Oxidative Stress In H9c2 Cells Research Articles

EMP1 knockdown mitigated high glucose-induced pyroptosis and oxidative stress in rat H9c2 cardiomyocytes by inhibiting the RAS/RAF/MAPK signaling pathway.

The purpose of this study was to investigate the mechanism of EMP1 action in high glucose (HG)-induced H9c2 cardiac cell pyroptosis and oxidative injury. Rat cardiomyocytes H9c2 were exposed to 33 mM glucose for 24, 48, or 72 h to induce cytotoxicity. EMP1-siRNA, NLRP3 agonist Nigericin, and pcNDA-RAS were used to treat H9c2 cells under HG conditions. Cell Counting Kit (CCK)-8 assay showed that cell proliferation was decreased following HG induction, which was rescued by EMP1 knockdown. Our results also suggested that EMP1 siRNA transfection significantly decreased the apoptosis and pyroptosis of HG-induced cells, as indicated by the reduction of NLRP3 IL-1β, ASC, GSDMD, cleaved-caspase1 and cleaved-caspase3 levels in HG-induced H9c2 cells. In addition, EMP1 knockdown alleviated HG-induced mitochondrial damage and oxidative stress in H9c2 cells. NLRP3 activation reversed the inhibitory effects of EMP1 knockdown on pyroptosis and oxidative stress in HG-induced H9c2 cells. Mechanistically, we found that EMP1 knockdown suppressed the RAS/RAF/MAPK signaling pathway in HG-induced H9c2 cells. RAS overexpression blocked the protective effect of EMP1 knockdown on HG-induced H9c2 cell apoptosis, pyroptosis, and oxidative injury. Our findings suggest that EMP1 knockdown treatment might provide a novel therapy for diabetic cardiomyopathy.

Role of the ROS/NLRP3/caspase-1 pathway in NiSO4-induced cellular pyroptosis and apoptosis in H9c2 cells

According to comprehensive research, the cardiovascular system is damaged by nickel exposure. The present study selected rat cardiomyocytes (H9c2 cells) and subjected them to varying doses of sodium nickel sulfate (NiSO4) for 24hours to better understand the mechanism of cardiovascular damage caused by NiSO4 exposure. The relevant indicators were detected employing biochemical analysis, real-time quantitative polymerase chain reaction (RT-qPCR), and western blot, and flow cytometry was used to detect the cell apoptosis rate. The study revealed that the survival rate of H9c2 cells fell significantly when the concentration of NiSO4 exposure rose. Moreover, it caused oxidative stress in H9c2 cells by raising the expression of reactive oxygen species and the concentration of LDH in the cell supernatants. After NiSO4 exposure, the levels of ASC, NLRP3, gasdermin D, and caspase-1 in H9c2 cells increased, suggesting that H9c2 cells underwent pyroptosis induced by NiSO4. In addition, NiSO4 exposure also led to inflammation, with increased levels of interleukin [IL]-18, IL-1β. After adding the antioxidant N-Acetyl-L-cysteine (NAC), the level of ROS indicated that the oxidative stress level in H9c2 cells was reduced, western blot inhibited inflammation, the level of pyroptosis was reduced, and the activity of the NLRP3/caspase1 signaling pathway was reduced. To examine the connection between pyroptosis and apoptosis, the cells were treated with the caspase1 inhibitor Z-YVAD-Fluoromethyl Ketone (Z-YVAD-FMK, YVAD), which resulted in a significant decrease in the rate of cell apoptosis as well as a reduction in the activity of the related protein in the signaling pathway, which in turn decreased the level of pyroptosis. NiSO4 could induce pyroptosis in H9c2 cells through the ROS/NLRP3/caspase-1 axis. Furthermore, NiSO4-induced apoptosis and pyroptosis were found to be reduced by the addition of the caspase-1 inhibitor.

Propofol Ameliorates Sepsis-Induced Myocardial Dysfunction via Anti-Apoptotic, Anti-Oxidative Properties, and mTOR Signaling.

Sepsis often leads to cardiomyopathy, contributing to increased mortality rates. 2,6-Diisopropylphenol (propofol), an anesthetic, has demonstrated efficacy in protecting cardiomyocytes from cell death caused by hypoxia and reoxygenation. This study examined the effects of propofol on sepsis-associated myocardial dysfunction and explored the underlying mechanism of action. Mice and rat cardiomyocytes (H9C2 cell line) were used to establish a sepsis-induced myocardial dysfunction model. Lipopolysaccharides (LPS)-treated mice and H9C2 cells were treated with propofol, with rapamycin used for mechanistic studies in H9C2 cells. Cardiac function was evaluated by echocardiographic measurements. Heart tissues were stained with hematoxylin and eosin, and heart weight/body weight ratio along with the levels of cardiac biomarkers were measured using Enzyme Linked Immunosorbent Assay (ELISA). Activation of the mammalian target of rapamycin (mTOR) pathway was assessed by western blotting. Apoptosis in heart tissues and H9C2 cells was evaluated using Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay, and cell viability was quantified using Cell Counting Kit (CCK)-8 assay. Oxidative stress in H9C2 cells was assessed by measuring reactive oxygen species (ROS) levels through immunofluorescence staining and malondialdehyde (MDA) and superoxide dismutase (SOD) levels using ELISA. Propofol reversed LPS-induced myocardial changes and cardiac dysfunction (p < 0.05). In mouse tissues and H9C2 cells, propofol reversed LPS-induced mTOR pathway inhibition and apoptosis (p < 0.001). Moreover, propofol alleviated oxidative stress in LPS-treated cells. The activation of the mTOR pathway by propofol, along with its inhibitory effects on oxidative stress and apoptosis in cardiomyocytes, was negated by rapamycin (p < 0.001). Propofol ameliorates sepsis-induced myocardial dysfunction triggered by LPS through the mTOR pathway, thereby promoting antioxidative stress and reducing cell apoptosis.

Potential Efficacy of Syringaresinol in the Treatment of Myocardial Injury: Inhibition of Pyroptosis and Oxidative Stress

This paper aims to investigate the possible effects of Syringaresinol on a myocardial ischemia/reperfusion injury cell model and uncover the underlying mechanism. A myocardial ischemia/reperfusion injury cell model was constructed using an oxygen-glucose deprivation/reperfusion-subjected H9c2 cell line. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-diphenytetrazoliumromide) and lactate dehydrogenases releasing assays showed the effects on cell viability. Immunoblot and flow cytometry assays showed the impact on cell pyroptosis. Enzyme-linked immunosorbent assay uncovered the effects on oxidative stress in H9c2 cells. Further, immunoblot and quantitative polymerase chain reaction assays were conducted to confirm the mechanism. Syringaresinol enhanced the survival and suppressed pyroptosis in H9c2 cells subjected to oxygen-glucose deprivation/reperfusion. Syringaresinol also inhibited oxidative stress in H9c2 cells subjected to oxygen-glucose deprivation/reperfusion, and mechanically, it suppressed the expression of HSP90, thus inhibiting myocardial ischemia/reperfusion injury. Syringaresinol ameliorated the pyroptosis and oxidative stress of oxygen-glucose deprivation/reperfusion in cardiomyocytes and alleviated cardiomyocyte damage.

ABHD5 protects cardiac function in alcoholic cardiomyopathy via HDAC4-NT to inhibit CAMKII/MEF2 axis.

Excessive intake of Alcohol is associated with a high incidence of alcoholic cardiomyopathy (ACM), which may impair cardiac function. In our study, we explored the Abhydrolase Domain Containing 5 (ABHD5) mechanism in ACM about histone deacetylase 4 (HDAC4) and CaM-CaMKII/MEF2 signaling pathway. Rat models of ACM were established in Wistar rats, and in vitro cell models were constructed in rat cardiomyocytes H9C2 utilizing 12-h of treatment of Alcohol (200 mM) to study the regulatory role of ABHD5 in ACM with the involvement of HDAC4 and CaM-CaMKII/MEF2 signaling pathway, as evidenced by determination of cardiac function, myocardial fibrosis, apoptosis of cardiomyocytes and oxidative stress condition. We found that both ABHD5 mRNA and protein expression was significantly lower in the ACM rats and rat cardiomyocytes H9C2. ACM rats with oe-ABHD5 injection showed repressed myocardial hypertrophy and myocardial fibrosis. Also, overexpression of ABHD5 reduced apoptosis and oxidative stress in H9C2 cells. Mechanistic studies demonstrated that ABHD5 via HDAC4-NT inhibits CAMKII/MEF2 axis. This study highlighted that ABHD5 decreased cardiac hypertrophy and myocardial fibrosis and limited cardiomyocyte apoptosis and oxidative stress injury in ACM.

Endogenous ligand of the APJ receptor Apelin-13 inhibits cell apoptosis and oxidative stress of cardiomyocytes.

The present study aimed to investigate the effect of Apelin-13 on nicotine-induced injuries of cardiomyocytes. To establish an H9c2 cell model of nicotine-induced apoptosis, H9c2 cells were divided into the control group, nicotine group, and Apelin-13+nicotine group. The apoptosis rate of H9c2 cells was then detected by flow cytometry. Later, the expressions of indicators related to apoptosis, oxidative stress, and inflammatory responses were measured via Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The results revealed that the expression of B-cell lymphoma-2 (Bcl-2) was remarkably down-regulated (P<0.01), while the apoptosis rate and the expressions of apoptosis-related proteins (Bcl-2-associated X protein (Bax) and cysteinyl aspartate specific proteinase-3 (Caspase-3)) were significantly up-regulated (P<0.01) in the nicotine group. However, the variation trends of Bcl-2, Bax, and Caspase-3 in the Apelin-13+nicotine group were contrary to those in the nicotine group (P<0.01). Additionally, the expressions of interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) obviously declined (P<0.01), while those of superoxide dismutase 1 (SOD1) and SOD2 dramatically rose in the Apelin-13+nicotine group (P<0.01). Furthermore, Apelin-13 treatment evidently elevated the expressions of phosphorylated protein kinase B (p-AKT) and phosphorylated phosphatidylinositol 3-kinase (PI3K). In conclusion, Apelin-13 inhibits nicotine-induced apoptosis and oxidative stress in H9c2 cells via the PI3K/AKT signaling pathway.

Pharmacological Activation of Rev-erbα Attenuates Doxorubicin-Induced Cardiotoxicity by PGC-1α Signaling Pathway.

Doxorubicin-induced cardiotoxicity has been closely concerned in clinical practice. Rev-erbα is a transcriptional repressor that emerges as a drug target for heart diseases recently. This study is aimed at investigating the role and mechanism of Rev-erbα in doxorubicin-induced cardiotoxicity. H9c2 cells were treated with 1.5 μM doxorubicin, and C57BL/6 mice were treated with a 20 mg/kg cumulative dose of doxorubicin to construct doxorubicin-induced cardiotoxicity models in vitro and in vivo. Agonist SR9009 was used to activate Rev-erbα. PGC-1α expression level was downregulated by specific siRNA in H9c2 cells. Cell apoptosis, cardiomyocyte morphology, mitochondrial function, oxidative stress, and signaling pathways were measured. SR9009 alleviated doxorubicin-induced cell apoptosis, morphological disorder, mitochondrial dysfunction, and oxidative stress in H9c2 cells and C57BL/6 mice. Meanwhile, PGC-1α and downstream signaling NRF1, TAFM, and UCP2 expression levels were preserved by SR9009 in doxorubicin-treated cardiomyocytes in vitro and in vivo. When downregulating PGC-1α expression level by specific siRNA, the protective role of SR9009 in doxorubicin-treated cardiomyocytes was attenuated with increased cell apoptosis, mitochondrial dysfunction, and oxidative stress. Pharmacological activation of Rev-erbα by SR9009 could attenuate doxorubicin-induced cardiotoxicity through preservation of mitochondrial function and alleviation of apoptosis and oxidative stress. The mechanism is associated with the activation of PGC-1α signaling pathways, suggesting that PGC-1α signaling is a mechanism for the protective effect of Rev-erbα against doxorubicin-induced cardiotoxicity.

Secreted APE1/Ref-1 inhibits doxorubicin-induced cardiotoxicity via suppressing ROS generation and p53 pathway

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This research is supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health &amp; Welfare and Basic Science Research Program of the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Republic of Korea. Background Doxorubicin is an anthracycline anticancer drug with a potent therapeutic effect against various malignancies. However, its use is limited due to cardiotoxicity, which can lead to heart failure. Oxidative stress is a key player in the development of anthracycline cardiotoxicity. Apurinic/apyrimidinic endonuclease 1/redox factor-1(APE1/Ref-1) is a multifunctional protein that regulates oxidative stress and anti-inflammatory function. Secreted APE1/Ref-1 has shown anti-oxidant and anti-inflammatory properties, but its therapeutic potential in various diseases is largely unknown. Purpose This study aims to test the protective effect of secreted APE1/Ref-1 in the doxorubicin-induced cardiomyopathy model in vivo and in vitro. Methods H9C2 cells were treated with doxorubicin(2uM) to induce cardiotoxicity. Adenovirus preprotrypsin leading sequence APE1/Ref-1(AdPPT-LS-APE1/Ref-1), a secretory APE1/Ref-1 adenovirus encoding human APE1/Ref-1 gene, was amplified in HEK293T cells. H9C2 cells were then treated with the supernatant of the amplified AdPPT-LS-APE1/Ref-1. In vivo model of doxorubicin-induced cardiotoxicity was established by injecting doxorubicin(15mg/kg) into C57BL/6 mice via the peritoneum. The conditioned media of HEK293T cells with AdPPT-LS-APE1/Ref-1 transfection was injected i.p. after 24h. The severity of cardiotoxicity was evaluated by echocardiography and plasma NT-proBNP. Cell survival, morphology, and ROS level were examined. The expression of cellular APE1/Ref-1, p53, Bax/Bcl-2, and Caspase-3 were analyzed by Western blot and ELISA. Redox activity was measured using the DTNB reduction assay. Results Doxorubicin caused apoptosis and oxidative stress in H9C2 cells and cardiomyopathy in mice. NT-proBNP level increased both in vitro(p&amp;lt;0.001) and in vivo(p&amp;lt;0.05) upon administering doxorubicin. Interestingly, doxorubicin increased p53 level in a dose-dependent manner in 48 hr, but the cellular APE1/Ref-1 level did not change. Secreted APE1/Ref-1 in the conditional media showed redox activity and decreased NT-proBNP in doxorubicin-treated H9C2 cells and mice(p&amp;lt;0.001 and p&amp;lt;0.05, respectively). Doxorubicin aggravated cell death(p&amp;lt;0.001) and oxidative tissue damage(p&amp;lt;0.001) in H9C2 cells, while pre-treatment of secreted APE1/Ref-1 inhibited cell death in H9C2 cells(p&amp;lt;0.05). Secreted APE1/Ref-1 significantly reduced the expression of p53(p&amp;lt;0.001) and its effectors, Bax/Bcl-2(p&amp;lt;0.05) and Caspase-3(p&amp;lt;0.05) in doxorubicin-treated H9C2 cells. It also reduced p53(p&amp;lt;0.05) along with Bax/Bcl-2(p&amp;lt;0.05) and Caspase-3(p&amp;lt;0.05) in doxorubicin-treated mice(n=4 in each group). Finally, secreted APE1/Ref-1 significantly decreased intracellular ROS production(p&amp;lt;0.001). Conclusion Secreted APE1/Ref-1 is protective against doxorubicin-induced cardiomyocyte apoptosis and oxidative stress by suppressing ROS generation and p53 pathway. APE1/Ref-1 showed therapeutic potential for the prevention and treatment of doxorubicin-induced cardiotoxicity.

DUSP12 ameliorates myocardial ischemia-reperfusion injury through HSPB8-induced mitophagy.

This study aimed to explore the role of dual specificity phosphatase 12 (DUSP12) in regulating myocardial ischemia-reperfusion (I/R) injury and the underlying mechanism. The expression of DUSP12 in myocardial tissuesand heat-shock protein beta-8(HSPB8) and mitophagy-related proteins in myocardial tissues and H9c2 cells were detected by western blot analysis. The serum creatine kinase isoenzymes (CK-MB)and lactate dehydrogenase (LDH), levels of reactive oxygen speciesand malondialdehyde,superoxide dismutase activity in myocardial tissuesand H9c2 cells, and caspase-3 activity in H9c2 cells were analyzed by corresponding assay kits. The infarct area in therat's heart was observed by triphenyl tetrazolium chloridestaining. The apoptosis of myocardial cells in myocardial tissues and H9c2 cells was detected by terminal-deoxynucleotidyl transferase dUTP-biotin nick-end labelingassay. The interaction between DUSP12 and HSPB8 was clarified by the coimmunoprecipitation assay. The transfection efficacy of si-HSPB8#1 and si-HSPB8#2 in H9c2 cells was confirmed by real-time quantitative-polymerasechain reactionand western blot analysis. As a result, DUSP12 expression was downregulated in I/R rats, which was promoted by lentivirus-expressing DUSP12. DUSP12 overexpression reduced the serumcreatine kinase isoenzymes (CK-MB) and LDH, decreased the infarct area in the rat's heart, and suppressed the apoptosis and oxidative stress in myocardial tissues. DUSP12 overexpression also upregulated the expression of HSPB8 to promote mitophagy. The coimmunoprecipitation assay indicated that DUSP12 could be combined with HSPB8. In addition, DUSP12 overexpression could inhibit hypoxia/reoxygenation-elicited apoptosis as well as oxidative stress in H9c2 cells by upregulating HSPB8 expression to promote mitophagy, which was countervailed by HSPB8 deficiency. In conclusion, DUSP12 overexpression decreased the apoptosis and oxidative stress in myocardial I/R injury through HSPB8-induced mitophagy.

Rutaecarpine ameliorates cardiomyocyte injury induced by high glucose by promoting TRPV1-mediated autophagy.

Diabetic cardiomyopathy (DCM) is a dominant factor contributing to diabetic death. Rutaecarpine has many cardiovascular biological effects and anti-high-glucose activity. Therefore, this paper aimed to investigate the impact of rutaecarpine on high glucose (HG)-elicited cardiomyocyte injury. Cell counting kit 8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU), TdT-mediated dUTP Nick-End Labeling (TUNEL) assays judged H9c2 cell activity and apoptosis, and oxidative stress was assessed by corresponding assay kits. The expression of apoptosis, oxidative stress, autophagy-associated factors and TRPV1 were examined with western blot. IF assay tested GFP-LC3 expression. As a result, rutaecarpine had no obvious effect on the viability of H9c2 cells while elevated HG-exposed H9c2 cell viability. Rutaecarpine inhibited the apoptosis and oxidative stress of H9c2 cells induced by HG. In addition, rutaecarpine activated TRPV1 to induce autophagy. However, inhibition of TRPV1 inactivated the autophagy, which drove HG-evoked H9c2 apoptosis and oxidative stress. In conclusion, rutaecarpine suppressed HG-stimulated H9c2 cell viability injury, apoptosis as well as oxidative stress via promoting TRPV1-mediated autophagy (Fig. 10, Ref. 40).

The potential protective effects of nintedanib against cardiac hypertrophy in vitro and in vivo

IntroductionCardiac hypertrophy is an independent risk factor for heart failure. However, the underlying mechanisms of cardiac hypertrophy are still unclear. Nintedanib is a Food and Drug Administration (FDA) approved therapeutic agent for the treatment of progressive fibrosing lung diseases.Material and methodsIn this study, we examined the effects of nintedanib on cardiac hypertrophy using an in vivo murine model with the transverse aortic constriction (TAC) operation and an in vitro cardiomyocytes model stimulated with Ang II.ResultsNintedanib has a protective effect on cardiac function in TAC mice with decreased heart rates, heart weight/body weight (HW/BW), and reduced plasma levels of creatine kinase-MB (CK-MB) and aspartate aminotransferase (AST). Wheat germ agglutinin (WGA) staining proved that the increased cardiomyocytes sizes in TAC mice were restored by nintedanib treatment. Nintedanib also reversed the decreased plasma levels of oxidative markers nuclear factor erythroid-2-related factor 2 (Nrf2), lipid peroxidation products thiobarbituric acid reactive substances (TBARS), and GSH, as well the increased homocysteine (Hcy) levels in TAC mice. In the in vitro cardiomyocytes model, cells were treated with nintedanib, followed by Ang II stimulation. Nintedanib improved Ang II induction-caused cell injury and oxidative stress in H9C2 cells, as shown by the decreased release of lactate dehydrogenase (LDH), and elevated mRNA levels of GPX1 and HO-1. Mechanistically, we prove that the protective effect of nintedanib is mediated by SIRT1.ConclusionsIn conclusion, this study demonstrates the protective effects of nintedanib on cardiac hypertrophy both in vivo and in vitro, which was attributed to its anti-oxidative activity through regulating SIRT1 expression.

Isolation and characterization of fucoidan from four brown algae and study of the cardioprotective effect of fucoidan from Sargassum wightii against high glucose-induced oxidative stress in H9c2 cardiomyoblast cells.

Oxidative stress plays a vital role in the initiation and progression of diabetic cardiomyopathy (DCM). Increased cardiac dysfunction and apoptosis in DCM are independent factors associated with hypertension or coronary artery disease. Fucoidan, a class of sulfated polysaccharides, is widely used as food supplements and reported to have various pharmacological properties. However, the pharmacological property of Indian seaweeds remains unexplored. The present study is focused on isolating and characterizing the fucoidan from four brown seaweeds such as Sargassum wightii (SwF), Sargassum swartzii (SsF), Sargassum polycystum (SpF), Turbinaria ornata (ToF), and aimed to investigate cardioprotective effect of fucoidan against High Glucose (HG) induced oxidative stress in H9c2 cells. The mild acid hydrolysis method was used to isolate crude fucoidan from four brown seaweeds purified by the FPLC system. The biochemical composition analysis showed that SwF had a high content of fucoidan and sulfate, followed by SsF, SpF, and ToF. Further, FTIR, XRD, NMR, and SEM analysis confirmed the isolated fucoidan structures. SwF showed higher DPPH activity compared to another isolated fucoidan. In vitro studies with SwF revealed significantly decreased cytotoxicity, prevented the loss of MMP, reduced lipid peroxidation, and increased cellular enzymatic and non-enzymatic activity. qRT-PCR results showed SwF significantly upregulated the Nrf2, HO-1, NQO1, and Bcl2 and down-regulated the Bax and Caspase-3 mRNA expression compared to HG-treated cells. In conclusion, SwF could be used to develop functional foods for diabetic-mediated CVD complications compared to another isolated fucoidan. PRACTICAL APPLICATIONS: Bioactive carbohydrates have gained significant interest among researchers to improve human health. The biomedical field showed great interest in seaweed research in managing various diseases. In particular, seaweeds contain many bioactive compounds because of their chemical and biological diversity. Despite the various beneficial effects of fucoidan in CVD, the therapeutic potential of Indian seaweeds remains largely unexplored. Hence, this study isolated fucoidan from four brown seaweeds and studied their bioactive properties. Results revealed that SwF showed higher free radical scavenging activity compared to another isolated fucoidan. Therefore, SwF was selected for the in vitro study. SwF increased the cytoprotection through increasing antioxidant levels against oxidative stress in H9c2 cells. Staining analysis showed SwF increased cellular protection via inhibiting ROS protection and increasing MMP. Overall, fucoidan from SwF could be developed as a functional food for CVD.

Exosomal miR-27b-3p Derived from Hypoxic Cardiac Microvascular Endothelial Cells Alleviates Rat Myocardial Ischemia/Reperfusion Injury through Inhibiting Oxidative Stress-Induced Pyroptosis via Foxo1/GSDMD Signaling.

Background Exosomes derived from cardiac microvascular endothelial cells (CMECs) under hypoxia can mediate cardiac repair functions and alleviate pyroptosis and oxidative stress during ischemia-reperfusion (I/R) injury. This study is aimed at investigating the effect and mechanism of miR-27b-3p underlying hypoxic CMECs-derived exosomes against I/R injury. Methods CMECs were isolated from the left ventricle of Sprague-Dawley rats, followed by culturing under hypoxic conditions or pretreatment with the miR-27b-3p inhibitor. CMECs-derived exosomes were added into H9C2 cells before hypoxia/reoxygenation (H/R) or injected into the rat heart before I/R injury. An in vivo I/R injury model was established by ligating and releasing the left anterior descending coronary artery. Expression of pyroptosis-related factors was detected using Western blot, and heart infarcted size was determined by the 2,3,5-triphenyl-2H-tetrazpinolium chloride staining method. Dual-Luciferase Reporter assays were performed to analyze the interactions of nmiR-27b-3p-forkhead box O1 (Foxo1) and Gasdermin D- (GSDMD-) Foxo1. Chromatin-immunoprecipitation (ChIP) assays were performed to validate the interactions between forkhead box O1 (Foxo1) and Gasdermin D (GSDMD) and Foxo1-mediated histone acetylation of GSDMD. Results CMECs were successfully identified from left ventricle of Sprague-Dawley rats. The expressions of Foxo1 and pyroptosis-related proteins (GSDMD, NLPR3, cleaved caspase 1, IL-1β, and IL-18) were upregulated in the rat heart after I/R injury. Treatment of CMEC-derived exosomes, especially that under hypoxic conditions, significantly reduced pyroptosis in the rat heart. miR-27b-3p was significantly upregulated in CMEC-derived exosomes under hypoxic conditions, and miR-27b-3p inhibition in exosomes alleviated its cytoprotection and inhibited oxidative stress in H9C2 cells. Treatment with Foxo1 overexpression plasmids aggravated in vitro H/R and in vivo I/R injury by upregulating pyroptosis-related proteins. Further experiments validated that miR-27b-3p negatively targeted Foxo1, which bound to the promoter region of GSDMD. Conclusions These results demonstrated a great therapeutic efficacy of miR-27b-3p overexpression in hypoxic CMEC-derived exosomes in preventing the development of myocardial damage post I/R injury through inhibiting Foxo1/GSDMD signaling-induced oxidative stress and pyroptosis.

Hyperglycemia-Induced Overexpression of PH Domain Leucine-Rich Repeat Protein Phosphatase 1 (PHLPP1) Compromises the Cardioprotective Effect of Ischemic Postconditioning Via Modulation of the Akt/Mst1 Pathway Signaling.

Ischemic postconditioning (IPostC) alleviates myocardial ischemia/reperfusion (IR) injury, but the protective effect is lost during diabetes. PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) is able to inactivate Akt. Our previous study found that PHLPP1 expression was upregulated in diabetic hearts. We presumed that the attenuation of myocardial injury by IPostC might be hindered by PHLPP1 overexpression in diabetic animals. Nondiabetic and diabetic mice were subjected to 45 min of ischemia followed by 2 h of reperfusion with or without IPostC. H9c2 cells were exposed to normal or high glucose and were subjected to 4 h of hypoxia followed by 4 h of reoxygenation with or without hypoxic postconditioning (HPostC). IPostC attenuated postischemic infarction, apoptosis, creatine kinase-MB, and oxidative stress, which were accompanied by increased p-Akt and decreased PHLPP1 expression and p-Mst1 in nondiabetic but not in diabetic mice. PHLPP1 knockdown or an Mst1 inhibitor reduced hypoxia/reoxygenation (HR)-induced cardiomyocyte damage in H9c2 cells exposed to normal glucose, but the effect was abolished by a PI3K/Akt inhibitor. HPostC attenuated HR-induced cardiomyocyte injury and oxidative stress accompanied by increased p-Akt as well as decreased PHLPP1 expression and p-Mst1 in H9c2 cells exposed to normal glucose but not high glucose. In addition, HPostC in combination with PHLPP1 knockdown or PHLPP1 knockdown alone reduced cell death and oxidative stress in H9c2 cells exposed to high glucose, which was hindered by PI3K/Akt inhibitor. IPostC prevented myocardial IR injury partly through PHLPP1/Akt/Mst1 signaling, and abnormalities in this pathway may be responsible for the loss of IPostC cardioprotection in diabetes.

Discovery of 1,3,4-oxadiazole derivatives containing a bisamide moiety as a novel class of potential cardioprotective agents

Myocardial injury is a nonnegligible problem in cardiovascular diseases and cancer therapy. The functional feature of N-containing heterocycles in the cardiovascular field has attracted much attention in recent years. Herein, we discovered a lead compound 12a containing 1,3,4-oxadiazole by extensive screening of anticancer derivatives containing nitrogen-heterocycle, which exhibited potential protective activity against oxidative stress in cardiomyocytes. Follow-up structure-activity relationship (SAR) studies also highlighted the role of substitution sites and bisamide moiety in enhancing the protective activity against oxidative stress. Specifically, compound 12d exhibited low cytotoxicity under high concentration and potent myocardial protection against oxidative stress in H9c2 cells. Preliminary mechanistic studies showed compound 12d could decrease the expression of cardiac hypertrophy and oxidative stress-related proteins/genes and reduce mitochondria-mediated cell apoptosis, thereby enhancing the cell vitality of injured cardiomyocytes. In this study, 1,3,4-oxadiazole may represent a novel pharmacophore that possesses potential myocardial protection and provides more choices for future optimization of cardiovascular drugs, especially for the treatment of onco-cardiology.

Inhibition of Sestrin2 overexpression in diabetic cardiomyopathy ameliorates cardiac injury via restoration of mitochondrial function.

Mitochondrial dysfunction-induced apoptosis plays a crucial role in the progression of diabetic cardiomyopathy (DCM). Sestrin2 is an important oxidative stress response protein and is involved in the maintenance of mitochondrial function, especially under stress. The aim of the present study was to investigate the role of Sestrin2 in DCM and to explore the underlying mechanisms. H9c2 cardiomyocytes were induced with high glucose (HG) medium (33 mmol/l glucose) for an in vitro DCM model. C57BL/6 mice were induced for the in vivo DCM model by intraperitoneal streptozotocin injection. H9c2 cardiomyocytes were exposed to HG and infected with lentiviruses to express Sestrin2 short hairpin RNA (shRNA). The study found that cell viability and mitochondrial function were impaired while cell apoptosis and oxidative stress were increased in DCM. Sestrin2 was significantly upregulated in myocardial tissues of DCM mice and H9c2 cardiomyocytes in HG conditions. Downregulation of Sestrin2 increased cell viability, decreased cell apoptosis, and attenuated oxidative stress in H9c2 cells exposed to HG. Moreover, HG-induced mitochondrial injury was alleviated by Sestrin2 silencing. In conclusion, our finding indicated that the inhibition of enhanced Sestrin2 expression ameliorates cardiac injury in DCM, which might be largely attributed to the restoration of mitochondrial function.

MicroRNA-21-5p acts via the PTEN/Akt/FOXO3a signaling pathway to prevent cardiomyocyte injury caused by high glucose/high fat conditions.

MicroRNAs (miRNAs or miRs) play important roles in cardiovascular disease. miR-21-5p is known to be involved in the regulation of cardiomyocyte injury under high glucose and high fat (HG-HF) conditions, but its mechanism of action remains unclear. In the present study, a cardiomyocyte cell line, H9c2, was treated with 33 mM glucose and 250 µM sodium palmitate for 24, 48, and 72 h to produce HG-HF injury. After treatment, miR-21-5p expression was detected by reverse transcription-quantitative PCR. A miR-21-5p mimic was then constructed and transfected into the cells and the potential molecular mechanism was investigated using Cell Counting Kit-8, TUNEL, flow cytometry and western blot assays. Expression of miR-21-5p was significantly downregulated by HG-HF treatment of H9c2 cells for 24, 48, and 72 h. In subsequent experiments, cells were treated for an intermediate period (48 h). Compared with the control group, HG-HF treatment significantly inhibited H9c2 proliferation and promoted apoptosis, while these effects were significantly reduced in the miR-21-5p mimic. Compared with the control group, HG-HF treatment significantly increased reactive oxygen species, while miR-21-5p mimic significantly reduced this effect. Compared with the control group, HG-HF treatment significantly increased the expression of the pro-apoptotic proteins Bax and phosphorylated (p)-Akt and decreased the expression of the anti-apoptotic proteins Bcl-2, p-PTEN, and p-FOXO3a, while overexpression of miR-21-5p significantly reduced these effects. The results revealed that miR-21-5p inhibited apoptosis and oxidative stress in H9c2 cells induced by HG-HF, likely through the PTEN/Akt/FOXO3a signaling 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

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.

Dexmedetomidine protects cardiomyocytes against hypoxia/reoxygenation injury via multiple mechanisms.

BackgroundMyocardial infarction (MI) is a serious cardiovascular disease associated with myocardial ischemia/reperfusion (I/R) injury. Dexmedetomidine (Dex), an α2‐adrenoceptor agonist, has been reported to protect against I/R injury. We examined the cardioprotective effects of Dex on cardiomyocytes under hypoxia/reoxygenation (H/R) conditions and explored the underlying mechanisms.Materials and methodsA H/R model was established to mimic the MI injury. The CCK‐8 assay was performed to measure cell viability. Cellular apoptosis was measured using the Annexin V fluorescein isothiocyanate (FITC)‐propidium iodide (PI) staining. The levels of interleukin (IL)‐1α and tumor necrosis factor (TNF)‐α, and the activity of lactate dehydrogenase (LDH) were measured using a commercial enzyme‐linked immunosorbent assay (ELISA) kit. Reactive oxygen species (ROS) were measured using the 2'‐7’ dichlorofluorescein diacetate (DCFH‐DA) staining assay. In addition, the levels of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD), catalase (CAT), and caspase‐3 were measured using a commercial kit. siRNA was used to silence Bcl‐2, catalase, or STAT3. Western blotting was used to measure the change in the levels of proteins.ResultsDex improved the cell viability and inhibited the inflammatory response in H9c2 cells exposed to H/R treatment. In addition, Dex inhibited apoptosis and alleviated the endoplasmic reticulum (ER) stress and oxidative stress in H9c2 cells under the H/R treatment. Mechanism investigation showed that Dex inhibited the intrinsic pathway of apoptosis. Moreover, Dex enhanced the activation of the JAK2/STAT3 signaling pathway in H/R‐treated H9c2 cells.ConclusionAltogether, our findings suggested Dex as a promising therapeutic agent for myocardial I/R.