The effect of astragaloside IV on a model of isoproterenol-induced hypertrophic injury in H9c2 cells.

Yiqi Huoxue Decoction modifies the expression of myocardial cytoskeleton-associated proteins by regulating the AMPK signaling pathway in H9c2 cells exposed to hypoxic conditions

Hypoxia plays an important role in many heart diseases. MicroRNA-9 (miR-9) has been reported to be involved in hypoxia-induced cell proliferation, injury and apoptosis in cardiomyocytes. However, the underlying mechanism still remains poorly understood. The expression levels of miR-9 and cyclin-dependent kinase 8 (CDK8) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relative protein expression was measured by Western blot. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase (LDH) measurement, flow cytometry assays were conducted to detect cell proliferation, the release of LDH and cell apoptosis, respectively. The potential relationship between miR-9 and CDK8 was predicted by online database, and confirmed by dual-luciferase reporter assay. We found that miR-9 was increased, while CDK8 was decreased in hypoxia-treated H9c2 cells. miR-9 down-regulation or CDK8 up-regulation promoted cell proliferation, while repressed cell damage and apoptosis in hypoxia-induced H9c2 cells. Moreover, CDK8 was identified to be target of miR-9, and CDK8 knockdown could reverse the effects of miR-9 inhibitor on cell proliferation, damage and apoptosis in hypoxia-treated H9c2 cells. Besides, miR-9 could regulate the Wnt/b-catenin pathway by targeting CDK8 in hypoxic-induced H9c2 cells. In conclusion, miR-9 repressed cell proliferation and promoted cell damage and apoptosis by binding to CDK8 through the Wnt/ β-catenin pathway in hypoxic-induced H9c2 cells, which provided a new direction for further studying the treatment of hypoxia-aroused heart diseases.

MicroRNA-210 aggravates hypoxia-induced injury in cardiomyocyte H9c2 cells by targeting CXCR4

The goal of this study was to explore the role of tumor associated long noncoding RNA expressed on chromosome 2 (TALNEC2) in protecting against myocardial ischemic injury, as well as its underlying molecular mechanism. We established a cell model of myocardial injury through treating H9c2 cells with hypoxia, and the expression level of TALNEC2 was analyzed. Further, in vitro studies investigated the functional role of TALNEC2 dysregulation in hypoxia injury by assessing cell proliferation, migration, invasion, and apoptosis. Moreover, the expression of miR-21 was determined after dysregulation of TALNEC2, and whether TALNEC2-regulated hypoxia injury in H9c2 cells via regulating miR-21 expression were explored. Furthermore, the regulatory relationship between TALNEC2 and Wnt/β-catenin pathway was also investigated. TALNEC2 was highly expressed in the serum from patients with myocardial ischemic compared with that in healthy persons. Hypoxia-induced injury in H9c2 cells. Overexpression of TALNEC2 aggravated hypoxia injury in H9c2 cells. TALNEC2 could negative regulate the miR-21 expression, and overexpression of TALNEC2 aggravated hypoxia injury by downregulation of miR-21. Moreover, miR-21 negatively regulated the PDCD4 expression, and PDCD4 was a target of miR-21. Further studies disclosed that the overexpression of TALNEC2 further activated the Wnt/β-catenin pathway in hypoxia-treated H9c2 cells, implying that the Wnt/β-catenin pathway was a downstream mechanism mediating the role of TALNEC2 in regulating hypoxia injury in H9c2 cells. These findings confirmed the key functions of TALNEC2 in regulating myocardial ischemic injury. Upregulation of TALNEC2 may aggravate hypoxia injury in H9c2 cells via regulating miR-21/PDCD4-medited activation of the Wnt/β-catenin pathway. TALNEC2 may serve as a promising therapeutic target in myocardial ischemia.

G protein-coupled protein receptor CXC chemokine receptor 4 (CXCR4) has been shown to be involved in the development of sepsis; however, it remains unclear whether CXCR4 participates in the septic myocardial injury. In our study, treatment with lipopolysaccharide (LPS) increased the expression of specificity protein 1 (SP1) and CXCR4 in H9c2 cells. Notably, a positive association between SP1 and CXCR4 expression was observed in LPS-treated H9c2 cells, and SP1 positively regulated CXCR4 expression in H9c2 cells. Moreover, silencing of SP1 or CXCR4 suppressed LPS-induced inflammation and cell apoptosis in H9c2 cells, as evidenced by the increase in cell viability and decrease in lactate dehydrogenase release, interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α levels, and caspase-3 activity. Additionally, overexpression of CXCR4 abolished the protective effects of SP1 silencing on LPS-induced injury in H9c2 cells. SP1 was also shown to enhance the promoter activity of CXCR4 by directly binding with the binding motif site – 109/–100 in CXCR4 promoter. Besides, downregulation of SP1 or CXCR4 blocked LPS-induced activation of the NF-кB signaling in H9c2 cells. Furthermore, inhibition of NF-кB signaling by DHMEQ abolished LPS-induced myocardial inflammation and apoptosis. In conclusion, silencing of SP1 protected H9c2 cells against LPS-induced injury by binding to the promoter of CXCR4 and suppressing the NF-κB signaling pathway. Hence, our findings provide evidence that manipulation of SP1 or CXCR4 may be an effective approach to promote prevention or recovery of septic myocardial injury, and thereby, may serve as a potential therapeutic strategy for sepsis.

The present study was carried out to observe the protective effects of αB-crystallin protein on hydrogen peroxide (H2O2)-induced injury in rat myocardial cells (H9c2) and to investigate the mechanisms of these protective effects at the cellular level, which could provide the experimental basis for future applications of αB-crystallin in the treatment of cardiovascular disease. Western blotting was used to measure the expression of αB-crystallin in cultured H9c2 cells in vitro. A αB-crystallin recombinant expression vector, pcDNA3.1-Cryab, was constructed to transfect H9c2 cells for the establishment of cells that stably expressed αB-crystallin. A tetrazolium-based colorimetric assay (MTT test) was used to measure changes in the viability of the H9c2 cells at 1, 2, 3 and 4 h after induced by 150 μM H2O2 to establish a model of H2O2 injury to cells. H2O2 was applied to H9c2 cells that were stably transfected with αB-crystallin, and the effect of αB-crystallin overexpression on the viability of myocardial cells subjected to H2O2-induced injury was measured by the MTT assay. The effect of αB-crystallin overexpression on the H2O2-induced injury of H9c2 cells was also analyzed by flow cytometry. The mitochondrial components and cytoplasmic components of H9c2 cells were separated, and western blotting was used to measure the effect of αB-crystallin overexpression on the release of cytochrome c from the mitochondria. Western blotting was also used to measure the effect of αB-crystallin overexpression on the expression of the anti-apoptosis protein Bcl-2 and components of the phosphatidylinositol 3-OH kinase (PI3K)/AKT pathway. The αB-crystallin recombinant expression vector pcDNA3.1-Cryab successfully transfected H9c2 cells, and H9c2 cells that were stably transfected with αB-crystallin were established after G418 selection. The measurements carried out by western blotting showed that αB-crystallin proteins are expressed in normal H9c2 cells, but the proteins' expression was much higher in pcDNA3.1-Cryab transfected cells (P < 0.01). The MTT assays showed that 4 h of H2O2 treatment induced significant injury in H9c2 cells (P < 0.01), but αB-crystallin overexpression can effectively antagonize the H2O2-induced injury to H9c2 cells (P < 0.05). The results of flow cytometry analysis showed that αB-crystallin overexpression can significantly reduce apoptosis in H2O2-injured H9c2 cells (P < 0.05). The results of western blotting showed that αB-crystallin overexpression in myocardial cells can reduce the H2O2-induced release of cytochrome c from the mitochondria (P < 0.05), antagonize the H2O2-induced downregulation of Bcl-2 (P < 0.05) and magnify the decrease in phosphorylated AKT levels induced by H2O2 injury (P < 0.05). The overexpression of αB-crystallin has a protective effect on H2O2-injured H9c2 cells, and αB-crystallin can play a protective role by reducing apoptosis, reducing the release of cytochrome c from the mitochondria and antagonizing the downregulation of Bcl-2 expression. The protective effects of αB-crystallin may be related to the PI3K/AKT pathway.

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.

Recent studies have revealed that geranylgeranyl pyrophosphate synthase (GGPPS), a key enzyme involved in protein prenylation, plays a critical role in postnatal heart growth by regulating cardiomyocyte size. However, the role of GGPPS in myocardial ischemia/reperfusion (MIR) injury is still not clear. The objective of this work was to investigate the effect of GGPPS on MIR injury in H9c2 cells subjected to hypoxia/reoxygenation (HR) to mimic MIR. Prior to HR, the cells were transfected with GGPPS, shGGPPS, or shGFP. The results showed that cell viability was reduced, and cell injury and cell apoptosis were increased as a result of overexpression of GGPPS. Knockdown of GGPPS improved cell viability, and decreased cell injury and cell apoptosis. Furthermore, overexpression of GGPPS increased Rac1 activity and ROS generation, while GGPPS silencing decreased Rac1 activity and ROS generation. Based on these findings, we propose that the alteration of GGPPS expression changed the Rac1 activity and ROS production, and finally led to the different severity of HR-induced injury in H9c2 cells. These findings indicate that GGPPS might be a potential target in preventing H9c2 cells from HR-induced injury.

Recently, microRNAs (miRNAs) have been shown to involve in the process of heart failure. This study aims to investigate the functional role of miR-147b in rat H9c2 cardiomyocytes and explore the underlying molecular mechanisms. Cell viability of H9c2 cells was detected by MTT assay. Cell apoptosis was detected by flow cytometry. Expression of miR-147b and KLF13 mRNA was detected by quantitative real-time PCR. The relationship between miR-147b and KLF13 was verified by dual-luciferase reporter assay. Protein levels were detected by western blot analysis. It was found that H2O2 inhibited cell viability and promoted cell apoptosis of H9c2 cells in a concentration-dependent manner. MiR-147b overexpression suppressed cell viability and increased apoptosis in H9c2 cells, while knock-down of miR-147b increased cell viability and reduced apoptosis in H2O2-treated H9c2 cells. Luciferase reporter assay and in vitro functional assay showed that KLF13 was a downstream target of miR-147b, and KLF13 knock-down suppressed cell viability and induced apoptosis in H9c2 cells. Enforced expression of KLF13 restored the effects of miR-147b overexpression on cell viability and apoptosis in H9c2 cells. MiR-147b modulated the expression levels of apoptosis-related proteins, and the effects of miR-147b overexpression on apoptosis-related proteins levels were prevented by enforced expression of KLF13 in H9c2 cells. The in vivo experiments showed that miR-147b was up-regulated, and KLF13 was down-regulated in the myocardial tissues from rats with chronic heart failure. Collectively, miR-147b inhibits viability and promotes cell apoptosis by targeting KLF13 in H9c2 cells, which may be associated with the pathogenesis of heart failure.

ObjectivesAngelica sinensis exerts various pharmacological effects, such as antioxidant and anti-apoptotic activity. This study aimed to investigate the active ingredients in A. sinensis with antioxidant properties and whether A. sinensis polysaccharide (ASP) protects H9c2 cells against oxidative and endoplasmic reticulum (ER) stress.MethodsThe ingredients of A. sinensis and their targets and related pathways were determined using web-based databases. Markers of oxidative stress, cell viability, apoptosis, and ER stress-related signalling pathways were measured in H9c2 cells treated with hydrogen peroxide (H2O2) and ASP.ResultsThe ingredient–pathway–disease network showed that A. sinensis exerted protective effects against oxidative injury through its various active ingredients on regulation of multiple pathways. Subsequent experiments showed that ASP pretreatment significantly decreased H2O2-induced cytotoxicity and apoptosis in H9c2 cells. ASP pretreatment inhibited H2O2-induced reactive oxygen species generation, lactic dehydrogenase release, and malondialdehyde production. ASP exerted beneficial effects by inducing activating transcription factor 6 (ATF6) and increasing ATF6 target protein levels, which in turn attenuated ER stress and increased antioxidant activity.ConclusionsOur findings indicate that ASP, a major water-soluble component of A. sinensis, exerts protective effects against H2O2-induced injury in H9c2 cells by activating the ATF6 pathway, thus ameliorating ER and oxidative stress.

This article explored the mechanism by which ginsenoside Re reduces hypoxia/reoxygenation(H/R) injury in H9c2 cells by regulating mitochondrial biogenesis through nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/peroxisome prolife-rator-activated receptor gamma coactivator-1α(PGC-1α) pathway. In this study, H9c2 cells were cultured in hypoxia for 4 hours and then reoxygenated for 2 hours to construct a cardiomyocyte H/R injury model. After ginsenoside Re pre-administration intervention, cell activity, superoxide dismutase(SOD) activity, malondialdehyde(MDA) content, intracellular reactive oxygen species(Cyto-ROS), and intramitochondrial reactive oxygen species(Mito-ROS) levels were detected to evaluate the protective effect of ginsenoside Re on H/R injury of H9c2 cells by resisting oxidative stress. Secondly, fluorescent probes were used to detect changes in mitochondrial membrane potential(ΔΨ_m) and mitochondrial membrane permeability open pore(mPTP), and immunofluorescence was used to detect the expression level of TOM20 to study the protective effect of ginsenoside Re on mitochondria. Western blot was further used to detect the protein expression levels of caspase-3, cleaved caspase-3, Cyto C, Nrf2, HO-1, and PGC-1α to explore the specific mechanism by which ginsenoside Re protected mitochondria against oxidative stress and reduced H/R injury. Compared with the model group, ginse-noside Re effectively reduced the H/R injury oxidative stress response of H9c2 cells, increased SOD activity, reduced MDA content, and decreased Cyto-ROS and Mito-ROS levels in cells. Ginsenoside Re showed a good protective effect on mitochondria by increasing ΔΨ_m, reducing mPTP, and increasing TOM20 expression. Further studies showed that ginsenoside Re promoted the expression of Nrf2, HO-1, and PGC-1α proteins, and reduced the activation of the apoptosis-related regulatory factor caspase-3 to cleaved caspase-3 and the expression of Cyto C protein. In summary, ginsenoside Re can significantly reduce I/R injury in H9c2 cells. The specific mechanism is related to the promotion of mitochondrial biogenesis through the Nrf2/HO-1/PGC-1α pathway, thereby increasing the number of mitochondria, improving mitochondrial function, enhancing the ability of cells to resist oxidative stress, and alleviating cell apoptosis.

Myocardial ischemia‒reperfusion injury is a severe cardiovascular disease, and its treatment and prevention are crucial for improving patient prognosis and reducing the economic burden. This study aimed to explore the impact of hydrogen (H 2 ) on hypoxia/reoxygenation (H/R) injury in H9c2 cells (derived from rat embryonic heart tissue) induced by hydrogen peroxide (H 2 O 2 ) and to elucidate its underlying mechanism. An H/R injury model was established in H9c2 cells via exposure to 15 μM H 2 O 2 for 3 hours, followed by incubation in a 5% CO 2 atmosphere at 37°C for 24 hours. Then, the cells were treated with H 2 (50%) for 6, 12 or 24 hours. The results demonstrated that H9c2 cells exposed to H 2 O 2 and subjected to H/R injury presented a marked decrease in the cell survival rate, accompanied by severe morphological alterations, such as curling and wrinkling, and elevated lactate dehydrogenase levels. Notably, H 2 mitigated H/R injury induced by H 2 O 2 in a time-dependent manner, improving the morphological damage observed in H9c2 cells and decreasing lactate dehydrogenase levels. Compared with the model group, treatment with H 2 increased the activities of antioxidant enzymes, including catalase, superoxide dismutase, and glutathione peroxidase, while concurrently reducing the level of malondialdehyde, an indicator of cellular damage. Furthermore, H 2 treatment downregulated the expression of inflammatory cytokines and inflammatory-related factors, specifically interleukin-6, high-mobility group box 1, tumor necrosis factor-alpha, and Toll-like receptor 4, in H9c2 cells post-H/R injury. Furthermore, H 2 treatment resulted in a marked decrease in the expression levels of proteins associated with the Wnt/C-X3-C-motif receptor 1 signaling pathway, such as β-catenin, glycogen synthase kinase-3 beta, adenomatous polyposis coli, and Wnt and C-X3-C-motif receptor 1. This observation suggests a potential mechanism for its protective effects against H/R injury. Therefore, H 2 exerts a protective effect against H/R injury in H9c2 cells induced by H 2 O 2 , potentially by inhibiting the activated Wnt/C-X3-C-motif receptor 1 signaling pathway. This inhibition, in turn, prevents the generation of oxidative stress, inflammatory cytokines, and inflammation-associated factors.

Acute myocardial infarction (AMI) is a serious cardiovascular disease that threatens human life. MicroRNA is considered to be an important participant in the pathophysiology of AMI. This article focused on the role of microRNA-495 (miR-495) in regulating apoptosis after myocardial infarction (MI) and its underlying mechanisms. H9c2 cells were cultured in an incubator containing 1% O2 to establish a cell model of MI. Quantitative reverse-transcription polymerase chain reaction (RT-PCR) was utilized to detect miR-495 expression in H9c2 cells. The effects of miR-495 and NFIB on hypoxia-treated H9c2 cells were observed by Western blot, lactate dehydrogenase (LDH) detection, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, flow cytometry, and terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling (TUNEL) staining. Luciferase reporter gene experiment was used to prove the regulatory relationship between miR-495 and NFIB. Hypoxia induced injury to H9c2 cells, which was manifested by decreased cell viability, increased LDH release, increased pro-apoptotic proteins (Bax, Cleaved Caspase-3) expression, decreased anti-apoptotic protein (Bcl-2) expression, and increased in the rate of apoptosis and TUNEL positive cells. MiR-495 expression was remarkably increased in H9c2 cells treated with hypoxia. Inhibiting miR-495 expression markedly alleviated the hypoxia-induced injury in H9c2 cells, while silencing NFIB aggravated the hypoxia-induced damage. In addition, NFIB was confirmed to be the target of miR-495. MiR-495 expression was increased in hypoxia-treated H9c2 cells. Silencing miR-495 could significantly inhibit hypoxia-induced apoptosis of H9c2 cells by targeting NFIB.

Myocardial infarction (MI) is one of the most serious cardiovascular diseases associated with myocardial ischemia/reperfusion (I/R) injury. Glaucocalyxin A (GLA) is a biologically active ent-kauranoid diterpenoid that has been found to ameliorate myocardial I/R injury in mice. However, the mechanism has not been fully investigated. In the present study, we aimed to investigate the effect of GLA on rat cardiomyocytes H9c2 cells exposed to hypoxia/reoxygenation (H/R). The results showed that GLA treatment improved cell viability of H/R-stimulated H9c2 cells. Administration with GLA suppressed the H/R-stimulated reactive oxygen species (ROS) production in H9c2 cells. GLA also elevated the activities of antioxidant enzymes, including superoxide dismutase and glutathione peroxidase in H/R-stimulated H9c2 cells. Moreover, GLA prevented H/R-stimulated cell apoptosis in H9c2 cells, as evidenced by increased bcl-2 expression, decreased bax expression, as well as reduced caspase-3 activity. Furthermore, GLA enhanced the activation of protein kinase B (Akt)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway in H9c2 cells exposed to H/R. Additionally, treatment with LY294002 reserved the protective effects of GLA on H/R-stimulated oxidative injury in H9c2 cells. In conclusion, these findings suggested that GLA protected H9c2 cells from H/R-stimulated oxidative damage, which was mediated by the Akt/Nrf2/HO-1 signaling pathway. Thus, GLA might be a promising therapeutic agent for the prevention and treatment of myocardial I/R.

Recombinant human brain natriuretic peptide regulates PI3K/AKT/mTOR pathway through lncRNA EGOT to attenuate hypoxia-induced injury in H9c2 cardiomyocytes