Nesfatin-1 protects H9c2 cardiomyocytes against cobalt chloride-induced hypoxic injury by modulating the MAPK and Notch1 signaling pathways

Diabetes aggravates myocardial ischemia-reperfusion (I/R) injury because of the combination effects of changes in glucose and lipid energy metabolism, oxidative stress, and systemic inflammatory response. Studies have indicated that myocardial I/R may coincide and interact with sepsis and inflammation. However, the role of LPS in hypoxia/reoxygenation (H/R) injury in cardiomyocytes under high glucose conditions is still unclear. Our objective was to examine whether lipopolysaccharide (LPS) could aggravate high glucose- (HG-) and hypoxia/reoxygenation- (H/R-) induced injury by upregulating ROS production to activate NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes. H9C2 cardiomyocytes were exposed to HG (30 mM) condition with or without LPS, along with caspase-1 inhibitor (Ac-YVAD-CMK), inflammasome inhibitor (BAY11-7082), ROS scavenger N-acetylcysteine (NAC), or not for 24 h, then subjected to 4 h of hypoxia followed by 2 h of reoxygenation (H/R). The cell viability, lactate dehydrogenase (LDH) release, caspase-1 activity, and intracellular ROS production were detected by using assay kits. The incidence of pyroptosis was detected by calcein-AM/propidium iodide (PI) double staining kit. The concentrations of IL-1β and IL-18 in the supernatants were assessed by ELISA. The mRNA levels of NLRP3, ASC, and caspase-1 were detected by qRT-PCR. The protein levels of NF-κB p65, NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18 were detected by western blot. The results indicated that pretreatment LPS with 1 μg/ml not 0.1 μg/ml could efficiently aggravate HG and H/R injury by activating NLRP3 inflammasome to mediate pyroptosis in H9C2 cells, as evidenced by increased LDH release and decreased cell viability in the cells, and increased expression of NLRP3, ASC, cleaved caspase-1 (p10), IL-1β, and IL-18. Meanwhile, Ac-YVAD-CMK, BAY11-7082, or NAC attenuated HG- and H/R-induced H9C2 cell injury with LPS stimulated by reversing the activation of NLRP3 inflammasome-mediated pyroptosis. In conclusion, LPS could increase the sensitivity of H9C2 cells to HG and H/R and aggravated HG- and H/R-induced H9C2 cell injury by promoting ROS production to induce NLRP3 inflammasome-mediated pyroptosis.

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.

Objective To research the protective effect of insulin(IN)on lipopolysaccharide(LPS)-induced impairments of rat cardiomyocytes H9c2, and the role of uncoupling protein 2(UCP2)in this process. Methods Using randomized controlled grouping, after cultured for 24 h, H9c2 cells were randomly divided into 5 groups as follows: control group, LPS stimulation group (LPS group), LPS+ 70 IU/L IN group(IN 70 IU/L group), LPS+ 350 IU/L IN group(IN 350 IU/L group), and LPS+ 700 IU/L IN group(IN 700 IU/L group). H9c2 cells in IN group were treated with 70 IU/L, 350 IU/L or 700 IU/L IN 15 min before LPS stimulation, and H9c2 cells in control group were treated with an equal volume of saline.After that, cells in group LPS and IN were treated with LPS for 24 h. Lactate dehydrogenase (LDH)in the culture was determined with LDH detecting assay kit.The activity of reactive oxygen species (ROS)and superoxide dismutase (SOD), and content of malonaldehyde (MDA)were determined by colorimetric detection.Cell viability was evaluated by cell count kit-8.The expressions of UCP2 in transcription and translation levels were detected through transcription polymerase chain reaction and Western blot respectively. Results The levels of LDH, MDA, and intracellular ROS in LPS group significantly increased compared with control group[LDH: (829.3±75.3)U/L vs (223.5±23.6)U/L, MDA: (60.90±5.73) nmol/mgprot vs (19.70±1.99)nmol/mgprot, ROS: (410.2±81.6)U/well vs (94.3±18.5)U/well, all P<0.05)], while the cell viability and SOD activity significantly decreased[cell viability: 0.822±0.058 vs 1.012±0.023, SOD: (49.20±5.81)U/mgprot vs (89.80±2.57)U/mgprot, all P<0.05]. And the mRNA and protein expressions of UCP2 in LPS stimulation group were up-regulated(1.867±0.130 vs 1.028±0.097, 0.288±0.018 vs 0.180±0.008, all P<0.05).350 IU/L and 700 IU/L IN intervention significantly decreased the levels of LDH, MDA and intracellular ROS[LDH: (568.2±35.7)U/L, (622.8±27.6)U/L vs (829.3±75.3)U/L, MDA: (29.20±4.20)nmol/mgprot, (42.10±2.32)nmol/mgprot vs (60.90±5.73)nmol/mgprot, ROS: (270.3±46.8)U/well, (301.5±16.9)U/well vs (410.2±81.6)U/well, all P<0.05], increased the cell survival and the levels of SOD activity[cell viability: 0.960±0.029, 0.906±0.039 vs 0.822±0.058, SOD: (75.20±2.21)U/mgprot, (61.20±3.38)U/mgprot vs (49.20±5.81)U/mgprot, all P<0.05]. And IN with 350 IU/L and 700 IU/L increased the mRNA and protein expression of UCP2(3.830±0.265, 2.855±0.215 vs 1.867±0.130, 0.464±0.215, 0.355±0.006 vs 0.288±0.018, all P<0.05). Compared with 70 IU/L and 700 IU/L IN group, 350 IU/L IN group had better results. Conclusions IN attenuates LPS-induced oxidative injury in H9c2 cells, which is probably mediated through up-regulating the expression of UCP2. Key words: Insulin; Lipopolysaccharide; H9c2 cell; UCP2; Oxidative stress

Objective To explore the anti-inflammatory effect of dexmedetomidine(Dex) in attenuating hypoxia/reoxgenation(H/R) injury of H9C2 cells. Methods Rat cardiomyocyte cell line H9C2 cells were cultured in vitro, and Na2S2O4 was used to establish the hypoxia and reoxygenation injury model on H9C2 cells. Cultured H9C2 cells were divided into three groups: normal control group (group C), the cells were cultured as usual, group H/R, the cells were subjected to 1 h hypoxia induced by 4 mmol/L Na2S2O4, then followed by 12 h reoxygenation by replaced the normal media, Dex preconditioning group (group D), Dex at various concentrations(0.1, 1.0, 10.0 μmol/L) was performed prior to Na2S2O4-induced H/R injury, the rest of the steps are essentially the same as the group H/R. The cell survival rate 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide(MTT) and lactate dehydrogenase(LDH) activity were detected after treatment, the cellular morphology was observed by inverted miscroscope, the TNF-α, IL-6, IL-1β mRNA expression were detected by reverse transcription-polymerase chain reaction(RT-PCR). Results Compared with the group C, the cell survival of group H/R decreased to(44±12)%(P<0.01), LDH activity increased significantly(P<0.01), compared with the group H/R, the cell survival rate and LDH activity of the group D were improved, Dex at 1 μmol/L concentration preconditioning significantly restored cell viability to(75±7)%(P<0.05) and reduced the activity of LDH significantly(P<0.05). The condition of cell necrosis, irregular cell arrangement, and the decline of cell membrane refractive index caused by H/R injury were partly reversed by Dex. Compared with the group C, the TNF-α, IL-6, IL-1β mRNA expression level of the group D and the group H/R increased significantly. Compared with group H/R, the TNF-α, IL-6, IL-1β mRNA expression levels of group D decreased significantly(P<0.05). Conclusions Dex preconditioning attenuates hypoxia and reoxygenation injury of H9C2 cells. The mechanism may be related to inhibiting inflammatory reaction. Key words: Dexmedetomidine; Proconditioning; Hypoxia/reoxygenation injury; Inflammatory response

The present study aimed to elucidate the function of long noncoding RNA GARS1-DT in hypoxia-induced injury in ex-vivo cardiomyocytes and explore its underlying mechanism. Hypoxic injury was confirmed in H9C2 cells by the determination of cell viability, migration, invasion, and apoptosis. GARS1-DT expression was estimated in H9C2 cells after hypoxia. We then measured the effects of GARS1-DT knockdown on hypoxia-induced H9C2 cells. The interaction between GARS1-DT and miR-212-5p was also investigated. Hypoxia treatment led to cell damage in H9C2 cardiomyocytes, accompanied with the upregulation of GARS1-DT expression. Transfection of GARS1-DT small interfering RNA remarkably attenuated hypoxia-induced injury by enhancing cell viability, migration, and invasion, and reducing apoptosis. Furthermore, GARS1-DT served as an endogenous sponge for miR-212-5p, and its expression was negatively regulated by GARS1-DT. The effects of GARS1-DT knockdown on hypoxia-induced injury were significantly abrogated by miR-212-5p silence. Besides, suppression of GARS1-DT activated PI3K/AKT pathway in hypoxia-treated H9C2 cells, which were reversed by inhibition of miR-212-5p. Our findings demonstrated the novel molecular mechanism of GARS1-DT/miR-212-5p/PI3K/AKT axis on the regulation of hypoxia-induced myocardial injury in H9C2 cells, which may provide potential therapeutic targets for acute myocardial infarction treatment.

Myocardial ischemia is accompanied with hypoxia injury in myocardial cells. Long noncoding RNAs (lncRNA) CAMK2D-associated transcript 1 (C2dat1) C2dat1) has been linked with several ischemic diseases. However, the investigation regarding its role in myocardial ischemia is relatively rare. The aim of this study was to examine the role of C2dat1 in hypoxia response in H9c2 cells. H9c2 cells were subjected to hypoxia to evoke cell damage. Expressions of C2dat1, miR-22, and VEGF in H9c2 cells were altered by transfection, and then cell survival, migration, and invasion were respectively assessed posttransfection. Regulatory relationship between C2dat1, miR-22, and VEGF, as well as the involvement of PI3K/AKT/mTOR and JAK/STAT3 pathways in H9c2 cells injury was then studied. C2dat1 upregulation ameliorated hypoxia injury in H9c2 cells due to the increased viability, migration, and invasion, as well as the decreased apoptosis. miR-22 was negatively regulated by C2dat1. The effects of C2dat1 on H9c2 cells injured by hypoxia were attenuated when miR-22 was overexpressed. VEGF was a target gene of miR-22, and VEGF exerted similar protective effects to C2dat1. Finally, we found that silence of C2dat1 deactivated PI3K/AKT/mTOR and JAK/STAT3 pathways via regulating miR-22 and its downstream gene VEGF. C2dat1-miR-22-VEGF axis could regulate hypoxia injury in H9c2 cells. C2dat1 alleviated hypoxia injury possibly via downregulating miR-22, then upregulating VEGF, which further enhancing the activation of PI3K/AKT/mTOR and JAK/STAT3 pathways.

The present study aimed to elucidate the role of Notch signaling in the development of myocardial infarction (MI) concomitant with diabetes in vivo and in vitro and evaluated the therapeutic effect of the Notch signaling in vitro. Streptozotocin-induced diabetic rats were subjected to 25 min of ischemia and 2 h of reperfusion. Cardiac troponin T (cTnT) and creatine kinase-MB (CK-MB) isoenzyme levels were detected. Infarct size was measured by 2,3,5-triphenyltetrazolium chloride staining. Myocardial apoptosis and fibrosis were examined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and Masson Trichrome staining, respectively. The mRNA and protein levels of Notch signaling components, including Notch1, Notch4, Delta-like 1, Jagged1, Mastermind-like protein 1 and p300, were quantified by reverse transcription-quantitative polymerase chain reaction and western blotting analyses, respectively. H9c2 cells were treated with/without 33 mM high glucose (HG) and/or subjected to hypoxia in the presence/absence of Jagged1. Cell viability and apoptosis were determined by MTT assay and Annexin V-fluorescein isothiocyanate/propidium iodide assay. Levels of the Notch signaling pathway members were examined. The present findings revealed that diabetes elevated CK-MB and cTnT, increased infarct size, induced myocardial apoptosis and inhibited the Notch signaling pathway in vivo after ischemia/reperfusion. Ischemia/reperfusion augmented the severity of MI in diabetic rats. Furthermore, HG reduced cell viability and induced cell apoptosis in H9c2 cells after hypoxia exposure, which was inhibited by Jagged1. We also found that HG inhibited Notch signaling in H9c2 cells after hypoxia, whereas Jagged1 exerted its cardioprotective effect on hypoxic injury (in HG environments or not) by activating the Notch signaling pathway. In conclusion, these findings suggest that diabetes promoted the progression of MI in vivo and in vitro via the inhibition of the Notch signaling pathway. Jagged1 may protect against MI in in vitro models by activating Notch signaling.

Dalspinin isolated from Spermacoce hispida (Linn.) protects H9c2 cardiomyocytes from hypoxic injury by modulating oxidative stress and apoptosis

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

Background. The intravenous anesthetic propofol is reported to be a cardioprotective agent against ischemic-reperfusion injury in the heart. However, the regulatory mechanism still remains unclear. Methods. In this study, we used H9c2 cell line under condition of oxygen glucose deprivation (OGD) followed by reperfusion (OGD/R) to induce in vitro cardiomyocytes ischemia-reperfusion injury. Propofol (5, 10, and 20 μM) was added to the cell cultures before and during the OGD/R phases to investigate the underlying mechanism. Results. Our data showed that OGD/R decreased cell viability, and increased lactate dehydrogenase leakage, and reactive oxygen species and malondialdehyde production in H9c2 cells, all of which were significantly reversed by propofol. Moreover, we found that propofol increased both the activities and protein expressions of superoxide dismutase and catalase. In addition, propofol increased FoxO1 expression in a dose-dependent manner and inhibited p-AMPK formation significantly. Conclusions. These results indicate that the propofol might exert its antioxidative effect through FoxO1 in H9c2 cells, and it has a potential therapeutic effect on cardiac disorders involved in oxidative stress.

Recovery of the blood supply is the most effective treatment against ischemic heart disease; however, it is also a major cause of myocardial ischemia/reperfusion injury in clinical therapy. Curcumin has been reported to possess beneficial effects against hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury by regulating cell proliferation, apoptosis and antioxidant enzyme activity. The aim of the present study was to investigate the molecular mechanisms underlying the effects of curcumin on H/R-injured cardiomyocytes. H9C2 cardiomyocytes were pretreated with curcumin, and then cultured under H/R conditions. The viability of H9C2 cells was measured using a Cell Counting kit-8 assay, and the levels of intracellular lactate dehydrogenase (LDH), malondialdehyde (MDA) and superoxide dismutase (SOD) were measured to assess cell injury. Levels of reactive oxygen species (ROS) and apoptosis were evaluated by flow cytometry. The expression levels of Notch intracellular domain (NICD) and numerous downstream genes were analyzed via reverse transcription-quantitative polymerase chain reaction and western blotting. The results revealed that curcumin protected H9C2 cells against H/R-induced injury, reversing the H/R-induced increases in LDH and MDA levels, and decreases in SOD levels. ROS levels in H/R-induced cells were also significantly downregulated by curcumin treatment (P<0.01), and the apoptotic rate was significantly decreased from 15.13% in the H/R group to 7.7% in the H/R + curcumin group (P<0.01). The expression levels of NICD, hairy and enhancer of split (Hes)-1, Hes-5 and hairy/enhancer-of-split related with YRPW motif protein 1 (Hey-1) were significantly decreased in H/R-treated cells following curcumin treatment. Treatment with Jagged1 attenuated the effects of curcumin on cell viability, ROS levels and apoptosis; the Notch pathway was also reactivated. The present study indicated that there was a role for the Notch pathway in the protective effects of curcumin against H/R-induced cardiomyocyte injury, suggesting that downregulation of the Notch pathway may alleviate H/R-induced injury in H9C2 cells.

Baicalin, a flavonoid glycoside separated from Scutellaria baicalensis, has cardioprotection against ischaemia/reperfusion (I/R) injury. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is considered as an endogenous protective mechanism against I/R injury depending on its anti-oxidant and anti-apoptotic characteristics. The present study demonstrates whether ALDH2 contributes to the cardioprotection of baicalin against hypoxia/reoxygenation (H/R)-inudced H9c2 cardiomyocytes injury. Our results observed that H/R treatment resulted in a significant decrease in cells viability and obvious increases in caspase-3 activity and apoptosis rate in H9c2 cells, while these alterations were evidently reversed by baicalin pretreatment. Simultaneously, baicalin mitigated H/R-induced the decreases in the levels of ALDH2 mRNA and protein as well as the activity of ALDH2 in H9c2 cells. However, we found that daidzin, an ALDH2 antagonist, remarkably attenuated baicalin-elicited inhibitory action on H/R-induced the downregulation of cells viability and Bcl-2 protein expression, and the upregulations of caspase-3 activity, apoptosis rate, cytochrome c and Bax proteins expressions in H9c2 cells. In addition, baicalin reversed H/R-induced oxidative stress as evidenced by the downregulation of malondialdehyde (MAD) and 4-hydroxy aldehydes (4-HNE) levels, the inhibition of endogenous reactive oxygen species (ROS) generation, and the downregulation of superoxide dismutase (SOD) activity induced by H/R treatment, while these effects were also blocked by daidzin. Furthermore, we found that Alda-1, an ALDH2 agonist, also abolished H/R-induced cytotoxicity, apoptosis, and oxidative stress, indicating that ALDH2 mediated H/R-induced H9c2 cell injury. Overall, these results suggested that baicalin prevents H/R-induced apoptosis and oxidative stress through enhancing ALDH activity and expression in H9c2 cardiomyocytes.

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.

Neuroinflammation mediated by the activated microglia is suggested to play a pivotal role in the pathogenesis of hypoxic brain injury; however, the underlying mechanism of microglia activation remains unclear. Here, we show that the canonical Notch signaling orchestrates microglia activation after hypoxic exposure which is closely associated with multiple pathological situations of the brain. Notch-1 and Delta-1 expression in primary microglia and BV-2 microglial cells was significantly elevated after hypoxia. Hypoxia-induced activation of Notch signaling was further confirmed by the concomitant increase in the expression and translocation of intracellular Notch receptor domain (NICD), together with RBP-Jκ and target gene Hes-1 expression. Chemical inhibition of Notch signaling with N-[N-(3,5-difluorophenacetyl)-1-alany1- S-phenyglycine t-butyl ester (DAPT), a γ-secretase inhibitor, effectively reduced hypoxia-induced upregulated expression of most inflammatory mediators. Notch inhibition also reduced NF-κB/p65 expression and translocation. Remarkably, Notch inhibition suppressed expression of TLR4/MyD88/TRAF6 pathways. In vivo, Notch signaling expression and activation in microglia were observed in the cerebrum of postnatal rats after hypoxic injury. Most interestingly, hypoxia-induced upregulation of NF-κB immunoexpression in microglia was prevented when the rats were given DAPT pretreatment underscoring the interrelationship between Notch signaling and NF-κB pathways. Taken together, we conclude that Notch signaling is involved in regulating microglia activation after hypoxia partly through the cross talk between TLR4/MyD88/TRAF6/NF-κB pathways. Therefore, Notch signaling may serve as a prospective target for inhibition of microglia activation known to be implicated in brain damage in the developing brain.

To evaluate the protective effect of tryptophan on an experimentally produced hypoxic myocardial injury via biochemical and pathological parameters. Materials and methods: A total of 26 rabbits were divided into 3 groups. Group 1 (n = 9) was only exposed to hypoxia. Group 2 (n = 10) was exposed to hypoxia and received L-tryptophan (200 mg/kg per day, orally for 5 days). Group 3 (n = 7) was the control group. Before the hypoxic injury and after the delivery of the medication, serum samples were taken for troponin-I, creatine kinase myocardial isoenzymes (CK-MB), lactate dehydrogenase (LDH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), malondialdehyde (MDA), and nitric oxide (NO) analysis, and then the rabbits were sacrificed. Next, the myocardium samples were taken and the myocardial NO, MDA, SOD, and GSH-Px enzyme activity levels were studied histopathologically. Results: In group 1, Serum GSH-Px and SOD activities were decreased. Conversely, troponin-I, CK-MB, and LDH were elevated. Severe cardiac injury was observed histopathologically. In group 2, serum troponin-I and SOD values were increased. Mild cardiac injury was demonstrated histopathologically. When groups 1 and 2 were compared, tissue NO and MDA levels in group 1 were higher compared to group 2, but GSH-Px level was found decreased in group 1. Conclusion: Our findings support that there is a clear effect of the free oxygen radicals and the lipid peroxidation products on hypoxic cardiac injury. In addition, L-tryptophan supplementation has a strong protective effect on hypoxic heart by antioxidant activity.