Cardiac Cell Injury Research Articles

Nrf2 activation and down-regulation of HMGB1 and MyD88 expression by amnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cells

Backgroundhuman Amniotic Membrane (hAM) extracts contain bioactive molecules such as growth factors and cytokines. Studies have confirmed the ability of hAM in reduction of post-operative dysfunction in patients with cardiac surgery. However, the function of Amniotic Membrane Proteins (AMPs), extracted from hAM, against hypoxia-induced H9c2 cells injury have never been investigated. In this study, we aimed to appraise the protective impact of AMPs on H9c2 cells under hypoxia condition. MethodsCardiomyocyte cells were pre-incubated with AMPs and subjected to 24 h hypoxia to elucidate its effects on expression of Nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1(HO-1). Furthermore, the high mobility group box-1 (HMGB1) and Myeloid differentiation primary response 88 (MyD88) expressions were detected by qPCR and western-blotting. The mitochondrial membrane potential (ΔΨm) was estimated by JC-1 using fluorescent microscopy and fluorimetry. Moreover, the cell apoptosis and intracellular calcium levels were measured by flow cytometry. ResultsPre-treatment of AMPs resulted in significant induction in cell viability and decreased the LDH release under hypoxic condition in H9c2 cells. Accordingly, these protective effects of AMPs were associated with a reduction in apoptosis rates and intracellular Ca2+, meanwhile, ΔΨm was increased. Pre-treatment with AMPs resulted in degradation of HMGB1 and MyD88 levels and depicted pro-survival efficacy of AMPs against hypoxia-induced cell damage through induction of HO-1 and Nrf2. ConclusionThe data indicated that AMPs mediated HO-1 regulation by Nrf2 activation and plays critical protective effects in hypoxia-induced H9c2 injury in vitro by the inhibition of myocardial HMGB1 and MyD88 inflammatory cascade.

Exosomal Mst1 transfer from cardiac microvascular endothelial cells to cardiomyocytes deteriorates diabetic cardiomyopathy

Diabetic cardiomyopathy (DCM) is characterized by cardiac microvascular endothelial cells (CMECs) injury and cardiomyocyte (CM) dysfunction. Exosomes mediated cellular communication between CMECs and CM has emerging roles in the pathogenesis of DCM, but the underlining mechanisms are unclear. Mammalian sterile 20-like kinase 1 (Mst1), a key component in Hippo pathway which participates in regulating organ size, apoptosis and autophagy, is involved in the development of DCM. We generated the endothelial-specific Mst1 transgenic mice (Tg-Mst1EC) and constructed diabetic model with streptozotocin (STZ). Interestingly, Tg-Mst1EC mice suffered from worse cardiac function and aggravated insulin resistance compared with non-transgenic (NTg) diabetic mice. The content of Mst1 protein was increased, while Mst1 mRNA had no significant change in CM isolated from diabetic Tg-Mst1EC mice. In vitro, CMECs-derived exosomes were taken up by CM and increased Mst1 protein content which inhibited autophagy, as well as enhanced apoptosis in high glucose (HG) cultured CM as evidenced by immunofluorescence and western blot analysis. In addition, Mst1 inhibited glucose uptake under diabetic condition by disrupting the glucose transporter type 4 (GLUT4) membrane translocation through decreasing the interaction between Daxx and GLUT4, as well as enhancing the association of Mst1 and Daxx. Our study exemplifies pleiotropic effects of Mst1-enriched exosomes released from CMECs on inhibiting autophagy, promoting apoptosis and suppressing the glucose metabolism in CM.

Protective role of soluble adenylyl cyclase against reperfusion-induced injury of cardiac cells

AimsDisturbance of mitochondrial function significantly contributes to the myocardial injury that occurs during reperfusion. Increasing evidence suggests a role of intra-mitochondrial cyclic AMP (cAMP) signaling in promoting respiration and ATP synthesis. Mitochondrial levels of cAMP are controlled by type 10 soluble adenylyl cyclase (sAC) and phosphodiesterase 2 (PDE2), however their role in the reperfusion-induced injury remains unknown. Here we aimed to examine whether sAC may support cardiomyocyte survival during reperfusion. Methods and resultsAdult rat cardiomyocytes or rat cardiac H9C2 cells were subjected to metabolic inhibition and recovery as a model of simulated ischemia and reperfusion. Cytosolic Ca2+, pH, mitochondrial cAMP (live-cell imaging), and cell viability were analyzed during a 15-min period of reperfusion. Suppression of sAC activity in cardiomyocytes and H9C2 cells, either by sAC knockdown, by pharmacological inhibition or by withdrawal of bicarbonate, a natural sAC activator, compromised cell viability and recovery of cytosolic Ca2+ homeostasis during reperfusion. Contrariwise, overexpression of mitochondria-targeted sAC in H9C2 cells suppressed reperfusion-induced cell death. Analyzing cAMP concentration in mitochondrial matrix we found that inhibition of PDE2, a predominant mitochondria-localized PDE isoform in mammals, during reperfusion significantly increased cAMP level in mitochondrial matrix, but not in cytosol. Accordingly, PDE2 inhibition attenuated reperfusion-induced cardiomyocyte death and improved recovery of the cytosolic Ca2+ homeostasis. ConclusionsAC plays an essential role in supporting cardiomyocytes viability during reperfusion. Elevation of mitochondrial cAMP pool either by sAC overexpression or by PDE2 inhibition beneficially affects cardiomyocyte survival during reperfusion.

Alterations in necroptosis during ALDH2‑mediated protection against high glucose‑induced H9c2 cardiac cell injury.

The aim of the present study was to investigate whether necroptosis occurs in high glucose (HG)-induced H9c2 cardiac cell injury and whether the activation of aldehyde dehydrogenase 2 (ALDH2) can inhibit necroptosis. H9c2 cardiac cells were treated with 35 mM glucose to establish a HG-induced cell injury model. Alda-1 (20 µM), a specific activator of ALDH2 and necrostatin-1 (Nec-1, 100 µM), an inhibitor of necroptosis were used to treat H9c2 cardiac cells under HG conditions. Cell viability was measured using a Cell Counting Kit-8 assay and reactive oxygen species (ROS) generation was measured by the dihydroethidium staining method. ALDH2 activity was measured at 450 nm. The mRNA and protein expression of ALDH2, necroptosis-associated genes, receptor-interacting protein (RIP)1, RIP3 and mixed lineage kinase domain like pseudokinase (MLKL), were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting. The expression of cleaved caspase-3 protein was also examined by western blotting. The results demonstrated that under HG conditions, cell viability, ALDH2 activity, mRNA and protein expression were decreased. Furthermore, ROS generation, mRNA and protein expression of RIP1, RIP3, MLKL and the protein expression of cleaved caspase-3 were increased. Treatment with Alda-1 or Nec-1 attenuated HG-induced downregulation of ALDH2 activity, mRNA and protein expression. In addition, RIP1, RIP3, MLKL mRNA, and protein expression were downregulated. Furthermore, Alda-1 but not Nec-1 decreased cleaved caspase-3 protein expression. Collectively these data indicated that activation of ALDH2 protected H9c2 cardiac cells against HG-induced injury, partly by inhibiting the occurrence of necroptosis.

Exogenous hydrogen sulfide ameliorates high glucose-induced myocardial injury & inflammation via the CIRP-MAPK signaling pathway in H9c2 cardiac cells

AimsHydrogen sulfide (H2S) is a novel signaling molecule with potent cytoprotective actions. In this study, we hypothesize that exogenous H2S may protect cardiac cells against high glucose (HG)-induced myocardial injury and inflammation with the involvement of the CIRP-MAPK signaling pathway. Main methodsH9c2 cardiac cells cultured under HG conditions were transfected with siRNA and different inhibitor for detecting the effects of sodium hydrogen sulfide (NaHS) (a H2S donor) on cell biological processes. The cardiac cell viability and LDH activity were determined by CCK-8 and LDH kit. ELISA was employed to measure the levels of inflammatory factors, while 2′,7′-dichlorofluorescein diacetate (DCFH-DA) to evaluate reactive oxygen species (ROS). Mitochondrial membrane potential (MMP) was identified by rhodamine 123 staining. TUNEL staining and Hoechst 33258 staining were employed to observe cardiac cell apoptosis. Besides, we determined the expression of CIRP-MAPK signaling pathway- and apoptosis-related factors by protein immunoblot analysis. Key findingsHG culturing induced toxicity, LDH, higher level of inflammatory factors, ROS, MMP, and apoptosis in cardiac cells, attenuated the viability of cardiac cells, and activated the CIRP-MAPK signaling pathway. Notably, CIRP silencing aggravated the above condition. H2S or blockade of the MAPK signaling pathway reversed the above conditions induced by HG. SignificanceThe present study provides evidence for the protective effect of exogenous H2S on HG-induced myocardial injury and inflammation in H9c2 cardiac cells and suggests that the activation of CIRP-MAPK signaling pathway might be one of the mechanisms underlying the protective effect of H2S.

Overexpression and pre-treatment of recombinant human Secretory Leukocyte Protease Inhibitor (rhSLPI) reduces an in vitro ischemia/reperfusion injury in rat cardiac myoblast (H9c2) cell.

One of the major causes of cardiac cell death during myocardial ischemia is the oversecretion of protease enzymes surrounding the ischemic tissue. Therefore, inhibition of the protease activity could be an alternative strategy for preventing the expansion of the injured area. In the present study, we investigated the effects of Secretory Leukocyte Protease Inhibitor (SLPI), by means of overexpression and treatment of recombinant human SLPI (rhSLPI) in an in vitro model. Rat cardiac myoblast (H9c2) cells overexpressing rhSLPI were generated by gene delivery using pCMV2-SLPI-HA plasmid. The rhSLPI-H9c2 cells, mock transfected cells, and wild-type (WT) control were subjected to simulated ischemia/reperfusion (sI/R). Moreover, the treatment of rhSLPI in H9c2 cells was also performed under sI/R conditions. The results showed that overexpression of rhSLPI in H9c2 cells significantly reduced sI/R-induced cell death and injury, intracellular ROS level, and increased Akt phosphorylation, when compared to WT and mock transfection (p <0.05). Treatment of rhSLPI prior to sI/R reduced cardiac cell death and injury, and intra-cellular ROS level. In addition, 400 ng/ml rhSLPI treatment, prior to sI, significantly inhibited p38 MAPK phosphorylation and rhSLPI at 400-1000 ng/ml could increase Akt phosphorylation.

RP105 ameliorates hypoxia̸reoxygenation injury in cardiac microvascular endothelial cells by suppressing TLR4̸MAPKs̸NF-κB signaling.

The radioprotective 105kDa protein (RP105) has been implicated in the pathological process of multiple cardiovascular diseases through its functional and physical interactions with Toll‑like receptor4 (TLR4). However, the effects of RP105 on cardiac microvascular endothelial cells (CMECs) in response to hypoxia̸reoxygenation(H̸R) injury have not been extensively investigated. The aim of the present study was to elucidate the potential roles of RP105 in the protection of CMECs against H̸R injury, and investigate the underlying mechanisms. CMECs isolated from Sprague‑Dawley rats were transduced with adenoviral vectors encoding RP105 or green fluorescent protein (GFP). At 48h post‑transfection, CMECs were subjected to hypoxia for 4h and reoxygenation for 2h (H̸R) to simulate the invivo ischemia̸reperfusion model. The mRNA and protein levels of RP105 were detected by reverse transcription‑quantitative polymerase chain reaction and western blot analysis, respectively. The effects of RP105 on CMEC proliferation, migration and apoptosis were measured by GFP‑8, Transwell chamber and flow cytometry assays, respectively. The secretion of interleukin (IL)‑6 and tumor necrosis factor (TNF)‑α in the culture medium was measured by ELISA. Moreover, the expression level of TLR4, p38mitogen‑activated protein kinase (MAPK), extracellular-signal-regulated kinase1̸2, c-Jun N-terminal kinase, nuclear factor (NF)‑κB̸p65, IL‑6, TNF‑α and intercellular adhesion melecule‑1 was evaluated by western blot analysis. The results demonstrated that RP105 was minimally expressed in CMECs subjected to H̸R injury. Overexpression of RP105 via adenoviral vectors was able to significantly protect CMECs against H̸R injury, as evidenced by the promotion of cell proliferation and migration, as well as the amelioration of inflammation and apoptosis. These beneficial effects were at least partly mediated through inhibition of TLR4̸MAPKs̸NF‑κB signaling. Therefore, RP105 may be a promising candidate for prevention against CMECs‑associated H̸R injury.

Antineoplastic Drug-Induced Cardiotoxicity: A Redox Perspective.

Antineoplastic drugs can be associated with several side effects, including cardiovascular toxicity (CTX). Biochemical studies have identified multiple mechanisms of CTX. Chemoterapeutic agents can alter redox homeostasis by increasing the production of reactive oxygen species (ROS) and reactive nitrogen species RNS. Cellular sources of ROS/RNS are cardiomyocytes, endothelial cells, stromal and inflammatory cells in the heart. Mitochondria, peroxisomes and other subcellular components are central hubs that control redox homeostasis. Mitochondria are central targets for antineoplastic drug-induced CTX. Understanding the mechanisms of CTX is fundamental for effective cardioprotection, without compromising the efficacy of anticancer treatments. Type 1 CTX is associated with irreversible cardiac cell injury and is typically caused by anthracyclines and conventional chemotherapeutic agents. Type 2 CTX, associated with reversible myocardial dysfunction, is generally caused by biologicals and targeted drugs. Although oxidative/nitrosative reactions play a central role in CTX caused by different antineoplastic drugs, additional mechanisms involving directly and indirectly cardiomyocytes and inflammatory cells play a role in cardiovascular toxicities. Identification of cardiologic risk factors and an integrated approach using molecular, imaging, and clinical data may allow the selection of patients at risk of developing chemotherapy-related CTX. Although the last decade has witnessed intense research related to the molecular and biochemical mechanisms of CTX of antineoplastic drugs, experimental and clinical studies are urgently needed to balance safety and efficacy of novel cancer therapies.

Triptolide reduces ischemia/reperfusion injury in rats and H9C2 cells via inhibition of NF‑κB, ROS and the ERK1/2 pathway.

Myocardial ischemia/reperfusion (I/R) induces cardiac cell injury; however, the mechanism underlying cardiac damage remains unclear. A previous study demonstrated that triptolide (TP) exerts protective effects against I/R in cerebral cells. The present study aimed to evaluate the protective effects of TP on cardiac cells, and investigated the potential mechanisms involved in I/R-induced damage. Rats and cardiac H9C2 cells undergoing I/R were pretreated with TP, and cell damage was assessed in vivo and in vitro. Hematoxylin and eosin and terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining were employed to evaluate I/R injury in rat cardiac tissue. Inflammatory factors, including tumor necrosis factor-α, interleukin (IL)-1β and IL-6, were detected by ELISA. Biochemical analyses were performed to evaluate the bioactivity of superoxide dismutase, malondialdehyde and catalase. In addition, viability of H9C2 cells was measured using the Cell Counting kit 8 assay. Flow cytometry was used to evaluate cell apoptosis and reactive oxygen species (ROS) generation. Furthermore, the expression levels of proteins associated with apoptosis, peroxide and inflammation were measured using western blot analysis. H9C2 cells were also treated with N-acetylcysteine and pyrrolidine dithiocarbamate, and cell injury was assessed after peroxidation or I/R. The results demonstrated that TP exerted a significant protective effect on cardiac cells in vivo and in vitro. TP reduced the inflammatory response, as determined by nuclear factor-κB inhibition. In addition, TP decreased ROS-mediated lipid peroxidation, and reduced ROS generation. TP also inhibited cell apoptosis by activating the extracellular signal-regulated kinase 1/2 pathway. In conclusion, TP may protect cardiac cells from I/R injury; the potential protective mechanisms of TP against I/R include anti-inflammatory action, antioxidation and apoptotic resistance.

Qiliqiangxin attenuates hypoxia-induced injury in primary ratcardiac microvascular endothelial cells via promoting HIF-1α-dependent glycolysis.

Protection of cardiac microvascular endothelial cells (CMECs) against hypoxia injury is an important therapeutic strategy for treating ischaemic cardiovascular disease. In this study, we investigated the effects of qiliqiangxin (QL) on primary rat CMECs exposed to hypoxia and the underlying mechanisms. Rat CMECs were successfully isolated and passaged to the second generation. CMECs that were pre‐treated with QL (0.5 mg/mL) and/or HIF‐1α siRNA were cultured in a three‐gas hypoxic incubator chamber (5% CO2, 1% O2, 94% N2) for 12 hours. Firstly, we demonstrated that compared with hypoxia group, QL effectively promoted the proliferation while attenuated the apoptosis, improved mitochondrial function and reduced ROS generation in hypoxic CMECs in a HIF‐1α‐dependent manner. Meanwhile, QL also promoted angiogenesis of CMECs via HIF‐1α/VEGF signalling pathway. Moreover, QL improved glucose utilization and metabolism and increased ATP production by up‐regulating HIF‐1α and a series of glycolysis‐relevant enzymes, including glucose transport 1 (GLUT1), hexokinase 2 (HK2), 6‐phosphofructokinase 1 (PFK1), pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA). Our findings indicate that QL can protect CMECs against hypoxia injury via promoting glycolysis in a HIF‐1α‐dependent manner. Lastly, the results suggested that QL‐dependent enhancement of HIF‐1α protein expression in hypoxic CMECs was associated with the regulation of AMPK/mTOR/HIF‐1α pathway, and we speculated that QL also improved HIF‐1α stabilization through down‐regulating prolyl hydroxylases 3 (PHD3) expression.

MicroRNA-106b overexpression alleviates inflammation injury of cardiac endothelial cells by targeting BLNK via the NF-κB signaling pathway.

We aim to investigate whether microRNA-106b (miR-106b) affects the inflammation injury of cardiac endothelial cells (ECs) by targeting B-cell linker (BLNK) via the NF-κB signaling pathway. Human cardiac microvascular endothelial cells (HCMECs) were assigned into the control, hypoxia/reoxygenation (H/R), negative control (NC), pyrrolidine dithiocarbamate (PDTC), miR-106b mimic, miR-106b inhibitor, and si-BLNK, and miR-106b inhibitor+si-BLNK groups. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were conducted for miR-106b expression and expressions of BLNK, interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, NF-κB, pIκBα, BTK, and PLC-γ2. Enzyme-linked immunosorbent assay was applied for levels of IL-6, IL-10, and TNF-α; cell counting Kit-8 assay for cell proliferation; and flow cytometry for cell cycle and ensuing apoptosis. In-vitro tube formation assay was performed for tube formation ability. Dual-luciferase reporter assay revealed that BLNK was verified as the target gene of miR-106b. Compared with the H/R and NC groups, the PDTC, miR-106b mimic, and si-BLNK groups had declined expressions of IL-6, IL-1β, TNF-α, BTK, PLC-γ2, NF-κB p105/p50, and pIκBα as well as levels of L-6 and TNF-α, but contrarily elevated levels of NF-κB p105/p50 and IL-10. The PDTC, miR-106b mimic, and si-BLNK groups had less cell number in G0 /G1 phase but higher cell count in both S and G2 phases, decreased cell apoptosis but increased proliferation and tube formation ability, while opposite trends were observed in the miR-106b inhibitor group. Our findings indicated that the overexpression of miR-106b alleviated the inflammation injury of cardiac ECs by targeting BLNK via the NF-κB signaling pathway.

Graphene Quantum Dots Protect against Copper Redox-Mediated Free Radical Generation and Cardiac Cell Injury.

In this work, we investigated the effects of graphene quantum dots (GQDs) on copper redox-mediated free radical generation and cell injury. Using electron paramagnetic resonance (EPR) spectrometry in conjunction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap, we found that GQDs at a concentration as low as 1 μg/ml significantly inhibited Cu(II)/H2O2-mediated hydroxyl radical formation. GQDs also blocked Cu(II)-catalyzed nucleophilic addition of H2O to DMPO to form a DMPO-OH adduct in the absence of H2O2, suggesting a potential for GQDs to inhibit copper redox activity. Indeed, we observed that the presence of GQDs prevented H2O2-mediated reduction of Cu(II) to Cu(I) though GQDs themselves also caused the reduction of Cu(II) to Cu(I). To further investigate the effects of GQDs on copper redox activity, we employed the Cu(II)/hydroquinone system in which copper redox activity plays an essential role in the oxidation of hydroquinone to semiquinone radicals with consequent oxygen consumption. Using oxygen polarography as well as EPR spectrometry, we demonstrated that the presence of GQDs drastically blocked the oxygen consumption and semiquinone radical formation resulting from the reaction of Cu(II) and hydroquinone. These results suggested that GQDs suppressed free radical formation via inhibiting copper redox activity. Lastly, using cultured human cardiomyocytes, we demonstrated that the presence of GQDs also protected against Cu(II)/H2O2-mediated cardiac cell injury as indicated by morphological changes (e.g., cell shrinkage and degeneration). In conclusion, our work shows, for the first time, the potential for using GQDs to counteract copper redox-mediated biological damage.

MicroRNA-495 Ameliorates Cardiac Microvascular Endothelial Cell Injury and Inflammatory Reaction by Suppressing the NLRP3 Inflammasome Signaling Pathway

Background/Aims: In recent years, microRNA-495 (miR-495) has been reported to be a tumor-suppressor miR that is down-modulated in cancers. However, its potential mechanism remains unknown. Therefore, this study aimed to demonstrate the role of miR-495 in cardiac microvascular endothelial cell (CMEC) injury and inflammatory reaction by mediating the pyrin domain-containing 3 (NLRP3) inflammasome signaling pathway. Methods: Overall, 40 mice were assigned into myocardial ischemia/reperfusion injury (MIR) and sham groups. After model establishment, the levels of troponin T (TnT), troponin I (TnI), N-terminal pro-B-type natriuretic peptide (NT-proBNP), creatine kinase isoenzyme MB (CK-MB), myoglobin (MYO), tumor necrosis factor-alpha (TNF-α), and interleukin 1beta (IL-1β) were detected by Enzyme-Linked Immunosorbent Assay (ELISA). Apoptosis was evaluated using Terminal deoxy (d)-UTP nick end labeling (TUNEL) staining, the level of NLRP3 protein was determined by immunohistochemical assay, and miR-495 was detected by in situ hybridization (ISH). The infarct size was determined using 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The expression of miR-495 and the mRNA and protein levels of NLRP3, TNF-α, IL-1β, IL-18 and caspase-1 were evaluated by RT-qPCR and western blot analysis. After transfection, the cells were treated with a miR-495 mimic, a miR-495 inhibitor, or siNLRP3. Cell proliferation was measured by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and cell cycle and apoptosis by flow cytometry. Results: Mice with myocardial I/R injury had elevated levels of TnT, TnI, NT-proBNP, CK-MB, MYO, TNF-α and IL-1β; enhanced cell apoptosis; increased expression of NLRP3, TNF-α, IL-1β, IL-18 and caspase-1; and decreased miR-495 expression. MiR-495 was confirmed to target NLRP3. Moreover, miR-495 reduced the mRNA and protein levels of NLRP3, TNF-α, IL-1β, IL-18 and caspase-1, inhibited cell apoptosis and decreased cells at the G₀/G₁ phase while improving cell proliferation and increasing cells at the S phase. However, the effects of NLRP4 were proved to be reciprocal. Conclusion: In conclusion, the current study indicated that miR-495 improved CMEC injury and inflammation by suppressing the NLRP3 inflammasome signaling pathway.

Effects of microrna-93 on mouse cardiac microvascular endothelial cells injury and inflammatory response by mediating SPP1 through the NF-ΚB pathway.

This study aims to investigate the regulative role of microRNA-93 (miR-93) in mouse cardiac microvascular endothelial cells (CMECs) injury and inflammatory response by negatively targeting SPP1 gene via the NF-κB signaling pathway. Healthy Balb/c mice were recruited to establish a mouse model with myocarditis using the CVB3 virus. Mice were grouped into normal, blank, negative control (NC), miR-93 inhibitor, miR-93 mimic, SPP1 short hairpin RNA (shRNA), and miR-93 mimic+SPP1 shRNA groups. Reverse transcription quantitative polymerase chain reaction and Western blot analysis were applied to determine the expressions of miR-93, SPP1, VEGFA, p50, p65, Bax, and Bcl-2. MTT assay was conducted to evaluate cell viability, annexin V-fluorescein isothiocyanate/propidium iodide double staining was conducted to examine cell apoptosis, enzyme-linked immunosorbent assay was conducted to measure secretion of inflammatory factors, and chemical colorimetry was conducted to determine NO secretion. SPP1 was a target gene of miR-93. Compared with the normal group, other six groups showed increased expressions of SPP1, p50, p65, VEGFA, and Bax, as well as cell apoptosis rate and secretion of cell inflammatory factors, and decreased expression of Bcl-2, cell viability, and NO secretion. Compared with the blank group, the miR-93 inhibitor group showed elevated expressions of SPP1, p50, p65, VEGFA, and Bax, as well as cell apoptosis rate and secretion of cell inflammatory factors, and reduced Bcl-2, cell viability, and NO secretion. While the miR-93 mimic and SPP1 shRNA groups displayed opposite results. Taking our results together, we conclude that upregulation of miR-93 reduces CMECs injury and inflammatory response by negatively targeting SPP1 via inactivating the NF-κB signaling pathway.

Early structural changes of the heart after experimental polytrauma and hemorrhagic shock.

Evidence is emerging that systemic inflammation after trauma drives structural and functional impairment of cardiomyocytes and leads to cardiac dysfunction, thus worsening the outcome of polytrauma patients. This study investigates the structural and molecular changes in heart tissue 4 h after multiple injuries with additional hemorrhagic shock using a clinically relevant rodent model of polytrauma. We determined mediators of systemic inflammation (keratinocyte chemoattractant, macrophage chemotactic protein 1), activated complement component C3a and cardiac troponin I in plasma and assessed histological specimen of the mouse heart via standard histomorphology and immunohistochemistry for cellular and subcellular damage and ongoing apoptosis. Further we investigated spatial and quantitative changes of connexin 43 by immunohistochemistry and western blotting. Our results show significantly increased plasma levels of both keratinocyte chemoattractant and cardiac troponin I 4 h after polytrauma and 2 h after induction of hypovolemia. Although we could not detect any morphological changes, immunohistochemical evaluation showed increased level of tissue high-mobility group box 1, which is both a damage-associated molecule and actively released as a danger response signal. Additionally, there was marked lateralization of the cardiac gap-junction protein connexin 43 following combined polytrauma and hemorrhagic shock. These results demonstrate a molecular manifestation of remote injury of cardiac muscle cells in the early phase after polytrauma and hemorrhagic shock with marked disruption of the cardiac gap junction. This disruption of an important component of the electrical conduction system of the heart may lead to arrhythmia and consequently to cardiac dysfunction.

SanggenonC protects against cardiomyocyte hypoxia injury by increasing autophagy.

Sanggenon C is isolated from Morus alba, a plant that has been used for anti-inflammatory purposes in Oriental medicine. Little is known about the effect of Sanggenon C on cardiomyocyte hypoxia injury. This study, using H9c2 rat cardiomyoblasts, was designed to determine the effects of Sanggenon C on cardiomyocyte hypoxia injury. Inflammatory cytokine levels were measured by reverse transcription-polymerase chain reaction, reactive oxygen species were measured by 2′,7′-dichlorofluorescin diacetate fluorescent probe, autophagy was detected using the LC3II/I ratio and cell apoptosis was detected by TUNEL staining. The molecular mechanisms underlying Sanggenon C-induced cyto-protection were also determined by western blotting, especially the possible involvement of autophagy and AMP-activated protein kinase (AMPK). Results indicated that samples pretreated with different concentrations of Sanggenon C (1, 10 and 100 µM) reduced the expression levels of pro-inflammatory cytokines, including tumor necrosis factor α, interleukin (IL)-1 and IL-6, under hypoxia. The beneficial effects of Sanggenon C were also associated with reduced levels of reactive oxygen species generation and increased levels of antioxidant nitric oxide and superoxide dismutase. Sanggenon C enhanced hypoxia-induced autophagy as evidenced by the increased expression levels of autophagy-associated proteins Beclin and autophagy related 5 as well as the decreased the accumulation of p62, and increased the LC3II/I ratio. Sanggenon C also reduced hypoxia-induced apoptosis as detected by TUNEL staining and the expression of Bcl-2 proteins. The beneficial effects of Sanggenon C were associated with enhanced activation level of AMPKα and suppressed hypoxia-induced mechanistic target of rapamycin (mTOR) and forkhead box O3a (FOXO3a) phosphorylation. The AMPK inhibitor Compound C (CpC) was used, and the anti-apoptotic and pro-autophagy effects of Sanggenon C in response to hypoxia were abolished by CpC. In conclusion, the current study demonstrated that Sanggenon C possessed direct cytoprotective effects against hypoxia injury in cardiac cells via signaling mechanisms involving the activation of AMPK and concomitant inhibition of mTOR and FOXO3a.

(-)-Epigallocatechin-3-gallate attenuates myocardial injury induced by ischemia/reperfusion in diabetic rats and in H9c2 cells under hyperglycemic conditions.

(−)-Epigallocatechin gallate (EGCG) exerts multiple beneficial effects on cardiovascular performance. In this study, we aimed to examine the effects of EGCG on diabetic cardiomyopathy during myocardial ischemia/reperfusion (I/R) injury. EGCG (100 mg/kg/day) was administered at week 6 for 2 weeks to diabetic rats following the induction of type 1 diabetes by streptozotocin (STZ). At the end of week 8, the animals were subjected to myocardial I/R injury. The EGCG-elicited structural and functional effects were analyzed. Additionally, EGCG (20 μM) was administered for 24 h to cultured cardiac H9c2 cells under hyperglycemic conditions (30 mM glucose) prior to hypoxia/reoxygenation (H/R) challenge, and its effects on oxidative stress were compared to H9c2 cells transfecteed with silent information regulator 1 (SIRT1) small interfering RNA (siRNA). In rats with STZ-induced diabetes, EGCG treatment ameliorated post-ischemic cardiac dysfunction, decreased the myocardial infarct size, apoptosis and cardiac fibrosis, and reduced the elevated lactate dehydrogenase (LDH) and malonaldehyde (MDA) levels, and attenuated oxidative stress. Furthermore, EGCG significantly reduced H/R injury in cardiac H9c2 cells exposed to high glucose as evidenced by reduced apoptotic cell death and oxidative stress. The protein expression levels of SIRT1 and manganese superoxide dismutase (MnSOD) were reduced in the diabetic rats and the H9c2 cells under hyperglycemic conditions, compared with the control rats following I/R injury and H9c2 cells under normal glucose conditions. EGCG pre-treatment significantly upregulated the levels of htese proteins in vitro and in vivo. However, treatment with EX527 and SIRT1 siRNA blocked the EGCG-mediated cardioprotective effects. Taken together, our data indicate that SIRT1 plays a critical role in the EGCG-mediated amelioration of I/R injury in diabetic rats, which suggests that EGCG may be a promising dietary supplement for the prevention of diabetic cardiomyopathy.

A novel damage mechanism: Contribution of the interaction between necroptosis and ROS to high glucose-induced injury and inflammation in H9c2 cardiac cells.

Recently, a novel mechanism known as 'programmed necrosis' or necroptosis has been shown to be another important mechanism of cell death in the heart. In this study, we investigated the role of necroptosis in high glucose(HG)-induced injury and inflammation, as well as the underlying mechanisms. In particular, we focused on the interaction between necroptosis and reactive oxygen species(ROS) in H9c2cardiac cells. Our results demonstrated that the exposure of H9c2cardiac cells to 35mM glucose(HG) markedly enhanced the expression level of receptor-interacting protein3(RIP3), a kinase which promotes necroptosis. Importantly, co-treatment of the cells with 100µM necrostatin-1(a specific inhibitor of necroptosis) and HG for 24h attenuated not only the increased expression level of RIP3, but also the HG-induced injury and inflammation, as evidenced by an increase in cell viability, a decrease in ROS generation, the attenuation of the dissipation of mitochondrial membrane potential and a decrese in the secretion levels of inflammatory cytokines, i.e.,interleukin(IL)-1β and tumor necrosis factor(TNF)-α. Furthermore, treatment of the cells with 1mM N-acetyl‑L‑cysteine(a scavenger of ROS) for 60min prior to exposure to HG significantly reduced the HG-induced increase in the RIP3expression level, as well as the injury and inflammatory response described above. Taken together, the findings of this study clearly demonstrate a novel damage mechanism involving the positive interaction between necroptosis and ROS attributing to HG-induced injury and inflammation in H9c2cardiac cells.

From Molecular Mechanisms to Clinical Management of Antineoplastic Drug-Induced Cardiovascular Toxicity: A Translational Overview.

Significance: Antineoplastic therapies have significantly improved the prognosis of oncology patients. However, these treatments can bring to a higher incidence of side-effects, including the worrying cardiovascular toxicity (CTX). Recent Advances: Substantial evidence indicates multiple mechanisms of CTX, with redox mechanisms playing a key role. Recent data singled out mitochondria as key targets for antineoplastic drug-induced CTX; understanding the underlying mechanisms is, therefore, crucial for effective cardioprotection, without compromising the efficacy of anti-cancer treatments. Critical Issues: CTX can occur within a few days or many years after treatment. Type I CTX is associated with irreversible cardiac cell injury, and it is typically caused by anthracyclines and traditional chemotherapeutics. Type II CTX is generally caused by novel biologics and more targeted drugs, and it is associated with reversible myocardial dysfunction. Therefore, patients undergoing anti-cancer treatments should be closely monitored, and patients at risk of CTX should be identified before beginning treatment to reduce CTX-related morbidity. Future Directions: Genetic profiling of clinical risk factors and an integrated approach using molecular, imaging, and clinical data may allow the recognition of patients who are at a high risk of developing chemotherapy-related CTX, and it may suggest methodologies to limit damage in a wider range of patients. The involvement of redox mechanisms in cancer biology and anticancer treatments is a very active field of research. Further investigations will be necessary to uncover the hallmarks of cancer from a redox perspective and to develop more efficacious antineoplastic therapies that also spare the cardiovascular system.

Combination of Panaxadiol and Panaxatriol Type Saponins and Ophioponins From Shenmai Formula Attenuates Lipopolysaccharide-induced Inflammatory Injury in Cardiac Microvascular Endothelial Cells by Blocking NF-kappa B Pathway.

Vascular inflammatory injury leads to vascular endothelial dysfunction, thereby resulting in a variety of cardiovascular diseases (CVDs). Thus, attenuating vascular inflammatory injury has great significance for the prevention and treatment of CVDs. In China, Shenmai formula, a well-known ancient Chinese prescription, has been widely used to treat CVDs, such as coronary atherosclerosis and viral myocarditis. In vivo study has demonstrated that the optimal combination of 3 major active components from Shenmai formula, panaxadiol and panaxatriol type saponins and ophioponins, in a ratio of 1:2:2 might exert significant cardioprotective effects and anti-inflammatory activities. The aim of this study was to investigate whether the combination may exert anti-inflammatory effects on lipopolysaccharide-induced inflammatory injury in cardiac microvascular endothelial cells by blocking nuclear factor-kappa B (NF-κB) pathway. We found that the combination could exert anti-inflammatory effects by inhibiting the mRNA and protein expression of interleukin-1, interleukin-6, tumor necrosis factor alpha, and intercellular adhesion molecule, as well as reducing the lactate dehydrogenase content in lipopolysaccharide-injured cardiac microvascular endothelial cells supernatant. Further experiments showed that the combination could suppress the NF-κB p65 expression and IκBα phosphorylation in these cells. These findings suggested that the combination inhibits vascular inflammatory injury by blocking NF-κB pathway, which proves a new molecular mechanism of the Shenmai formula to treat CVDs.