Sotagliflozin Ameliorates LPS‑Induced Cardiomyocyte Injury by Regulating the PI3K‑Akt and Extracellular Matrix-Related Pathways.

MiR-208a aggravates H2O2-induced cardiomyocyte injury by targeting APC

Astragaloside IV prevents lipopolysaccharide-induced injury in H9C2 cardiomyocytes

Upregulation of PTPN1 aggravates endotoxemia-induced cardiac dysfunction through inhibiting mitophagy

This study aims to elucidate the role and molecular mechanism of microRNA-21 (miR-21) in LPS-induced inflammatory injury in H9c2 cardiomyocytes. H9c2 cardiomyocytes were treated with lipopolysaccharide (LPS) to establish an in vitro model. The expression of miR-21 was quantified using RT-qPCR, while protein levels were assessed via Western blot analysis. The impact of miR-21 on inflamma-tory response, cell proliferation, and apoptosis in LPS-treated H9c2 cells was evalu-ated using ELISA, CCK-8/EdU assays, and flow cytometry. TargetScan predictions and dual-luciferase reporter assays were employed to identify potential miR-21 tar-gets. The regulatory effects of miR-21 on inflammation, proliferation, and apop-tosis in cells were further examined following transfection with phospholipase D1 (PLD1) overexpression constructs or signal transducer and activator of transcription 3 (STAT3) activation. The expression levels of miR-21, PLD1, and p-STAT3 were significantly elevated in LPS-treated H9c2 cells. Knockdown of miR-21 markedly inhibited the LPS-induced inflammatory response, enhanced cell proliferation, and reduced apoptosis in H9c2 cells. PLD1 and STAT3 were confirmed as direct targets of miR-21. Overexpression of PLD1 or activation of STAT3 significantly reversed the protective effects of miR-21 downregulation in LPS-treated H9c2 cells. Downregu-lation of miR-21 protects cardiomyocytes against LPS-induced inflammatory injury and apoptosis by inhibiting PLD1 expression and STAT3 phosphorylation.

Objective To evaluate the influence of preconditioning with and anti-myosin-monoclonal antibody (mAb2G4)-nuclear factor-kappa B decoy oligodeoxynucleotide (ODN)-lipofectamine (lip) on hypoxia-reoxygenation (H/R) injury in H9c2 cardiomyocytes. Methods H9c2 cardiomyocytes were seeded in 6-well plate at the density of 1×105/ml (2 ml/well), and were divided into 3 groups (n=9 each) using a random number table: control group (group C), H/R group and mAb2G4-ODN-lip group (group MOL). The cells underwent 2 h of hypoxia in an air-tight bag, followed by 1 h reoxygenation. In MOL group, the cells were treated with mAb2G4-ODN-lip (2 μg ODN) for 4 h and then cultured in the common culture medium for 8 h before hypoxia. At the end of reoxygenation, proliferation of cells was measured using MTT assay, and the cells and supernatant of the culture medium were collected to determine the activity of lactate dehydrogenas (LDH), content of malondialdehyde (MDA), concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (by ELISA). The rate of proliferation inhibition was calculated. Results Compared with group C, the rate of proliferation inhibition, LDH activity, MDA content, and concentrations of TNF-α and IL-6 were significantly increased in the other two groups. Compared with group H/R, the rate of proliferation inhibition, LDH activity, MDA content, and concentrations of TNF-α and IL-6 were significantly decreased in MOL group. Conclusion mAb2G4-ODN-lip can mitigate H/R injury in H9c2 cardiomyocytes. Key words: NF-kappa B; Decoy oligodeoxynucleotide; Cationic liposome; Myocytes, cardiac; Cell hypoxia; Oxygen

Polo-like kinase 2 (PLK2) is associated with cardiac fibrosis in patients with atrial fibrillation. However, the role of PLK2 in sepsis-induced cardiac injury has not been fully elucidated. We hypothesize that PLK2 may participate in the progression of sepsis-induced cardiac injury. We established a cardiac injury model in C57BL6 mice by injecting lipopolysaccharide (LPS). PLK2 was overexpressed in mice using an adeno-associated virus 9 vector. Cardiac function was evaluated using echocardiography 12 hours after the LPS injection. H9c2 cells were transfected with a PLK2 small interfering RNA. PLK2 was downregulated in the hearts of LPS-treated mice and LPS-stimulated H9c2 cardiomyocytes. Mice in the LPS group presented aggravated cardiac injury and a reduced survival rate with increased cardiac inflammatory responses and oxidative stress. Moreover, PLK2 relieved LPS-induced cardiac injury and increased the survival rate of the mice by weakening the inflammatory response and decreasing oxidative stress. Moreover, the LPS-induced increase in ferroptosis was inhibited by PLK2 overexpression.LPS caused an increase in inflammation and cell injury in H9c2 cardiomyocytes, and PLK2 silencing aggravated LPS-induced cell injury, inflammation, and oxidative stress. Furthermore, PLK2 overexpression increased the expression levels of the antiferroptotic proteins solute carrier family 7 member 11, glutathione peroxidase 4, and ferritin in heart tissue. PLK2 increased the nuclear NRF2 expression level. Moreover, the overexpression of PLK2 increased the phosphorylation of glycogen synthase kinase 3β and promoted the nuclear translocation of NRF2. NRF2 overexpression relieved cardiac injury in mice induced with LPS. However, NRF2 mitigated the deteriorating effects of PLK2 knockdown in the mouse heart. PLK2 ameliorated LPS-induced cardiac injury by blocking cardiomyocyte ferroptosis through NRF2 regulation.

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

The aim of this study was to investigate the mechanism of luteolin regulating lipoxygenase pathway against oxygen-glucose deprivation/reperfusion(OGD/R) injury in H9 c2 cardiomyocytes. First, Discovery Studio 2019 was used for the molecular docking of luteolin with three key enzymes including lipoxygenase 5(ALOX5), lipoxygenase 12(ALOX12), and lipoxygenase 15(ALOX15) in lipoxygenase pathway. The docking results showed that luteolin had high docking score and similar functional groups with the original ligand. From this, H9 c2 cardiomyocytes were cultured in vitro, and then the injury model of H9 c2 cardiomyocytes was induced by deprivation of oxygen-glucose for 8 h, and rehabilitation of oxygen-glucose for 12 h. Cell viability was detected by tetrazolium(MTT) colorimetry. H9 c2 cardiomyocytes were observed with a fluorescence inverted microscope, and colorimetry was used to detect the level of lactate dehydrogenase(LDH) in cell supernatant. The results showed that luteolin could significantly protect the morphology of H9 c2 cells, significantly improve the survival rate of H9 c2 cardiomyocytes in OGD/R injury model, reduce the level of LDH in cell supernatant, inhibit cytotoxicity, and maintain the integrity of cell membrane. The inflammatory cytokines interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with the model group, luteolin can significantly reduce the release of IL-6 and TNF-α. Western blot was employed to detect the protein levels of ALOX5, ALOX12, and ALOX15 in lipoxygenase pathway. After luteolin intervention, the protein levels of ALOX5, ALOX12, and ALOX15 were significantly down-regulated compared with those in model group. These results indicate that luteolin can inhibit the release of IL-6 and TNF-α by restraining the activation of lipoxygenase pathway, thereby playing a protective role in the cardiomyocyte injury model induced by OGD/R.

BackgroundClinical research has demonstrated that alprostadil has an anti-inflammatory effect; however, to date, its molecular mechanisms remain unclear. This study aimed to examine the anti-inflammatory activity and related mechanisms of alprostadil in lipopolysaccharide (LPS)-treated H9c2 cells.MethodsCell morphology was observed under an inverted light microscope, while cell viability was assessed with the 3‑(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assays (ELISA) were conducted to study biochemical indicators of cellular damage, such as released lactate dehydrase (LDH) and troponin, and inflammatory cytokine levels including interleukin-1β (IL-1β), IL-6, IL-17, and tumor necrosis factor-α (TNF-α). The mRNA expression levels of Wnt5a, c‑jun N‑terminal kinase (JNK), and nuclear factor kappa B (NF-κB) were further investigated by real-time quantitative polymerase chain reaction (RT-PCR). The effects of alprostadil on the Wnt5a/JNK/NF-κB pathway in H9c2 cells was examined by Western blotting.ResultsAlprostadil increased the cell viability of LPS-stimulated H9c2 cells, reduced LDH and troponin production, and attenuated IL-1β, IL-6, IL-17, and TNF-α secretion. Moreover, alprostadil reduced the mRNA expression of Wnt5a, JNK, and NF-κB and decreased the expression of Wnt5a, NF-κB, and the ratio of p‑JNK/JNK in H9c2 cells treated with LPS. The siWnt5a or JNK inhibitor SP600125 significantly augmented the inhibitory effects of alprostadil on the Wnt5a/JNK/NF-κB pathway.ConclusionOur results show that alprostadil has anti-inflammatory effects and could attenuate LPS-induced injury in H9c2 cardiomyocytes via the Wnt5a/JNK/NF-κB pathway.

Prokineticin 2 (PK2) was reported to be decreased in the hearts of end-state heart failure patients. Our study aimed to explore the effects of PK2 on hypoxia/reoxygenation (H/R) injury and the underlying mechanism. H9c2 cardiomyocytes were treated with 5 nM PK2 in the presence or absence of 5 mM dual phosphatidylinositol 3-kinase (PI3K)/the mammalian target of rapamycin (mTOR) inhibitor (BEZ235) for 24 h and then subjected to H/R treatment. Cell viability and lactate dehydrogenase (LDH) release were evaluated by CCK-8 and LDH release assays, respectively. Apoptosis was determined by flow cytometry analysis. Oxidative stress was assessed by measuring superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities and malondialdehyde (MDA) content. Results showed that H/R treatment decreased PK2 expression and inactivated the Akt/mTOR pathway in H9c2 cardiomyocytes. PK2 treatment activated the Akt/mTOR pathway in H/R-exposed H9c2 cardiomyocytes. H/R stimulation suppressed cell viability, increased LDH release, induced apoptosis and oxidative stress in H9c2 cardiomyocytes, while these effects were neutralised by treatment with PK2. However, the inhibitory effects of PK2 on H/R-induced injury in H9c2 cardiomyocytes were abolished by the addition of BEZ235. In conclusion, PK2 relieved H/R-induced injury in H9c2 cardiomyocytes by activation of the Akt/mTOR pathway.

BackgroundThe microRNA (miR)‐17~92 is one of the best‐characterized polycistronic miRNA clusters, which encodes six individual miRNAs and contributes to various cellular and biological processes. In particular, miR‐17‐3p (a passenger miRNA of miR‐17) has been shown to indirectly inhibit Phosphatase and tensin homolog (PTEN) and attenuate myocardial ischemia/reperfusion (I/R) injury, but the underneath mechanism is unclear. Inhibition of PTEN can upregulate Akt/mTOR signaling and therefore suppresses the excessive autophagy, which may be a mechanism that underlies myocardial I/R injury. We, thus, hypothesized that miR‐17‐3p may protect against hypoxia/reoxygenation (H/R) induced cell injury by inhibiting post‐hypoxic excessive autophagy in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.Methods and resultsIn rat H9C2 cardiomyocytes, H/R (6 hours hypoxia followed by 6 hours reoxygenation) significantly enhanced the expression of miR‐17‐3p (P <0.05 vs. Control), which was concomitant by increased lactic acid dehydrogenase (LDH) leakage (cell injury marker, P<0.05 vs. Control) and the levels of p62 and the ratio of LC3II/I (autophagy markers, P<0.05 vs. Control), suggesting that miR‐17‐3p may be involved in the pathogenesis of H/R induced autophagic cell death. To explore the potential role of miR‐17‐3p in H/R‐induced cell injury, H9C2 cells were transfected with the miR‐17‐3p agomir or its negative control. The overexpression of miR‐17‐3p can significantly attenuate H/R‐induced cell injury (reduced LDH level, P <0.05 vs. H/R) and inhibit H/R‐induced excessive autophagy (decreased levels of p62 and LC3II/I, P <0.05 vs. H/R). Given the important role of PTEN‐Akt‐mTOR signaling in autophagy, the effects of miR‐17‐3p on the key events in the PTEN‐Akt‐mTOR signaling cascade during H/R were examined. As anticipated, miR‐17‐3p overexpression significantly down‐regulated PTEN expression and up‐regulated the levels of phosphorylated Akt1 (Thr308) and mTOR (P<0.05 vs. H/R), indicating that the miR‐17‐3p may protect against excessive post‐hypoxic autophagic cell death in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.ConclusionUpregulating PTEN‐Akt‐mTOR axis and the subsequent inhibition of excessive autophagy may represent the major mechanism whereby miR‐17‐3p attenuates H/R injury in H9C2 cardiomyocytes.Support or Funding InformationStudied supported by RGC/GRF grants (17158616M, 17117217 M)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Polysaccharide of Atractylodes macrocephala Koidz alleviate lipopolysaccharide-induced liver injury in goslings via the p53 and FOXO pathways

Lipopolysaccharide (LPS)-induced autophagy is involved in sepsis-associated myocardial injury with increased PKCβ2 activation. We previously found hyperglycemia-induced PKCβ2 activation impaired the expression of caveolin-3 (Cav-3), the dominant isoform to form cardiomyocytes caveolae which modulate eNOS signaling to confer cardioprotection in diabetes. However, little is known about the roles of PKCβ2 in autophagy and Cav-3/eNOS signaling in cardiomyocytes during LPS exposure. We hypothesize LPS-induced PKCβ2 activation promotes autophagy and impairs Cav-3/eNOS signaling in LPS-treated cardiomyocytes. H9C2 cardiomyocytes were treated with LPS (10µg/mL) in the presence or absence of PKCβ2 inhibitor CGP53353 (CGP, 1µM) or autophagy inhibitor 3-methyladenine (3-MA, 10µM). LPS stimulation induced cytotoxicity overtime in H9C2 cardiomyocytes, accompanied with excessive PKCβ2 activation. Selective inhibition of PKCβ2 with CGP significantly reduced LPS-induced cytotoxicity and autophagy (measured by LC-3II, Beclin-1, p62 and autophagic flux). In addition, CGP significantly attenuated LPS-induced oxidative injury, and improved Cav-3 expression and eNOS activation, similar effects were shown by the treatment of autophagy inhibitor 3-MA. LPS-induced myocardial injury is associated with excessive PKCβ2 activation, which contributes to elevated autophagy and impaired Cav-3/eNOS signaling. Selective inhibition of PKCβ2 improves Cav-3/eNOS signaling and attenuates LPS-induced injury through inhibiting autophagy in H9C2 cardiomyocytes.

Fibroblast growth factor (FGF)-21, a member of the family of FGFs, exhibits protective effects against myocardial ischemia and ischemia/reperfusion injury; it is also an enhancer of autophagy. However, the mechanisms underlying the protective role of FGF-21 against cardiomyocyte hypoxia/reoxygenation (H/R) injury remain unclear. The present study aimed to investigate the effect of FGF-21 on H9c2 cardiomyocyte injury induced by H/R and the mechanism associated with changes in autophagy. Cultured H9c2 cardiomyocytes subjected to hypoxia were treated with a vehicle or FGF-21 during reoxygenation. The viability of H9c2 rat cardiomyocytes was measured using Cell Counting Kit-8 and trypan blue exclusion assays. The contents of creatine kinase (CK) and creatine kinase isoenzymes (CK-MB), cardiac troponin I (cTnT), cardiac troponin T (cTnI) and lactate dehydrogenase (LDH) in culture medium were detected with a CK, CK-MB, cTnT, cTnI and LDH assay kits. The protein levels were examined by western blot analysis. Autophagic flux was detected by Ad-mCherry-GFP-LC3B autophagy fluorescent adenovirus reagent. The results indicated that FGF-21 alleviated H/R-induced H9c2 myocardial cell injury and enhanced autophagic flux during H/R, and that this effect was antagonized by co-treatment with 3-methyladenine, an autophagy inhibitor. Furthermore, FGF-21 increased the expression levels of Beclin-1 and Vps34 proteins, but not of mechanistic target of rapamycin. These data indicate that FGF-21 treatment limited H/R injury in H9c2 cardiomyocytes by promoting autophagic flux through upregulation of the expression levels of Beclin-1 and Vps34 proteins.

Sepsis is a life-threatening organ dysfunction syndrome triggered by infection and uncontrolled inflammatory responses, with sepsis-associated myocardial dysfunction being a leading cause of mortality and morbidity. Metformin, a widely prescribed antihyperglycemic drug, has shown emerging protective effects beyond glucose control. In this study, we investigated the protective role of metformin against LPS-induced injury and inflammation in H9C2 and AC16 cardiomyocytes and explored the underlying mechanisms. LPS stimulation significantly reduced cell viability, promoted apoptosis, and upregulated proinflammatory cytokines (TNFα, IL-6, and IL-1β) in H9C2 and AC16 cells. Metformin treatment markedly alleviated these effects, indicating its protective role against LPS-induced cytotoxicity and inflammation. Mechanistically, metformin significantly upregulated microRNA miR-497-5p, which directly suppressed β-transducin repeat containing E3 ubiquitin-protein ligase (BTRC) expression, leading to inhibition of IκBα degradation and NF-κB pathway activation. Importantly, miR-497-5p knockdown or BTRC overexpression partially reversed the protective effects of metformin, restoring NF-κB signaling and inflammatory cytokine production. These findings collectively demonstrate that metformin protects H9C2 and AC16 cardiomyocytes from LPS-induced injury through miR-497-5p/BTRC axis-mediated suppression of NF-κB activation and highlight the functional importance of miR-497-5p and BTRC in this regulatory process. SIGNIFICANCE STATEMENT: This study highlights the protective effects of metformin against LPS-induced H9C2 and AC16 cell injury and inflammation, revealing a novel mechanism involving miR-497-5p/BTRC axis-mediated NF-κB pathway inhibition. These findings offer insights into potential therapeutic strategies for sepsis-associated myocardial dysfunction.