppressed the cell viability and promoted apoptosis, inflammatory response and oxidative stress levels in H9c2 cells. However, the knockdown of ANGPTL2 could reverse the above phenomena in H/R-stimulated-H9c2 cells through activation of Nrf2/HO-1 pathway. HIF1A transcriptionally activated ANGPTL2 expression. The effect of knockdown of ANGPTL2 on H/R triggered-H9c2 cells was weakened by HIF1A overexpression. In conclusion, knockdown of HIF1A downregulated ANGPTL2 to alleviate H/R injury in HG-induced H9c2 cells by activating the Nrf2/HO-1 pathway.

KLF7 promotes LPS induced apoptosis, inflammation, and oxidative stress in H9c2 cells by activating NF-κB pathway

KLF7 promotes LPS induced apoptosis, inflammation, and oxidative stress in H9c2 cells by activating NF-κB pathway

Arsenic trioxide-induced cardiotoxicity triggers ferroptosis in cardiomyoblast cells.

Arsenic trioxide (ATO) has been found to be effective in acute promyelocytic leukemia. However, ATO-induced severe cardiotoxicity limits its clinical application. To date, the mechanisms of ATO-induced cardiotoxicity remain unclear. It is hypothesized that ferroptosis may trigger ATO-induced cardiotoxicity; however, this has not yet been investigated. To clarify this hypothesis, rat cardiomyocyte H9c2 cells were treated with ATO with or without ferrostain-1 (Fer-1). The results indicated that ATO exposure induced H9c2 cell death and apoptosis, and the ferroptosis inhibitor Fer-1, administered for 24h before ATO exposure, suppressed ATO-induced cell death, and apoptosis, as determined by Annexin V-APC/7-AAD apoptosis assay. Furthermore, Fer-1 displayed a cardioprotective effect through inhibiting the ATO-induced production of intracellular reactive oxygen species, improving the ATO-induced loss of the mitochondrial membrane potential, alleviating hyperactive endoplasmic reticulum stress, and alleviating the ATO-induced impairment in autophagy in H9c2 cells. Overall, the cardioprotective effect of Fer-1 against ATO-induced cell injury implies that ATO may trigger ferroptosis to induce cardiotoxicity. These findings lay the foundation for exploring the potential value of ferroptosis inhibitors against ATO-induced cardiotoxicity in the future.

Effect of Ginsenoside Rb2 on a Myocardial Cell Model of Coronary Heart Disease through Nrf2/HO-1 Signaling Pathway.

The ginsenoside Rbs are the primary active compounds of Panax ginseng and ginsenoside Rb2 is a renowned component among the Rbs. This study aimed to investigate the potential effects of ginsenoside Rb2 on coronary heart disease (CHD). H9c2 cells were exposed to H2O2 to establish CHD model in vitro. Gene expression was determined by quantitative realtime PCR (qPCR) and Western blot. Cellular functions were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assays. We found that Ginsenoside Rb2 promoted cell proliferation while suppressed oxidative stress and apoptosis of H9c2 cells induced by H2O2 exposure. Mechanistically, Ginsenodise Rb2 involves in the regulation of nuclear factor, erythroid 2 like 2 (Nrf2)/heme oxygenase (HO)-1 signaling pathway. Inactivation of Nrf2/HO-1 signaling pathway reversed the effects of ginsenoside Rb2 on H9c2 cells. Taken together, ginsenoside Rb2 exhibited a cardioprotective effect in vitro. The underlying mechanism of ginsenoside Rb2 in H9c2 cells could be standardized to Nrf2/HO-1 signaling pathway, inhibiting cell apoptosis and regaining cell proliferation. The present study has proposed a novel mechanism of ginsenoside Rb2 in the cardioprotective effect.

RETRACTED ARTICLE: Dexmedetomidine attenuates ischemia and reperfusion-induced cardiomyocyte injury through p53 and forkhead box O3a (FOXO3a)/p53-upregulated modulator of apoptosis (PUMA) signaling signaling

Statement of Retraction Feng Yun Yang, Lu Zhang, Yan Zheng, He Dong- 2022, Dexmedetomidine attenuates ischemia and reperfusion-induced cardiomyocyte injury through p53 and forkhead box O3a (FOXO3a)/p53-upregulated modulator of apoptosis (PUMA) signaling signaling, Bioengineered 13(1), doi: 10.1080/21655979.2021.2017611 Since publication, significant concerns have been raised about the integrity of the data and reported results in the article. When approached for an explanation, the authors did not provide their original data or any necessary supporting information. As verifying the validity of published work is core to the integrity of the scholarly record, we are therefore retracting the article. All authors listed in this publication have been informed. The authors have agreed to retract the article. Moreover, the authors have subseqeuntly informed us that they identified a further issue and requested retraction. We have been informed in our decision-making by our editorial policies and the COPE guidelines. The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.

Upregulation of serum and glucocorticoid-regulated kinase 1 (SGK1) ameliorates doxorubicin-induced cardiotoxic injury, apoptosis, inflammation and oxidative stress by suppressing glucose regulated protein 78 (GRP78)-mediated endoplasmic reticulum stress

ABSTRACT The clinical application of doxorubicin (Dox) in tumor chemotherapy is limited by time-dependent and dose-dependent cardiotoxicity. Hence, there is an urgent need to elucidate doxorubicin cardiotoxicity and to solve the difficult problem in clinical application. It has been verified that serum and glucocorticoid-regulated kinase 1 (SGK1) possess cardioprotective effects. Here, H9c2 cells were treated with 1 μM doxorubicin for 24 h to establish doxorubicin cardiotoxicity, so as to determine the biological role of SGK1 in doxorubicin cardiomyopathy and to elucidate the underlying molecular mechanism. SGK1 level in doxorubicin-treated H9c2 cells was assessed by performing Western blot assay and RT-qPCR. CCK-8 assay and TUNEL staining were employed to evaluate the cell viability and cell apoptosis. Besides, apoptosis-related proteins were measured by Western blot assay to analyze cell apoptosis. Additionally, the release of TNF-α, IL-1β, IL-6, and IL-10 and the levels of ROS, MDA, and SOD were detected to reflect inflammation and oxidative stress. Moreover, Western blot assay was adopted for determination of ERS-associated proteins. Results revealed that SGK1 was downregulated in doxorubicin-treated H9c2 cells. Upregulation of SGK1 alleviated doxorubicin-induced cardiotoxic injury, cell apoptosis, inflammation and oxidative stress in H9c2 cells. Moreover, SGK1 overexpression mitigated doxorubicin-induced ERS in H9c2 cells. The suppressing effects of SGK1 on doxorubicin-induced cardiotoxic injury, apoptosis, inflammation, oxidative stress and ERS in H9c2 cells were partially abolished upon GRP78 overexpression. To conclude, upregulation of SGK1 may alleviate doxorubicin cardiotoxicity by repressing GRP78-mediated ERS.

MiR-21-5p-expressing bone marrow mesenchymal stem cells alleviate myocardial ischemia/reperfusion injury by regulating the circRNA_0031672/miR-21-5p/programmed cell death protein 4 pathway.

BACKGROUNDFor patients with coronary heart disease, reperfusion treatment strategies are often complicated by ischemia/reperfusion (I/R) injury (IRI), leading to serious organ damage and malfunction. The miR-21/programmed cell death protein 4 (PDCD4) pathway is involved in the IRI of cardiomyocytes; however, the aberrant miR-21 expression remains unexplained. Therefore, this study aimed to explore whether circRNA_0031672 downregulates miR-21-5p expression during I/R and to determine whether miR-21-5p-expressing bone marrow mesenchymal stem cells (BMSCs) reduce myocardial IRI.METHODSCircRNA_0031672, miR-21-5p, and PDCD4 expressions were evaluated in the I/R rat model and hypoxia/re-oxygenation (H/R)-treated H9C2 cells. Their interactions were subsequently investigated using luciferase reporter and RNA pulldown assays. Methyltransferase-like 3, a methyltransferase catalyzing N6-methyladenosine (m6A), was overexpressed in H9C2 cells to determine whether m6A modification influences miR-21-5p targeting PDCD4. BMSCs stably expressing miR-21 were co-cultured with H9C2 cells to investigate the protective effect of BMSCs on H9C2 cells upon H/R.RESULTSI/R downregulated miR-21-5p expression and upregulated circRNA_0031672 and PDCD4 expressions. CircRNA_0031672 knockdown increased miR-21-5p expression, but repressed PDCD4 expression, indicating that circRNA_0031672 competitively bound to miR-21-5p and prevented it from targeting PDCD4 mRNA. The m6A modification regulated PDCD4 expression, but had no effect on miR-21-5p targeting PDCD4. The circRNA_0031672/miR-21-5p/PDCD4 axis regulated myocardial cells viability and apoptosis after H/R treatment; co-culture with miR-21-5p-expressing BMSCs restored miR-21-5p abundance in H9C2 cells and further reduced H9C2 cells apoptosis induced by H/R.CONCLUSIONSWe identified a novel circRNA_0031672/miR-21-5p/PDCD4 signaling pathway that mediates the apoptosis of cardiomyocytes and successfully alleviates IRI in myocardial cells by co-culture with miR-21-5p-expressing BMSCs, offering novel insights into the IRI pathogenesis in cardiovascular diseases.

Cardiotoxicity of patulin was found in H9c2 cells

Patulin (PAT) is a kind of mycotoxins that is universally found at rotten fruits, especially apples and apple products. Previous studies have shown that PAT has hepatotoxicity and nephrotoxicity. However, cardiotoxicity of PAT is rarely reported. Present study aimed at investigate the cardiotoxicity and relevant mechanisms of PAT on H9c2 cells. Cytotoxicity of PAT were evaluated by MTT assay and LDH. Hoechst 33258 staining was used to examine the nuclear morphology and AV/PI double staining was employed for apoptosis on H9c2 cells. Expression level of Caspase-3, Caspase-9, Bax, Bcl-2 were quantified to verify the potential mechanism of mitochondrial apoptosis pathway. The tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and interleukin 6 (IL-6) were quantified to determine the inflammatory response by using ELISA assay. ROS, SOD, MDA, GSH levels were measured to determine the oxidative stress status. Results demonstrated that PAT significantly induced cell injury, as evidenced by the down-regulated of cell viability, and the increase of LDH release. Hoesst33258 staining and flow cytometry showed that apoptosis rate was elevated by PAT. PAT treatment up-regulated the expression of Caspase-3, Caspase-9, Bax level and down-regulated the expression of Bcl-2 level. TNF-α, IL-1β, IL-6 levels showed that PAT increased the pro-inflammatory response. As PAT concentration increased, intracellular MDA, ROS content were elevated, while GSH content and the activity of SOD were significantly decreased. Thus, it is concluded that PAT may induce apoptosis of H9c2 cells through oxidative stress.

Network Pharmacology-Based Investigation and Experimental Exploration of the Antiapoptotic Mechanism of Colchicine on Myocardial Ischemia Reperfusion Injury.

Background: The beneficial effects of colchicine on cardiovascular disease have been widely reported in recent studies. Previous research demonstrated that colchicine has a certain protective effect on ischemic myocardium and has the potential to treat myocardial ischemia reperfusion injury (MIRI). However, the potential targets and pharmacological mechanism of colchicine to treat MIRI has not been reported. Methods: In this study, we used network pharmacology and experimental verification to investigate the pharmacological mechanisms of colchicine for the treatment of MIRI. Potential targets of colchicine and MIRI related genes were screened from public databases. The mechanism of colchicine in the treatment of MIRI was determined by protein-protein interaction (PPI), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Additionally, we evaluated the effect of colchicine on H9C2 cell activity using CCK-8 assays, observed the effect of colchicine on H9C2 cell apoptosis via flow cytometry, and further verified the expression of key targets after colchicine treated by Western blot. Results: A total of 626 target genes for colchicine and 1549 MIRI disease targets were obtained. 138 overlapping genes were determined as potential targets of colchicine in treating MIRI. the PPI network analysis demonstrated that the targets linked to MIRI were ALB, TNF, ACTB, AKT1, IL6, TP53, IL1B, CASP3 and these targets showed nice affinity with colchicine in molecular docking experiments. The results of GO analysis and KEGG pathway enrichment demonstrated that the anti-MIRI effect of colchicine involves in apoptotic signaling pathway. Further tests suggested that colchicine can protect H9C2 cell from Hypoxia/Reoxygenation (H/R) injury through anti-apoptotic effects. Western blot results demonstrated that colchicine can inhibited MIRI induced apoptosis of H9C2 cell by enhancing the decreased levels of Caspase-3 in myocardial injure model induced by H/R and activating the PI3K/AKT/eNOS pathway. Conclusions: we performed network pharmacology and experimental evaluation to reveal the pharmacological mechanism of colchicine against MIRI. The results from this study could provide a theoretical basis for the development and clinical application of colchicine.

Galectin‑1 alleviates myocardial ischemia‑reperfusion injury by reducing the inflammation and apoptosis of cardiomyocytes

Myocardial ischemia-reperfusion injury (MIRI) is one of the leading causes of morbidity and mortality worldwide, for which there is no effective treatment. The present study aimed to assess novel methods of clinical MIRI treatment by studying the effects of galectin-1 (Gal-1) on MIRI. Male 2-month-old Sprague Dawley rats and the rat cardiomyocyte cell line H9c2 were utilized in the present study. A rat model of MIRI was constructed by ligating the left anterior descending coronary artery, which was subsequently treated with Gal-1. Differences in myocardial injury were then assessed by hematoxylin and eosin (H&E) staining. In addition, the levels of inflammation and apoptosis in rat myocardial tissue were determined by immunohistochemistry staining. Hypoxia-reoxygenation was used to construct a model of MIRI in H9c2 cells. The effect of Gal-1 on the apoptosis and viability of H9c2 cells was also verified by flow cytometry and a Cell Counting Kit-8 assay. The results of H&E staining revealed that Gal-1 alleviated MIRI. Echocardiography demonstrated that Gal-1 improved cardiac function in rats following MIRI. In addition, MIRI increased levels of inflammation and apoptosis in rat myocardial tissues, with Gal-1 treatment reversing this effect. In cellular experiments, Gal-1 served anti-inflammatory and anti-apoptotic effects in hypoxic/reoxygenated cardiomyocytes. In conclusion, Gal-1 served a significant protective effect on the myocardial tissue after ischemia-reperfusion by reducing the level of inflammation and apoptosis in cardiomyocytes.

Substance P prevents doxorubicin‑induced cardiomyocyte injury by regulating apoptosis and autophagy: Invitro and invivo evidence.

The function of substance P (SP) in myocardial ischemia is well understood, but its effects on congestive heart failure are unclear. The present study aimed to use in vitro and in vivo approaches to investigate the effects of SP on doxorubicin-induced cardiomyocyte injury. Pathological changes, apoptosis, cardiomyocyte ultrastructure and molecular mechanisms were evaluated in vitro and in vivo. The effects of SP on cell viability of H9c2 myocardial cells were evaluated using the Cell Counting Kit-8 and flow cytometry. B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Beclin-1 and microtubule-associated protein 1A/1B-light chain 3 (LC3) were detected by western blotting. Heart failure in rats was established by intraperitoneal injection of doxorubicin. The in vitro data demonstrated that SP at concentrations of 1 µg/ml inhibited doxorubicin-induced apoptosis of H9c2 cells. Administration of doxorubicin reduced Bcl-2, Beclin-1 and LC3 expression levels in H9c2 cells, while having no effect on Bax levels. Administration of SP to these doxorubicin-treated cells did not affect Bcl-2 or Bax expression, but further reduced Beclin-1 while inhibiting the reduction in LC3 expression. In vivo, food intake was significantly increased in rats in the SP group compared with the model group. Cardiomyocytes in the heart-failure group underwent dysfunctional autophagy as ascertained by transmission electron microscopy. Compared with the heart-failure group, these pathological changes, including loss of striations and vacuolation, were inhibited by SP treatment, which promoted Bax expression, reduced Beclin-1 expression and inhibited the reduction in LC3 expression. Taken together, SP reduced cardiomyocyte apoptosis in doxorubicin-induced cardiomyocyte injury, likely by promoting autophagy, which suggested that SP is a potential therapeutic target for doxorubicin-induced heart failure.

Cox-2 Antagonizes the Protective Effect of Sevoflurane on Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis through Inhibiting the Akt Pathway.

Objective To uncover the protective role of sevoflurane on hypoxia/reoxygenation-induced cardiomyocyte apoptosis through the protein kinase B (Akt) pathway. Methods An in vitro hypoxia/reoxygenation (H/R) model was established in cardiomyocyte cell line H9c2. Sevoflurane (SEV) was administrated in H9c2 cells during the reoxygenation period. Viability, layered double hydroxide (LDH) release, and apoptosis in H9c2 cells were determined to assess H/R-induced cell damage. Relative levels of apoptosis-associated genes were examined. Moreover, phosphorylation of Akt was determined. Results H/R injury declined viability and enhanced LDH release and apoptotic rate in H9c2 cells. Cyclooxygenase-2 (Cox-2) was upregulated following H/R injury, which was partially reversed by SEV treatment. In addition, SEV treatment reversed changes in viability and LDH release owing to H/R injury in H9c2 cells, which were further aggravated by overexpression of Cox-2. The Akt pathway was inhibited in H9c2 cells overexpressing Cox-2. Conclusions Sevoflurane protects cardiomyocyte damage following H/R via the Akt pathway, and its protective effect was abolished by overexpression of Cox-2.

Epigallocatechin-3-gallate protects cardiomyocytes from hypoxia-reoxygenation damage via raising autophagy related 4C expression

ABSTRACT Myocardial ischemia/reperfusion (I/R) injury is a serious issue during the therapy of myocardial infarction. Herein, we explored the beneficial influence of Epigallocatechin-3-gallate (EGCG) on hypoxia/reoxygenation (H/R)-stimulated cardiomyocyte H9c2 cells damage, along with possible internal molecular mechanism related autophagy related 4C (ATG4C). H9c2 cells were subjected to H/R stimulation and/or EGCG treatment. ATG4C mRNA expression was measured via q-PCR assay. ATG4C overexpression plasmid (OE-ATG4C) was transfected to arise ATG4C level. Cell viability, apoptosis, reactive oxygen species (ROS) production, ATP level were tested via CCK-8 assay, Annexin V-FITC/PI staining, DCFH-DA staining and ATP Assay Kit, respectively. Western blotting was performed to test Cleaved-caspase 3, Cleaved-caspase 9, cytochrome C, and LC3B protein levels. H/R stimulation resulted in H9c2 cell viability loss, promoted cell apoptosis, and ROS overproduction, as well as lowered ATP level in cells. EGCG treatment alleviated H/R-resulted H9c2 cell viability loss, cell apoptosis, ROS overproduction, and reduction of ATP level. Moreover, H/R stimulation reduced the ATG4C expression in H9c2 cells, while EGCG raised the ATG4C expression. Overexpression of ATG4C strengthened the beneficial influence of EGCG on H/R-stimulated H9c2 cell viability, apoptosis and ROS production. Besides, ATG4C overexpression weakened the H/R-stimulated H9c2 cell autophagy via reducing LC3B II/I expression. EGCG exerted beneficial influence on H/R-stimulated cardiomyocytes, which protected cardiomyocytes from H/R-stimulated viability loss, apoptosis, and ROS overproduction via enhancing ATG4C expression.

MiR-190-5p Alleviates Myocardial Ischemia-Reperfusion Injury by Targeting PHLPP1.

Objective Myocardial ischemia-reperfusion (I/R) injury (MIRI) refers to the more serious myocardial injury after blood flow recovery, which seriously affects the prognosis of patients with ischemic cardiomyopathy. This study explored the new targets for MIRI treatment by investigating the effects of miR-190-5p and its downstream target on the structure and function of myocardial cells. Methods We injected agomir miR-190-5p into the tail vein of rats to increase the expression of miR-190-5p in rat myocardial cells and made an I/R rat model by coronary artery occlusion. We used 2,3,5-triphenyl tetrazolium chloride staining, lactate dehydrogenase (LDH) detection, echocardiography, and hematoxylin-eosin (HE) staining to determine the degree of myocardial injury in I/R rats. In addition, we detected the expression of inflammatory factors and apoptosis-related molecules in rat serum and myocardial tissue to determine the level of inflammation and apoptosis in rat myocardium. Finally, we determined the downstream target of miR-190-5p by Targetscan system and dual luciferase reporter assay. Results The expression of miR-190-5p in an I/R rat myocardium was significantly lower than that in normal rats. After treatment of I/R rats with agomir miR-190-5p, the ischemic area of rat myocardium and the concentration of LDH decreased. The results of echocardiography and HE staining also found that overexpression of miR-190-5p improved the structure and function of rat myocardium. miR-190-5p was also found to improve the viability of H9c2 cells in vitro and reduce the level of apoptosis of H9c2 cells. The results of Targetscan system and dual luciferase reporter assay found that miR-190-5p targeted to inhibit pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1). In addition, inhibition of PHLPP1 was found to improve the viability of H9c2 cells. Conclusion Therefore, miR-190-5p can reduce the inflammation and apoptosis of myocardium by targeting PHLPP1, thereby alleviating MIRI.