HG-induced Cardiomyocyte Injury Research Articles

Hmgcs2 is the hub gene in diabetic cardiomyopathy and is negatively regulated by Hmgcs2, promoting high glucose-induced cardiomyocyte injury.

Diabetic cardiomyopathy (DCM) represents a major cause of heart failure and a large medical burden worldwide. This study screened the potentially regulatory targets of DCM and analyzed their roles in high glucose (HG)-induced cardiomyocyte injury. Through GEO database, we obtained rat DCM expression chips and screened differentially expressed genes. Rat cardiomyocytes (H9C2) were induced with HG. 3-hydroxy-3-methylglutarylcoenzyme A synthase 2 (Hmgcs2) and microRNA (miR)-363-5p expression patterns in cells were measured by real-time quantitative polymerase chain reaction or Western blot assay, with the dual-luciferase assay to analyze their binding relationship. Then, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, lactate dehydrogenase assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, enzyme-linked immunosorbent assay, and various assay kits were applied to evaluate cell viability, cytotoxicity, apoptosis, inflammation responses, and oxidative burden. Hmgcs2 was the vital hub gene in DCM. Hmgcs2 was upregulated in HG-induced cardiomyocytes. Hmgcs2 downregulation increased cell viability, decreased TUNEL-positive cell number, reduced HG-induced inflammation and oxidative stress. miR-363-5p is the upstream miRNA of Hmgcs2. miR-363-5p overexpression attenuated HG-induced cell injury. Hmgcs2 had the most critical regulatory role in DCM. We for the first time reported that miR-363-5p inhibited Hmgcs2 expression, thereby alleviating HG-induced cardiomyocyte injury.

Dexmedetomidine ameliorates diabetic cardiomyopathy by inhibiting ferroptosis through the Nrf2/GPX4 pathway

ObjectiveDexmedetomidine (DEX) has been shown to have anti-apoptotic effects in diabetes mellitus, but its role in mitigating diabetic cardiomyopathy (DCM) through ferroptosis regulation is unclear.MethodsAn in vitro DCM model was established using H9C2 cells induced with high glucose (HG) and treated with DEX at varying doses and a nuclear factor erythroid 2-realated factor 2 (Nrf2) specific inhibitor ML385. Cell viability was evaluated using the MTT method after treatment with DEX or mannitol (MAN), and the dosage of DEX used in subsequent experimentation was determined. The effects of HG-induced high osmotic pressure were assessed using MAN as a control. Cell apoptosis was evaluated using flow cytometry. Protein levels of Bcl2, Bax, nuclear Nrf2, and glutathione peroxidase 4 (GPX4) were measured using Western blot. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) levels, Fe2+ concentration and reactive oxygen species (ROS) levels were measured using corresponding kits and dichlorodihydrofluorescein diacetate, respectively.ResultsTreatment with DEX or MAN had no effect on H9C2 cell viability. HG induction reduced H9C2 cell viability, increased cell apoptosis, upregulated levels of Bax, Fe2+, MDA, and ROS, and downregulated Bcl2 protein levels, SOD activity, and protein levels of nuclear Nrf2 and GPX4. DEX inhibited HG-induced H9C2 cell apoptosis, promoted Nrf2 nuclear translocation, and activated the Nrf2/GPX4 pathway. Inhibition of Nrf2 partially reversed the protective effects of DEX against HG-evoked H9C2 cell injury.ConclusionOur findings demonstrate that DEX attenuates HG-induced cardiomyocyte injury by inhibiting ferroptosis through the Nrf2/GPX4 pathway, providing potential therapeutic targets for DCM treatment.

Differentially expressed tRNA-derived fragments and their roles in primary cardiomyocytes stimulated by high glucose.

Diabetic cardiomyopathy (DCM) is a serious complication of diabetes mellitus that can cause malignant arrhythmia and sudden death and is associated with cardiomyocyte dysfunction induced by hyperglycemia. Emerging evidence has revealed that transfer RNA-derived fragments (tRFs), a novel class of noncoding RNAs, play a crucial role in a variety of pathophysiologic processes, including cell death, cell growth and proliferation. However, it remains unknown whether and how tRFs are involved in cardiomyocyte dysfunction during the progression of DCM. In this study, we found that cardiomyocyte abnormalities were induced by high glucose (HG) treatment, as demonstrated by a decrease in cell viability and autophagy activation as well as an increase in cell death and proinflammatory cytokine release. Moreover, HG treatment resulted in differential expression of tRFs in cardiomyocytes, of which 4 upregulated and 1 downregulated tRFs were observed compared with the control group. The differential expression of 4 upregulated tRFs was primarily involved in cardiac dysfunction-related processes, such as autophagy, AGE-RAGE signaling pathway in diabetic complications, MAPK signaling pathway, insulin signaling pathway, FoxO signaling pathway, insulin resistance and peroxisome pathways based on Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Furthermore, we found that tRF-5014a, the most significantly upregulated tRF among all tested tRFs, negatively regulated the expression of the autophagy-related protein ATG5. Importantly, inhibition of tRF-5014a not only abolished autophagy inactivation but also attenuated the decrease in cell viability and increase in cell death as well as proinflammatory cytokine release under HG conditions. These findings suggest that tRFs may contribute to HG-induced cardiomyocyte injury during DCM progression.

Long-chain noncoding RNA-GAS5/hsa-miR-138-5p attenuates high glucose-induced cardiomyocyte damage by targeting CYP11B2.

Objective: Diabetic cardiomyopathy (DCM) is one of the complications experienced by patients with diabetes. In recent years, long noncoding RNAs (lncRNAs) have been investigated because of their role in the progression of various diseases, including DCM. The purpose of the present study was to explore the role of lncRNA GAS5 in high glucose (HG)-induced cardiomyocyte injury and apoptosis.Materials and methods: We constructed HG-induced AC16 cardiomyocytes and a streptozotocin (STZ)-induced rat diabetes model. GAS5 was overexpressed and knocked out at the cellular level, and GAS5 was knocked down by lentiviruses at the animal level to observe its effect on myocardial injury. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of GAS5. Cell proliferation and apoptosis after GAS5 knockout were detected by CCK-8, TUNEL, and flow cytometry assays. ELISA was used to detect the changes in myocardial enzyme content in cells and animal myocardial tissues during the action of GAS5 on myocardial injury.Results: GAS5 expression was up-regulated in HG-treated AC16 cardiomyocytes and the rat diabetic myocardial injury model. The down-regulation of GAS5 could inhibit HG-induced myocardial damage. This work proved that the down-regulation of GAS5 could reverse cardiomyocyte injury and apoptosis by targeting miR-138 to down-regulate CYP11B2.Conclusion: We confirmed for the first time that the down-regulation of GAS5 could reverse CYP11B2 via the miR-138 axis to reverse HG-induced cardiomyocyte injury. This research might provide a new direction for explaining the developmental mechanism of DCM and potential targets for the treatment of myocardial injury.

Mitochondrial Fission and Mitophagy Coordinately Restrict High Glucose Toxicity in Cardiomyocytes.

Hyperglycemia-induced mitochondrial dysfunction plays a key role in the pathogenesis of diabetic cardiomyopathy. Injured mitochondrial segments are separated by mitochondrial fission and eliminated by autophagic sequestration and subsequent degradation in the lysosome, a process termed mitophagy. However, it remains poorly understood how high glucose affects the activities of, and the relationship between, mitochondrial fission and mitophagy in cardiomyocytes. In this study, we determined the functional roles of mitochondrial fission and mitophagy in hyperglycemia-induced cardiomyocyte injury. High glucose (30 mM, HG) reduced mitochondrial connectivity and particle size and increased mitochondrial number in neonatal rat ventricular cardiomyocytes, suggesting an enhanced mitochondrial fragmentation. SiRNA knockdown of the pro-fission factor dynamin-related protein 1 (DRP1) restored mitochondrial size but did not affect HG toxicity, and Mdivi-1, a DRP1 inhibitor, even increased HG-induced cardiomyocyte injury, as shown by superoxide production, mitochondrial membrane potential and cell death. However, DRP1 overexpression triggered mitochondrial fragmentation and mitigated HG-induced cardiomyocyte injury, suggesting that the increased mitochondrial fission is beneficial, rather than detrimental, to cardiomyocytes cultured under HG conditions. This is in contrast to the prevailing hypothesis that mitochondrial fragmentation mediates or contributes to HG cardiotoxicity. Meanwhile, HG reduced mitophagy flux as determined by the difference in the levels of mitochondria-associated LC3-II or the numbers of mitophagy foci indicated by the novel dual fluorescent reporter mt-Rosella in the absence and presence of the lysosomal inhibitors. The ability of HG to induce mitochondrial fragmentation and inhibit mitophagy was reproduced in adult mouse cardiomyocytes. Overexpression of Parkin, a positive regulator of mitophagy, or treatment with CCCP, a mitochondrial uncoupler, induced mitophagy and attenuated HG-induced cardiomyocyte death, while Parkin knockdown had opposite effects, suggesting an essential role of mitophagy in cardiomyocyte survival under HG conditions. Strikingly, Parkin overexpression increased mitochondrial fragmentation, while DRP1 overexpression accelerated mitophagy flux, demonstrating a reciprocal activation loop that controls mitochondrial fission and mitophagy. Thus, strategies that promote the mutual positive interaction between mitochondrial fission and mitophagy while simultaneously maintain their levels within the physiological range would be expected to improve mitochondrial health, alleviating hyperglycemic cardiotoxicity.

Inhibition of miR-223 attenuates the NLRP3 inflammasome activation, fibrosis, and apoptosis in diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is an independent and specific cardiomyopathy, which is associated with cardiac failure in diabetic patients. Currently, the pathogenesis of DCM is a popular research topic in the investigation of cardiovascular diseases. MicroRNAs (miRNAs) have been identified as the latent therapeutic targets for DCM. However, the functions and complex mechanisms of miRNAs in DCM have not been clarified. The cardiomyocyte injury model was established using high glucose (HG) ingestion, and the DCM rat model was established using 30 mg/kg streptozotocin. MicroRNA-223 (miR-223) expression was determined using qRT-PCR; the levels of NLRP3 inflammasome, fibrosis, and apoptosis-related genes and proteins were analyzed using qRT-PCR and western blot assays. Besides the morphological changes and fibrosis of myocardial tissues were evaluated using H&E and Masson staining. We discovered that miR-223 was highly expressed in the HG-induced cardiomyocyte injury model, and miR-223 inhibitor could further relieve the myocardial fibrosis and apoptosis, and inhibit NLRP3 inflammasome of HG-induced H9c2 cells. Additionally, we found that inhibition of miR-223 had obvious positive effects on the cardiac dysfunction and reduced the elevation of blood sugar in the DCM model rats. We found that the miRNA-223 inhibitor could improve the morphological structure and the degree of fibrosis in myocardial tissues in the DCM model rats. Moreover, we verified that inhibition of miR-223 could suppress the NLRP3 inflammasome activation, and alleviate myocardial fibrosis and apoptosis of the DCM model rats. In conclusion, our results suggested that miR-223 might be an underlying therapeutic target for DCM by reducing NLRP3 inflammasome activation, fibrosis, and apoptosis.

Hyperglycemia-induced cardiomyocyte death is mediated by lysosomal membrane injury and aberrant expression of cathepsin D

Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. Previous studies have shown an association between lysosomal dysfunction and diabetic heart injury. The present study examined if mimicking hyperglycemia in cultured cardiomyocytes could induce lysosomal membrane permeabilization (LMP), leading to the release of lysosome enzymes and subsequent cell death. High glucose (HG) reduced the number of lysosomes with acidic pH as shown by a fluorescent pH indicator. Also, HG induced lysosomal membrane injury as shown by an accumulation of Galectin3-RFP puncta, which was accompanied by the leakage of cathepsin D (CTSD), an aspartic protease that normally resides within the lysosomal lumen. Furthermore, CTSD expression was increased in HG-cultured cardiomyocytes and in the hearts of 2 mouse models of type 1 diabetes. Either CTSD knockdown with siRNA or inhibition of CTSD activity by pepstatin A markedly diminished HG-induced cardiomyocyte death, while CTSD overexpression exaggerated HG-induced cell death. Together, these results suggested that HG increased CTSD expression, induced LMP and triggered CTSD release from the lysosomes, which collectively contributed to HG-induced cardiomyocyte injury.

Activation of TGR5 Partially Alleviates High Glucose-Induced Cardiomyocyte Injury by Inhibition of Inflammatory Responses and Oxidative Stress.

High glucose- (HG-) induced cardiomyocyte injury is the leading cause of diabetic cardiomyopathy, which is associated with the induction of inflammatory responses and oxidative stress. TGR5 plays an important role in the regulation of glucose metabolism. However, whether TGR5 has cardioprotective effects against HG-induced cardiomyocyte injury is unknown. Neonatal mouse cardiomyocytes were isolated and incubated in a HG medium. Protein and mRNA expression was detected by western blotting and RT-PCR, respectively. Cell apoptosis was determined by Hoechst 33342 staining and flow cytometry. After treatment of cells with HG, TGR5-selective agonist INT-777 reduced the increase in expression of proinflammatory cytokines and NF-κB, whereas pretreatment of cells with TGR5 shRNA significantly reduced the inhibitory effects of INT-777. We also found that INT-777 increased the protein expression of Nrf2 and HO-1. In the presence of TGR5 shRNA, the expression of Nrf2 and HO-1 was reduced, indicating that TGR5 may exert an antioxidant effect partially through the Nrf2/HO-1 pathway. Furthermore, INT-777 treatment inhibited HG-induced ROS production and apoptosis that were attenuated in the presence of TGR5 shRNA or ZnPP (HO-1 inhibitor). Activation of TGR5 has cardioprotective effects against HG-induced cardiomyocyte injury and could be a pharmacological target for the treatment of diabetic cardiomyopathy.

Knockdown of long non-coding RNA LUCAT1 reverses high glucose-induced cardiomyocyte injury via targeting CYP11B2.

Diabetic cardiomyopathy (DCM) is one of the major complications in patients with diabetes mellitus. Recently, long noncoding RNAs (lncRNAs) have been well concerned for their roles in the progression of multiple diseases, including DCM. In this research, we aimed to explore the role of lncRNA LUCAT1 in cardiomyocyte injury and apoptosis induced by high glucose (HG) in vitro. High glucose-induced (HG-induced) AC16 cardiomyocytes transfected with LUCAT1 shRNA were constructed. LUCAT1 expression was detected by real-time quantitative polymerase chain reaction (RT-qPCR). Subsequently, cell proliferation and cell apoptosis were detected after LUCAT1 knockdown in HG-induced AC16 cells. Moreover, RT-qPCR and Western blot assay were performed to explore the potential underlying mechanism of LUCAT1 in DCM. The expression of LUCAT1 was significantly upregulated in HG-treated AC16 cardiomyocytes. Moreover, knockdown of LUCAT1 could reverse cardiomyocyte injury and apoptosis through downregulating CYP11B2. We first demonstrated that knockdown of LUCAT1 could reverse HG-induced cardiomyocyte injury by down-regulating CYP11B2. Our findings might offer a new direction for interpreting the mechanism of DCM development.

Phloretin protects against cardiac damage and remodeling via restoring SIRT1 and anti-inflammatory effects in the streptozotocin-induced diabetic mouse model.

Diabetic cardiomyopathy increases the risk of heart failure independent of coronary artery disease and hypertension. Phloretin (PHL) shows anti-inflammatory effects in macrophages. In this study, we explored the protective effects of PHL on high glucose (HG)-induced injury in diabetic cardiomyopathy in vivo and in vitro. Using streptozotocin-induced diabetic mouse model and incubating cardiac cells line under a HG environment, PHL were evaluated of the activities of anti-inflammation and anti-fibrosis. In the study, PHL treatment ameliorated cardiomyocyte inflammation injury, and reduced fibrosis in vivo and in vitro. PHL also improved cardiac biochemical criterions after 8 weeks of induction of diabetes in C57BL/6 mice. Molecular docking results indicated that silent information regulator 2 homolog 1 (SIRT1) bound to PHL directly and that SIRT1 expression was upregulated in the PHL-treated group in HG-induced H9C2 cells. Protective effect of PHL was been eliminated in silence SIRT1 H9C2 cells. Taken together, these results suggested that PHL suppressed HG-induced cardiomyocyte injury via restoring SIRT1 expression.

Protective effect of angiotensin-(1-7) against hyperglycaemia-induced injury in H9c2 cardiomyoblast cells via the PI3K̸Akt signaling pathway.

Angiotensin-(1-7) [Ang-(1-7)], a heptapeptide mainly generated from cleavage of AngI and AngII, possesses physiological and pharmacological properties, including anti-inflammatory and antidiabetic properties. Activation of the phosphoinositide 3-kinase and protein kinase B (PI3K/Akt) signaling pathway has been confirmed to participate in cardioprotection against hyperglycaemia-induced injury. The aim of the present study was to test the hypothesis that Ang-(1-7) protects H9c2 cardiomyoblast cells against high glucose (HG)-induced injury by activating the PI3K/Akt pathway. To examine this hypothesis, H9c2 cells were treated with 35 mmol/l (mM) glucose (HG) for 24 h to establish a HG-induced cardiomyocyte injury model. The cells were co-treated with 1 μmol/l (μM) Ang-(1-7) and 35 mM glucose. The findings of the present study demonstrated that exposure of H9c2 cells to HG for 24 h markedly induced injury, as evidenced by an increase in the percentage of apoptotic cells, generation of reactive oxygen species and level of inflammatory cytokines, as well as a decline in cell viability and mitochondrial luminosity. These injuries were significantly attenuated by co-treatment of the cells with Ang-(1-7) and HG. In addition, PI3K/Akt phosphorylation was suppressed by HG treatment, but this effect was abolished when the H9c2 cells were co-treated with Ang-(1-7) and HG. Furthermore, the cardioprotection of Ang-(1-7) against HG-induced injury in H9c2 cardiomyoblasts was highly attenuated in the presence of either D-Ala7-Ang-(1-7) (A-779, an antagonist of the Mas receptor) or LY294002 (an inhibitor of PI3K/Akt). In conclusion, the present study provided new evidence that Ang-(1-7) protects H9c2 cardiomyoblasts against HG-induced injury by activating the PI3K/Akt signaling pathway.

Abstract 42: Lysosomal Membrane Permeabilization Contributes to Hyperglycemia-Induced Cardiomyocytes Dysfunction

Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. Previous studies have shown an association between lysosomal dysfunction and diabetic heart failure. Lysosomal membrane permeabilization (LMP) is a prominent feature of lysosomal dysfunction. We examined if high glucose (HG) induces LMP in cardiomyocyte. Immunofluorescent microscopy showed the release of cathepsin D (CTSD) from lysosome under HG, suggesting that HG induces LMP. To determine whether LMP and the ensuing CTSD expression play a role in HG-induced cardiomyocyte injury, we treated cells with siRNA targeting CTSD or low dose of CTSD inhibitor pepstatinA (0.5ng/mL). Remarkably, both CTSD down-regulation approaches reduced HG-induced myocyte injury, as shown by elevated cleavage of Caspase3 and by propidium iodide staining. These results suggest that LMP and the ensuing CTSD expression mediate HG-induced cardiotoxicity. In addition, by using LMP/lysophagy reporter tfGalectin3, we found that HG-induced LMP was accompanied by impaired lysophagy, suggesting a failed elimination of damaged lysosomes by lysophagy, which may exacerbate HG-induced LMP. Indeed, the lysophagy inducer L-leucyl-L-leucine methyl ester (LLOMe) stimulated lysophagy flux, attenuated HG-induced LMP, and restored the beating frequency of HG-treated cardiomuyocytes. Together, these findings demonstrate a novel mechanism by which high glucose impairs lysophagy, induces LMP and triggers the release of CTSD, which collectively contribute to hyperglycemic cardiotoxicity.

Heat shock protein 90/Akt pathway participates in the cardioprotective effect of exogenous hydrogen sulfide against high glucose-induced injury to H9c2 cells.

It has been reported that exogenous hydrogen sulfide(H2S) protects against high glucose(HG)-induced cardiac injury and has a modulatory effect on heat shock protein(HSP) and Akt, which play a cardioprotective role. In this study, we examined whether the HSP90/Akt pathway contributes to the protective effects of exogenous H2S against HG-induced injury to H9c2 cardiac cells. Our results revealed that the exposure of H9c2 cardiac cells to 35mM glucose(HG) for 1to 24h decreased the expression of HSP90 and markedly reduced the expression level of phosphorylated (p)-Akt in a time-dependent manner. Co-exposure of the cells to HG and geldanamycin(GA; an inhibitor of HSP90) aggravated the inhibition of the p-Akt expression level by HG. Of note, treatment of the cells with 400µM NaHS(a donor of H2S) for 30min prior to exposure to HG significantly attenuated the HG-induced decrease in the expression levels of both HSP90 and p-Akt, along with inhibition of HG-induced cell injury, as indicated by the increase in cell viability and superoxide dismutase(SOD) activity, and by a decrease in the number of apoptotic cells, reactive oxygen species(ROS) generation, as well as by the decreased dissipation of mitochondrial membrance potential(MMP). Importantly, treatment of the cells with GA or LY294002(an inhibitor of Akt) prior to exposure to NaHS and HG considerably blocked the cardioprotective effects of NaHS against the HG-induced injury mentioned above. On the whole, the findings of this study demonstrate that the inhibition of the HSP90/Akt pathway may be an important mechanism responsible for HG-induced cardiomyocyte injury. We also provide novel evidence that exogenous H2S protects H9c2 cells against HG-induced injury by activating the HSP90/Akt pathway.

Exogenous hydrogen sulfide alleviates high glucose-induced cardiotoxicity via inhibition of leptin signaling in H9c2 cells

Hydrogen sulfide (H₂S) protects cardiomyoblasts against high glucose (HG)-induced injury by inhibiting the activation of p38 mitogen-activated protein kinase (MAPK). This study aims to determine whether the leptin-p38 MAPK pathway is involved in HG-induced injury and whether exogenous H2S prevents the HG-induced insult through inhibition of the leptin-p38 MAPK pathway in H9c2 cells. H9c2 cells were treated with 35 mM glucose (HG) for 24 h to establish a HG-induced cardiomyocyte injury model. Cell viability; mitochondrial membrane potential (ΔΨ m); apoptosis; reactive oxygen species (ROS) level; and leptin, leptin receptor, and p38 MAPK expression level were measured by the methods indicated. The results showed pretreatment of H9c2 cells with NaHS before exposure to HG led to an increase in cell viability, decrease in apoptotic cells, ROS generation, and a loss of ΔΨ m. Exposure of H9c2 cells to 35 mM glucose for 24 h significantly upregulated the expression levels of leptin and leptin receptors. The increased expression levels of leptin and leptin receptors were markedly attenuated by pretreatment with 400 μM NaHS. In addition, the HG-induced increase in phosphorylated (p) p38 MAPK expression was ameliorated by pretreatment with 50 ng/ml leptin antagonist. In conclusion, the present study has demonstrated for the first time that the leptin-p38 MAPK pathway contributes to the HG-induced injury in H9c2 cells and that exogenous H₂S protects H9c2 cells against HG-induced injury at least in part by inhibiting the activation of leptin-p38 MAPK pathway.

Naringin Inhibits ROS‐activated MAPK Pathway in High Glucose‐induced Injuries in H9c2 Cardiac Cells

Naringin, an active flavonoid isolated from citrus fruit extracts, exhibits biological and pharmacological properties, such as antioxidant activity and antidiabetic effect. Mitogen-activated protein kinase (MAPK) signalling pathway has been shown to participate in hyperglycaemia-induced injury. The present study tested the hypothesis that naringin protects against high glucose (HG)-induced injuries by inhibiting MAPK pathway in H9c2 cardiac cells. To examine this, the cells were treated with 35mM glucose (HG) for 24hr to establish a HG-induced cardiomyocyte injury model. The cells were pre-treated with 80μM naringin for 2hr before exposure to HG. The findings of this study showed that exposure of H9c2 cells to HG for 24hr markedly induced injuries, as evidenced by a decrease in cell viability, increases in apoptotic cells and reactive oxygen species (ROS) production, as well as dissipation of mitochondrial membrance potential (MMP). These injuries were significantly attenuated by the pre-treatment of cells with either naringin or SB203580 (a selective inhibitor of p38 MAPK) or U0126 (a selective inhibitor of extracellular signal regulated kinase 1/2, ERK1/2) or SP600125 (a selective inhibitor of c-jun N-termanal kinase, JNK) before exposure to HG, respectively. Furthermore, exposure of cells to HG increased the phosphorylation of p38 MAPK, ERK1/2 and JNK. The increased activation of MAPK pathway was ameliorated by pre-treatment with either naringin or N-acetyl-L-cysteine (NAC), a ROS scavenger, which also reduced HG-induced cytotoxicity and apoptosis, leading to increase in cell viability and decrease in apoptotic cells. In conclusion, our findings provide new evidence for the first time that naringin protects against HG-induced injuries by inhibiting the activation of MAPK (p38 MAPK, ERK1/2 and JNK) and oxidative stress in H9c2 cells.

Exogenous hydrogen sulfide protects H9c2 cardiac cells against high glucose-induced injury by inhibiting the activities of the p38 MAPK and ERK1/2 pathways

Hyperglycemia is a risk factor for the development of diabetic cardiovascular complications, which are associated with the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we demonstrate the inhibitory effects of exogenous hydrogen sulfide (H₂S) on the activation of the MAPK pathway. The aim of the present study was to determine whether exogenous H₂S prevents high glucose (HG)-induced injury by inhibiting the activation of the p38 MAPK and extracellular signal-regulated kinase (ERK)1/2 (members of MAPK) pathways in cardiomyoblasts (H9c2 cells). The findings of the present study demonstrated that the treatment of H9c2 cells with HG (35 mM glucose) for 24 h not only significantly induced injury, including cytotoxicity, apoptosis, overproduction of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (MMP), but also upregulated the expression levels of phosphorylated (p)-p38 MAPK and p-ERK1/2. The increased expression levels of p-p38 MAPK and p-ERK1/2 were markedly reduced by pre-treatment of the H9c2 cells with 400 µM sodium hydrogen sulfide (NaHS; a donor of H2S) prior to exposure to 35 mM glucose. Importantly, pre-treatment of the cells with 400 µM NaHS or 3 µM SB203580 (a selective inhibitor of p38 MAPK) or 15 µM U0126 (a selective inhibitor of ERK1/2) attenuated the HG-induced cardiomyocyte injury, leading to an increase in cell viability and a decrease in the number of apoptotic cells, preventing ROS generation, as well as the loss of MMP. In addition, pre-treatment of the cells with 1,000 µM N‑acetyl‑L‑cysteine (a ROS scavenger) prior to exposure to HG ameliorated the HG-induced cytotoxicity. Taken together, the data from the present study demonstrate for the first time, to our knowledge, that exogenous H2S exerts a protective effect against HG‑induced injury by inhibiting the activation of the p38 MAPK and ERK1/2 pathways and preventing oxidative stress in H9c2 cells.