Photobiomodulation improves cell survival and death parameters in cardiomyocytes exposed to hypoxia/reoxygenation.

INTRODUCTION: Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Ischemic heart disease is one of the most harmful conditions to cellular structure and function. After reperfusion treatment, a spectrum of adverse effects becomes evident, encompassing altered cell viability, heightened oxidative stress, activated autophagy, and increased apoptosis. Photobiomodulation (PBM) has been utilized in experimental models of cardiac hypoxia to enhance mitochondrial response and ameliorate biochemical changes in injured tissue. However, the effects of PBM on cultured cardiomyocytes subjected to hypoxia/reoxygenation are not yet well established. METHOD: H9C2 cardiomyocytes were exposed to hypoxia with concentrations of 300 µM CoCl2 for 24 h, followed by 16 h of reoxygenation through incubation in a normoxic medium. Treatment was conducted using GaAIAs Laser (850 nm) after hypoxia at an intensity of 1 J/cm2. Cells were divided into three groups: Group CT (cells maintained under normoxic conditions), Group HR (cells maintained in hypoxia and reoxygenation conditions without treatment), Group HR + PBM (cells maintained in hypoxia and reoxygenation conditions that underwent PBM treatment). Cell viability was analyzed using MTT, and protein expression was assessed by western blot. One-way ANOVA with the Tukey post hoc test was used for data analysis. Differences were significant when p < 0.05. RESULTS: PBM at an intensity of 1 J/cm2 mitigated the alterations in cell survival caused by hypoxia/reoxygenation. Additionally, it significantly increased the expression of proteins Nrf2, HSP70, mTOR, LC3II, LC3II/I, and Caspase-9, while reducing the expression of PGC-1α, SOD2, xanthine oxidase, Beclin-1, LC3I, and Bax. CONCLUSION: PBM at intensities of 1 J/cm2 reverses the changes related to oxidative stress, mitochondrial biogenesis, autophagy, and apoptosis caused by hypoxia and reoxygenation in a culture of cardiomyocytes.

[MiR-224-5p overexpression inhibits oxidative stress by regulating the PI3K/Akt/FoxO1 axis to attenuate hypoxia/reoxygenation-induced cardiomyocyte injury].

OBJECTIVES: To investigate the regulatory role of miRNA-224-5p in hypoxia/reoxygenation (H/R) -induced H9c2 cardiomyocyte injury. METHODS: Plasma samples were collected from 160 patients with acute myocardial infarction and 80 healthy controls(HC) to measure miRNA-224-5p levels and other biochemical parameters. In cultured H9c2 cells with H/R injury, the effects of transfection with miR-224-5p mimics or a negative control sequence on cell viability, malondialdehyde (MDA) content, and superoxide dismutase 2 (SOD2) and lactate dehydrogenase (LDH) activities were tested. Dual luciferase reporter gene assay was performed to verify the targeting relationship between miR-224-5p and PTEN. Bioinformatics methods were used to analyze the potential mechanisms of the target genes. The expression of miRNA-224-5p in the treated cells was detected with qRT-PCR, the protein expressions of PTEN, Bcl-2, Bax, cleaved caspase-3, SOD2, p-PI3K/PI3K, p-Akt/Ak and p-FoxO1/FoxO1 were determined using Western blotting, and cell apoptosis was analysed with flow cytometry. RESULTS: The levels of blood glucose, C-reactive protein, CK, CK-MB and cTnI were significantly higher in the AMI group compared with the HC group (P < 0.05). The expression level of miR-224-5p was significantly lowered in patients with STEMI and NSTEMI and in H9c2 cells with H/R injury. The viability of H9c2 cells decreased time-dependently following H/R injury. PTEN was a target gene of miR-224-5p, and the PI3K/Akt pathway was the most significantly enriched pathway. H9c2 cells with H/R injury showed significantly decreased SOD2 activity, increased LDH activity and MDA content, increased cell apoptosis, decreased protein expression levels of p-PI3K, p-Akt, p-FoxO1, SOD2, and Bcl-2, and increased expressions of PTEN, Bax, and cleaved caspase-3. These changes were obviously attenuated by trasnfection of the cells with miR-224-5p mimics prior to H/R exposure. CONCLUSION: MiR-224-5p overexpression upregulates the expression of the antioxidant gene SOD2 through the PI3K/Akt/FoxO1 axis to relieve H/R-induced oxidative stress and reduce apoptosis of H9c2 cells.

Effect of tubastatin A on NLRP3 inflammasome activation in macrophages under hypoxia/reoxygenation conditions.

BACKGROUND: There are currently no effective drugs to mitigate the ischemia/reperfusion injury caused by fluid resuscitation after hemorrhagic shock (HS). The aim of this study was to explore the potential of the histone deacetylase 6 (HDAC6)-specific inhibitor tubastatin A (TubA) to suppress nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation in macrophages under hypoxia/reoxygenation (H/R) conditions. METHODS: The viability of RAW264.7 cells subjected to H/R after treatment with different concentrations of TubA was assessed using a cell-counting kit-8 (CCK8) assay. Briefly, 2.5 µmol/L TubA was used with RAW264.7 cells under H/R condition. RAW264.7 cells were divided into three groups, namely the control, H/R, and TubA groups. The levels of reactive oxygen species (ROS) in the cells were detected using fluorescence microscopy. The protein expression of HDAC6, heat shock protein 90 (Hsp90), inducible nitric oxide synthase (iNOS), NLRP3, gasdermin-D (GSDMD), Caspase-1, GSDMD-N, and Caspase-1 p20 was detected by western blotting. The levels of interleukin-1ß (IL-1ß) and IL-18 in the supernatants were detected using enzyme-linked immunosorbent assay (ELISA). RESULTS: HDAC6, Hsp90, and iNOS expression levels were significantly higher (P<0.01) in the H/R group than in the control group, but lower in the TubA group than in the H/R group (P<0.05). When comparing the H/R group to the control group, ROS levels were significantly higher (P<0.01), but significantly reduced in the TubA group (P<0.05). The H/R group had higher NLRP3, GSDMD, Caspase-1, GSDMD-N, and Caspase-1 p20 expression levels than the control group (P<0.05), however, the TubA group had significantly lower expression levels than the H/R group (P<0.05). IL-1ß and IL-18 levels in the supernatants were significantly higher in the H/R group compared to the control group (P<0.01), but significantly lower in the TubA group compared to the H/R group (P<0.01). CONCLUSION: TubA inhibited the expression of HDAC6, Hsp90, and iNOS in macrophages subjected to H/R. This inhibition led to a decrease in the content of ROS in cells, which subsequently inhibited the activation of the NLRP3 inflammasome and the secretion of IL-1ß and IL-18.

Hypoxia Postconditioning Attenuates Hypoxia-Induced Inflammation and Endothelial Barrier Dysfunction.

INTRODUCTION: In recent years, a number of studies have demonstrated that hypoxia reoxygenation (HR) induced by ischemia postconditioning (IPC) reduces endothelial barrier dysfunction and inflammation in various models. When HR occurs, the P38 mitogen-activated protein kinase (P38 MAPK) breaks down the endothelial barrier. But no study has clearly clarified the effect of hypoxia postconditioning (HPC) on P38 MAPK in human dermal microvascular endothelial cells. Therefore, we investigated the function of HPC on P38 MAPK during HR in vitro. METHODS: Human dermal microvascular endothelial cells were cultured in a hypoxic incubator for 8 h. Then cells were reperfused for 12 h (reoxygenation) or postconditioned by 5 min of reoxygenation and 5 min of re-hypoxia 3 times followed by 11.5 h reoxygenation. SB203580 was used as an inhibitor of P38 MAPK. Cell counting kit-8 assay kits were employed to detect cell activity. The corresponding levels of IL-6, IL-8 and IL-1ß were examined via Enzyme-Linked ImmunoSorbent Assay. The endothelial barrier was evaluated using fluorescein isothiocyanate-dextran leakage assay. Western blot was used to detect claudin-5, phosphorylation of P38 MAPK (P-P38 MAPK) and P38 MAPK expression. Claudin-5 localization was studied by immunofluorescence. RESULTS: HR induced endothelial barrier hyperpermeability, elevated inflammation levels, and increased the P-P38 MAPK. But HPC reduced cell injury and maintained the integrity of the endothelial barrier while inhibiting P-P38 MAPK and increasing expression of claudin-5. HPC redistributed claudin-5 in a continuous and linear pattern on the cell membrane. CONCLUSIONS: HPC protects against HR induced downregulation and redistribution of claudin-5 by inhibiting P-P38 MAPK.

Enhancing Heart Transplantation: Utilizing Gas-Loaded Nanocarriers to Mitigate Cold/Hypoxia Stress.

Gas-loaded nanocarriers (G-LN) show promise in improving heart transplantation (HTx) outcomes. Given their success in reducing cell death during normothermic hypoxia/reoxygenation (H/R) in vitro, we tested their integration into cardioplegic solutions and static cold storage (SCS) during simulated HTx. Wistar rat hearts underwent four hours of SCS with four G-LN variants: O2- or N2-cyclic-nigerosyl-nigerose-nanomonomers (CNN), and O2- or N2-cyclic-nigerosyl-nigerose-nanosponges (CNN-NS). We monitored physiological-hemodynamic parameters and molecular markers during reperfusion to assess cell damage/protection. Hearts treated with nanomonomers (N2-CNN or O2-CNN) showed improvements in left ventricular developed pressure (LVDP) and a trend towards faster recovery of the rate pressure product (RPP) compared to controls. However, nanosponges (N2-CNN-NS or O2-CNN-NS) did not show similar improvements. None of the groups exhibited an increase in diastolic left ventricular pressure (contracture index) during reperfusion. Redox markers and apoptosis/autophagy pathways indicated an increase in Beclin 1 for O2-CNN and in p22phox for N2-CNN, suggesting alterations in autophagy and the redox environment during late reperfusion, which might explain the gradual decline in heart performance. The study highlights the potential of nanomonomers to improve early cardiac performance and mitigate cold/H/R-induced stunning in HTx. These early improvements suggest a promising avenue for increasing HTx success. Nevertheless, further research and optimization are needed before clinical application.

ADAM10 Alleviates Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Activating the Notch Signaling Pathway.

The occurrence of myocardial ischemia/reperfusion injury is commonly observed during cardiac surgery; however, there remains a dearth of effective therapeutic strategies to mitigate this injury. The a disintegrin and metallopeptidase domain 10 (ADAM10) is a transmembrane protein anchored on the cell membrane surface, and its precise mechanism of action in myocardial ischemia/reperfusion injury remains incompletely understood. This study aims to investigate the impact of ADAM10 on cardiomyocyte injury induced by hypoxia/reoxygenation (H/R) and elucidate the underlying mechanisms. The ADAM10 overexpression plasmid was transfected into H9c2 cells, which were subsequently treated with the Notch signaling pathway inhibitor DAPT and cultured under H/R conditions. Cell proliferation activity was assessed using the CCK-8 assay. The levels of LDH, SOD, and MDA were quantified through colorimetric analysis. The levels of ROS and the rate of apoptosis were measured using flow cytometry. The morphological changes in the nucleus of H9c2 cells were observed by employing Hoechst 33258 staining. The mRNA expression levels of ADAM10, Notch1, NICD, and Hes1 in H9c2 cells were determined using qRT-PCR. The expressions of Notch signaling pathway and apoptosis-related proteins were analyzed by Western blot. Overexpression of ADAM10 provided protection to H9c2 cells against injury induced by H/R, leading to an increase in SOD levels and alleviation of oxidative stress caused by the accumulation of ROS and the decrease of SOD activity. Meanwhile, overexpression of ADAM10 inhibited apoptosis in H9c2 cells exposed to H/R by regulating the expression of apoptosis-related proteins, such as Bax, Bcl-2 and Cleaved-caspase-3. Additionally, overexpression of ADAM10 facilitated the activation of the Notch1 signaling pathway in H9c2 cells exposed to H/R by upregulating the protein expression of Notch1, NICD, and Hes1. However, the protective effect of ADAM10 on H/R-induced H9c2 cells was partially reversed by DAPT. Our findings demonstrate that ADAM10 exerts protective effects in H/R-induced H9c2 cells by suppressing oxidative stress and apoptosis via the activation of the Notch signaling pathway.

In Vitro Hypoxia/Reoxygenation Induces Mitochondrial Cardiolipin Remodeling in Human Kidney Cells.

Renal ischemia/reperfusion is a serious condition that not only causes acute kidney injury, a severe clinical syndrome with high mortality, but is also an inevitable part of kidney transplantation or other kidney surgeries. Alterations of oxygen levels during ischemia/reperfusion, namely hypoxia/reoxygenation, disrupt mitochondrial metabolism and induce structural changes that lead to cell death. A signature mitochondrial phospholipid, cardiolipin, with many vital roles in mitochondrial homeostasis, is one of the key players in hypoxia/reoxygenation-induced mitochondrial damage. In this study, we analyze the effect of hypoxia/reoxygenation on human renal proximal tubule epithelial cell (RPTEC) cardiolipins, as well as their metabolism and mitochondrial functions. RPTEC cells were placed in a hypoxic chamber with a 2% oxygen atmosphere for 24 h to induce hypoxia; then, they were replaced back into regular growth conditions for 24 h of reoxygenation. Surprisingly, after 24 h, hypoxia cardiolipin levels substantially increased and remained higher than control levels after 24 h of reoxygenation. This was explained by significantly elevated levels of cardiolipin synthase and lysocardiolipin acyltransferase 1 (LCLAT1) gene expression and protein levels. Meanwhile, hypoxia/reoxygenation decreased ADP-dependent mitochondrial respiration rates and oxidative phosphorylation capacity and increased reactive oxygen species generation. Our findings suggest that hypoxia/reoxygenation induces cardiolipin remodeling in response to reduced mitochondrial oxidative phosphorylation in a way that protects mitochondrial function.

Panax quinquefolius saponins and panax notoginseng saponins attenuate myocardial hypoxia-reoxygenation injury by reducing excessive mitophagy.

OBJECTIVE: Panax quinquefolius saponins (PQS) and Panax notoginseng saponins (PNS) are key bioactive compounds in Panax quinquefolius L. and Panax notoginseng, commonly used in the treatment of clinical ischemic heart disease. However, their potential in mitigating myocardial ischemia-reperfusion injury remains uncertain. This study aims to evaluate the protective effects of combined PQS and PNS administration in myocardial hypoxia/reoxygenation (H/R) injury and explore the underlying mechanisms. METHODS: To investigate the involvement of HIF-1α/BNIP3 mitophagy pathway in the myocardial protection conferred by PNS and PQS, we employed small interfering BNIP3 (siBNIP3) to silence key proteins of the pathway. H9C2 cells were categorized into four groups: control, H/R, H/R + PQS + PNS, and H/R + PQS + PNS+siBNIP3. Cell viability was assessed by Cell Counting Kit-8, apoptosis rates determined via flow cytometry, mitochondrial membrane potential assessed with the JC-1 fluorescent probes, intracellular reactive oxygen species detected with 2',7'-dichlorodihydrofluorescein diacetate, mitochondrial superoxide production quantified with MitoSOX Red, and autophagic flux monitored with mRFP-GFP-LC3 adenoviral vectors. Autophagosomes and their ultrastructure were visualized through transmission electron microscopy. Moreover, mRNA and protein levels were analyzed via real-time PCR and Western blotting. RESULTS: PQS + PNS administration significantly increased cell viability, reduced apoptosis, lowered reactive oxygen species levels and mitochondrial superoxide production, mitigated mitochondrial dysfunction, and induced autophagic flux. Notably, siBNIP3 intervention did not counteract the cardioprotective effect of PQS + PNS. The PQS + PNS group showed downregulated mRNA expression of HIF-1α and BNIP3, along with reduced HIF-1α protein expression compared to the H/R group. CONCLUSIONS: PQS + PNS protects against myocardial H/R injury, potentially by downregulating mitophagy through the HIF-1α/BNIP3 pathway.

Ghrelin alleviates intestinal ischemia-reperfusion injury by activating the GHSR-1α/Sirt1/FOXO1 pathway.

Ischemia-reperfusion (IR) injury is primarily characterized by the restoration of blood flow perfusion and oxygen supply to ischemic tissue and organs, but it paradoxically leads to tissue injury aggravation. IR injury is a challenging pathophysiological process that is difficult to avoid clinically and frequently occurs during organ transplantation, surgery, shock resuscitation, and other processes. The major causes of IR injury include increased levels of free radicals, calcium overload, oxidative stress, and excessive inflammatory response. Ghrelin is a newly discovered brain-intestinal peptide with anti-inflammatory and antiapoptotic effects that improve blood supply. The role and mechanism of ghrelin in intestinal ischemia-reperfusion (IIR) injury remain unclear. We hypothesized that ghrelin could attenuate IIR-induced oxidative stress and apoptosis. To investigate this, we established IIR by using a non-invasive arterial clip to clamp the root of the superior mesenteric artery (SMA) in mice. Ghrelin was injected intraperitoneally at a dose of 50 µg/kg 20 min before IIR surgery, and [D-Lys3]-GHRP-6 was injected intraperitoneally at a dose of 12 nmol/kg 20 min before ghrelin injection. We mimicked the IIR process with hypoxia-reoxygenation (HR) in Caco-2 cells, which are similar to intestinal epithelial cells in structure and biochemistry. Our results showed that ghrelin inhibited IIR/HR-induced oxidative stress and apoptosis by activating GHSR-1α. Moreover, it was found that ghrelin activated the GHSR-1α/Sirt1/FOXO1 signaling pathway. We further inhibited Sirt1 and found that Sirt1 was critical for ghrelin-mediated mitigation of IIR/HR injury. Overall, our data suggest that pretreatment with ghrelin reduces oxidative stress and apoptosis to attenuate IIR/HR injury by binding with GHSR-1α to further activate Sirt1.

Sevoflurane postconditioning ameliorates cerebral hypoxia/reoxygenation injury in zebrafish involving the Akt/GSK-3ß pathway activation and the microtubule-associated protein 2 promotion.

Sevoflurane postconditioning has been shown to provide neuroprotection against cerebral hypoxia-ischemia injury, but the mechanisms remain elusive. Microtubule-associated protein 2 (MAP2) is implicated in early neuronal hypoxia-ischemia injury. This study aimed to investigate whether the neuroprotective effects of sevoflurane postconditioning are related to the Akt/GSK-3ß pathway and its downstream target MAP2 in zebrafish hypoxia/reoxygenation (H/R) model. Sevoflurane postconditioning or GSK-3ß inhibitor TDZD-8 were used to treat H/R zebrafish. The cerebral infarction, neuronal apoptosis, and mitochondrial changes were evaluated using TTC staining, TUNEL staining, and transmission electron microscopy, respectively. The distribution of MAP2 in the brain was determined by immunofluorescence imaging. The levels of Akt, p-Akt, GSK-3ß, p-GSK-3ß, and MAP2 proteins were evaluated by Western blotting. The neurobehavioral recovery of zebrafish was assessed based on optokinetic response behavior. Our results indicated that sevoflurane postconditioning and TDZD-8 significantly reduced the cerebral infarction area, suppressed cell apoptosis, and improved mitochondrial integrity in zebrafish subjected to H/R. Furthermore, sevoflurane postconditioning and TDZD-8 elevated the ratios of p-Akt/Akt and p-GSK-3ß/GSK-3ß. However, the neuroprotective effect of sevoflurane postconditioning was effectively abolished upon suppression of MAP2 expression. In conclusion, sevoflurane postconditioning ameliorated cerebral H/R injury and facilitated the restoration of neurobehavioral function through the activation of Akt/GSK-3ß pathway and promotion of MAP2 expression.

[Identification of cardioprotective substances in Panax ginseng/P. notoginseng based on mitochondrial morphological characteristics and UPLC-Triple-TOF-MS].

Panax ginseng is reputed to be capable of replenishing healthy Qi and bolstering physical strength, and P. notoginseng can resolve blood stasis and alleviate pain. P. ginseng and P. notoginseng are frequently employed to treat ischemic heart diseases caused by blockages in the heart vessels. Mitochondrial dysfunction often coexists with abnormal mitochondrial morphology, and mitochondrial plasticity and dynamics play key roles in cardiovascular diseases. In this study, primary neonatal rat cardiomyocytes were exposed to 4 hours of hypoxia(H) followed by 2 hours of reoxygenation(R). MitoTracker Deep Red and Hoechst 33342 were used to label mitochondria and nuclei, respectively. Fluorescence images were then acquired using ImageXpress Micro Confocal. Automated image processing and parameter extraction/calculation were carried out using ImagePro Plus. Subsequently, representative parameters were selected as indicators to assess alterations in mitochondrial morphology and function. The active compounds of P. ginseng and P. notoginseng were screened out and identified based on the UPLC-Triple-TOF-MS results and mitochondrial morphometric parameters. The findings demonstrated that RS-2, RS-4, SQ-1, and SQ-4 significantly increased the values of three key morphometric parameters, including mitochondrial length, branching, and area, which might contribute to rescuing morphological features of myocardial cells damaged by H/R injury. Among the active components of the two medicinal herbs, 20(R)-ginsenoside Rg_3, ginsenoside Re, and gypenoside ⅩⅦ exhibited the strongest protective effects on mitochondria in cardiomyocytes. Specifically, 20(R)-ginsenoside Rg_3 might upregulate expression of optic atrophy 1(OPA1) and mitofusin 2(MFN2), and ginsenoside Re and gypenoside ⅩⅦ might selectively upregulate OPA1 expression. Collectively, they promoted mitochondrial membrane fusion and mitigated mitochondrial damage, thereby exerting protective effects on cardiomyocytes. This study provides experimental support for the discovery of novel therapeutic agents for myocardial ischemia-reperfusion injury from P. ginseng and P. notoginseng and offers a novel approach for large-scale screening of bioactive compounds with cardioprotective effects from traditional Chinese medicines.

miR-652-3p Suppressed the Protective Effects of Isoflurane Against Myocardial Injury in Hypoxia/Reoxygenation by Targeting ISL1.

This research is concentrated on investigating the role and mechanism of miR-652-3p in the protective effects of isoflurane (ISO) against myocardial ischemia-reperfusion (I/R) injury. H9c2 cells underwent pretreatment with varying concentrations of ISO, and subsequently, a hypoxia/reoxygenation (H/R) model was constructed. The levels of miR-652-3p, ISL LIM homeobox 1 (ISL1), and inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were evaluated through reverse transcription polymerase chain reaction (RT-qPCR). Enzyme-linked immunosorbent assay was employed to investigate concentrations of myocardial injury markers, such as creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI). Cell counting kit-8 was used to evaluate cell viability, while flow cytometry was utilized to measure apoptosis. Additionally, a dual luciferase reporter assay was conducted to validate the targeting relationship between ISL1 and miR-652-3p. Herein, we confirmed that the level of miR-652-3p was gradually increased with prolonged hypoxia; nevertheless, this increase was suppressed by ISO pretreatment (P < 0.05). Additionally, ISO pretreatment prevented the decrease in cell viability, increase in apoptosis, and overproduction of IL-6, TNF-α, CK-MB, and cTnI induced by H/R (P < 0.05). However, the inhibitory effects of ISO were counteracted by the increased levels of miR-652-3p (P < 0.05). ISL1 is a potential target of miR-652-3p. H/R induction suppressed ISL1 levels compared to the control, but ISO treatment increased its expression (P < 0.05). Overexpression of ISL1 inhibited the elimination of the protective effect of ISO on myocardial damage induced by the elevation of miR-652-3p (P < 0.05). The findings of this research confirm that miR-652-3p attenuated the protective effect of ISO on cardiomyocytes in myocardial ischemia by targeting ISL1.

Esketamine alleviates hypoxia/reoxygenation injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.

OBJECTIVE: This study aimed to investigate the effect of Esketamine (ESK) on the Hypoxia/Reoxygenation (H/R) injury of cardiomyocytes by regulating TRPV1 and inhibiting the concentration of intracellular Ca2+. METHODS: The H/R injury model of H9c2 cardiomyocytes was established after 4h hypoxia and 6h reoxygenation. H9c2 cells were treated with different concentrations of ESK or TRPV1 agonist capsaicin (10 µM) or TRPV1 inhibitor capsazepine (1 µM). Cell viability was detected by CCK-8 method, and apoptosis by flow cytometry. Intracellular Ca2+ concentration was evaluated by Fluo-4 AM. LDH, MDA, SOD, and GSH-Px were detected with corresponding commercial kits. TRPV1 and p-TRPV1 proteins were detected by Western blot. RESULTS: After H/R, H9c2 cell viability decreased, apoptosis increased, intracellular Ca2+ concentration increased, LDH and MDA levels increased, SOD and GSH-Px levels decreased, and p-TRPV1 expression increased. ESK treatment rescued these changes induced by H/R. After up-regulating TRPV1, the protective effect of ESK on H/R injury of H9c2 cells was weakened, while down-regulating TRPV1 could further protect against H/R injury. CONCLUSION: ESK alleviates H/R injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.

Targeting the Ferroptosis and Endoplasmic Reticulum Stress Signaling Pathways by CBX7 in Myocardial Ischemia/reperfusion Injury.

Ferroptosis and endoplasmic reticulum stress (ERS) are common events in the process of myocardial ischemia/reperfusion injury (IRI). The suppression of chromobox7 (CBX7) has been reported to protect against ischemia/reperfusion injury, This research is purposed to expose the impacts and mechanism of CBX7 in myocardial IRI. CBX7 expression was detected using RT-qPCR and western blotting analysis. CCK-8 assay detected cell viability. Inflammatory response and oxidative stress were detected by ELISA, DCFH-DA probe and related assay kits. Flow cytometry analysis and caspase3 activity assay were used to detect cell apoptosis. C11-BODIPY 581/591 staining and ferro-orange staining were used to detect lipid reactive oxygen species (ROS) and Fe2+ level, respectively. Western blotting was used to detect the expression of proteins associated with apoptosis, ferroptosis and ERS. In the hypoxia/reoxygenation (H/R) model of rat cardiomyocytes H9c2, CBX7 was highly expressed. CBX7 interference significantly protected against inflammatory response, oxidative stress, apoptosis, ferroptosis and ERS induced by H/R in H9c2 cells. Moreover, after the pretreatment with ferroptosis activator erastin or ERS agonist Tunicamycin (TM), the protective effects of CBX7 knockdown on the inflammation, oxidative stress and apoptosis in H/R-induced H9c2 cells was partially abolished. To summarize, CBX7 down-regulation may exert anti-ferroptosis and anti-ERS activities to alleviate H/R-stimulated myocardial injury.

miR-30a-5p attenuates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by regulating PTEN protein expression and activating PI3K/Akt signaling pathway.

BACKGROUND: This study was designed to investigate the mechanism by which miR-30a-5p mediates cardiomyocyte apoptosis after acute myocardial infarction (AMI) induced by hypoxia/reoxygenation (H/R). METHODS: Differentially expressed miRNAs were analyzed by RNA high-throughput sequencing in acute myocardial infarction (ST-elevation myocardial infarction) patients versus healthy individuals (controls). The H/R model was used to assess the regulatory mechanism of miRNAs in AMI. Lentivirus-associated vectors were used to overexpress or knock down miR-30a-5p in cellular models. The pathological mechanisms of miR-30a-5p regulating the development of acute myocardial infarction were serially explored by qPCR, bioinformatics, target gene prediction, dual luciferase, enzyme-linked immunosorbent assays (ELISAs) and Western blotting. RESULTS: The results showed that the expression of miR-30a-5p was significantly increased in AMI patients and H9C2 cells. Hypoxia decreased cardiomyocyte survival over time, and reoxygenation further reduced cell survival. Bax and Phosphatase and tensin homolog (PTEN)were suppressed, while Bcl-2 was upregulated. Additionally, miR-30a-5p specifically targeted the PTEN gene. According to the GO and KEGG analyses, miR-30a-5p may participate in apoptosis by interacting with PTEN. The miR-30a-5p mimic decreased the expression of apoptosis-related proteins and the levels of the proinflammatory markers IL-1ß, IL-6, and TNF-α by activating the PTEN/PI3K/Akt signaling pathway. Conversely, anti-miR-30a-5p treatment attenuated these effects. Additionally, silencing PTEN and anti-miR-30a-5p had opposite effects on H/R-induced cell apoptosis. CONCLUSIONS: miR-30a-5p plays a crucial role in cardiomyocyte apoptosis after hypoxia-induced acute myocardial infarction. Our findings provide translational evidence that miR-30a-5p is a novel potential therapeutic target for AMI.

A potent and selective activator of large-conductance Ca2+-activated K+ channels induces preservation of mitochondrial function after hypoxia and reoxygenation by handling of calcium and transmembrane potential.

AIMS: Ischaemic heart disease remains a significant cause of mortality globally. A pharmacological agent that protects cardiac mitochondria against oxygen deprivation injuries is welcome in therapy against acute myocardial infarction. Here, we evaluate the effect of large-conductance Ca2+-activated K+ channels (BKCa) activator, Compound Z, in isolated mitochondria under hypoxia and reoxygenation. METHODS: Mitochondria from mice hearts were obtained by differential centrifugation. The isolated mitochondria were incubated with a BKCa channel activator, Compound Z, and subjected to normoxia or hypoxia/reoxygenation. Mitochondrial function was evaluated by measurement of O2 consumption in the complexes I, II, and IV in the respiratory states 1, 2, 3, and by maximal uncoupled O2 uptake, ATP production, ROS production, transmembrane potential, and calcium retention capacity. RESULTS: Incubation of isolated mitochondria with Compound Z under normoxia conditions reduced the mitochondrial functions and induced the production of a significant amount of ROS. However, under hypoxia/reoxygenation, the Compound Z prevented a profound reduction in mitochondrial functions, including reducing ROS production over the hypoxia/reoxygenation group. Furthermore, hypoxia/reoxygenation induced a large mitochondria depolarization, which Compound Z incubation prevented, but, even so, Compound Z created a small depolarization. The mitochondrial calcium uptake was prevented by the BKCa activator, extruding the mitochondrial calcium present before Compound Z incubation. CONCLUSION: The Compound Z acts as a mitochondrial BKCa channel activator and can protect mitochondria function against hypoxia/reoxygenation injury, by handling mitochondrial calcium and transmembrane potential.

RP105 Attenuates Ischemia/Reperfusion-Induced Oxidative Stress in the Myocardium via Activation of the Lyn/Syk/STAT3 Signaling Pathway.

Although our previous studies have established the crucial role of RP105 in myocardial ischemia/reperfusion injury (MI/RI), its involvement in regulating oxidative stress induced by MI/RI remains unclear. To investigate this, we conducted experiments using a rat model of ischemia/reperfusion (I/R) injury. Adenovirus carrying RP105 was injected apically at multiple points, and after 72 h, the left anterior descending coronary artery was ligated for 30 min followed by 2 h of reperfusion. In vitro experiments were performed on H9C2 cells, which were transfected with recombinant adenoviral vectors for 48 h, subjected to 4 h of hypoxia, and then reoxygenated for 2 h. We measured oxidative stress markers, including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, as well as malondialdehyde (MDA) concentration, using a microplate reader. The fluorescence intensity of reactive oxygen species (ROS) in myocardial tissue was measured using a DHE probe. We also investigated the upstream and downstream components of the signal transducer and activator of transcription 3 (STAT3). Upregulation of RP105 increased SOD and GSH-Px activities, reduced MDA concentration, and inhibited ROS production in response to I/R injury in vivo and hypoxia reoxygenation (H/R) stimulation in vitro. The overexpression of RP105 led to a decrease in the myocardial enzyme LDH in serum and cell culture supernatant, as well as a reduction in infarct size. Additionally, left ventricular fraction (LVEF) and fractional shortening (LVFS) were improved in the RP105 overexpression group compared to the control. Upregulation of RP105 promoted the expression of Lyn and Syk and further activated STAT phosphorylation, which was blocked by PP2 (a Lyn inhibitor). Our findings suggest that RP105 can inhibit MI/RI-induced oxidative stress by activating STAT3 via the Lyn/Syk signaling pathway.

[Ginsenoside Re regulates mitochondrial biogenesis through Nrf2/HO-1/PGC-1α pathway to reduce hypoxia/reoxygenation injury in H9c2 cells].

This article explored the mechanism by which ginsenoside Re reduces hypoxia/reoxygenation(H/R) injury in H9c2 cells by regulating mitochondrial biogenesis through nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/peroxisome prolife-rator-activated receptor gamma coactivator-1α(PGC-1α) pathway. In this study, H9c2 cells were cultured in hypoxia for 4 hours and then reoxygenated for 2 hours to construct a cardiomyocyte H/R injury model. After ginsenoside Re pre-administration intervention, cell activity, superoxide dismutase(SOD) activity, malondialdehyde(MDA) content, intracellular reactive oxygen species(Cyto-ROS), and intramitochondrial reactive oxygen species(Mito-ROS) levels were detected to evaluate the protective effect of ginsenoside Re on H/R injury of H9c2 cells by resisting oxidative stress. Secondly, fluorescent probes were used to detect changes in mitochondrial membrane potential(ΔΨ_m) and mitochondrial membrane permeability open pore(mPTP), and immunofluorescence was used to detect the expression level of TOM20 to study the protective effect of ginsenoside Re on mitochondria. Western blot was further used to detect the protein expression levels of caspase-3, cleaved caspase-3, Cyto C, Nrf2, HO-1, and PGC-1α to explore the specific mechanism by which ginsenoside Re protected mitochondria against oxidative stress and reduced H/R injury. Compared with the model group, ginse-noside Re effectively reduced the H/R injury oxidative stress response of H9c2 cells, increased SOD activity, reduced MDA content, and decreased Cyto-ROS and Mito-ROS levels in cells. Ginsenoside Re showed a good protective effect on mitochondria by increasing ΔΨ_m, reducing mPTP, and increasing TOM20 expression. Further studies showed that ginsenoside Re promoted the expression of Nrf2, HO-1, and PGC-1α proteins, and reduced the activation of the apoptosis-related regulatory factor caspase-3 to cleaved caspase-3 and the expression of Cyto C protein. In summary, ginsenoside Re can significantly reduce I/R injury in H9c2 cells. The specific mechanism is related to the promotion of mitochondrial biogenesis through the Nrf2/HO-1/PGC-1α pathway, thereby increasing the number of mitochondria, improving mitochondrial function, enhancing the ability of cells to resist oxidative stress, and alleviating cell apoptosis.

Mitochondrial responses to constant and cyclic hypoxia depend on the oxidized fuel in a hypoxia-tolerant marine bivalve Crassostrea gigas.

Sessile benthic organisms like oysters inhabit the intertidal zone, subject to alternating hypoxia and reoxygenation (H/R) episodes during tidal movements, impacting respiratory chain activities and metabolome compositions. We investigated the effects of constant severe hypoxia (90 min at ~ 0% O2 ) followed by 10 min reoxygenation, and cyclic hypoxia (5 cycles of 15 min at ~ 0% O2 and 10 min reoxygenation) on isolated mitochondria from the gill and the digestive gland of Crassostrea gigas respiring on pyruvate, palmitate, or succinate. Constant hypoxia suppressed oxidative phosphorylation (OXPHOS), particularly during Complex I-linked substrates oxidation. It had no effect on mitochondrial reactive oxygen species (ROS) efflux but increased fractional electron leak (FEL). In mitochondria oxidizing Complex I substrates, exposure to cyclic hypoxia prompted a significant drop after the first H/R cycle. In contrast, succinate-driven respiration only showed significant decline after the third to fifth H/R cycle. ROS efflux saw little change during cyclic hypoxia regardless of the oxidized substrate, but Complex I-driven FEL tended to increase with each subsequent H/R cycle. These observations suggest that succinate may serve as a beneficial stress fuel under H/R conditions, aiding in the post-hypoxic recovery of oysters by reducing oxidative stress and facilitating rapid ATP re-synthesis. The impacts of constant and cyclic hypoxia of similar duration on mitochondrial respiration and oxidative lesions in the proteins were comparable indicating that the mitochondrial damage is mostly determined by the lack of oxygen and mitochondrial depolarization. The ROS efflux in the mitochondria of oysters was minimally affected by oxygen fluctuations indicating that tight regulation of ROS production may contribute to robust mitochondrial phenotype of oysters and protect against H/R induced stress.

[Tetrahydropalmatine inhibiting mitophagy through ULK1/FUNDC1 pathway to alleviate hypoxia/reoxygenation injury in H9c2 cells].

This study explored the specific mechanism by which tetrahydropalmatine(THP) inhibited mitophagy through the UNC-51-like kinase 1(ULK1)/FUN14 domain containing 1(FUNDC1) pathway to reduce hypoxia/reoxygenation(H/R) injury in H9c2 cells. This study used H9c2 cells as the research object to construct a cardiomyocyte H/R injury model. First, a cell viability detection kit was used to detect cell viability, and a micro-method was used to detect lactate dehydrogenase(LDH) leakage to evaluate the protective effect of THP on H/R injury of H9c2 cells. In order to evaluate the protective effect of THP on mitochondria, the chemical fluorescence method was used to detect intracellular reactive oxygen species, intramitochondrial reactive oxygen species, mitochondrial membrane potential, and autophagosomes, and the luciferin method was used to detect intracellular adenosine 5'-triphosphate(ATP) content. Western blot was further used to detect the ratio of microtubule-associated protein 1 light chain 3(LC3) membrane type(LC3-Ⅱ) and slurry type(LC3-Ⅰ) and activated cleaved caspase-3 expression level. In addition, ULK1 expression level and its phosphorylation degree at Ser555 site, as well as the FUNDC1 expression level and its phosphorylation degree of Ser17 site were detected to explore its specific mechanism. The results showed that THP effectively reduced mitochondrial damage in H9c2 cells after H/R. THP protected mitochondria by reducing the level of reactive oxygen species in cells and mitochondria, increasing mitochondrial membrane potential, thereby increasing cellular ATP production, enhancing cellular activity, reducing cellular LDH leakage, and finally alleviating H/R damage in H9c2 cells. Further studies have found that THP could reduce the production of autophagosomes, reduce the LC3-Ⅱ/LC3-Ⅰ ratio, and lower the expression of the apoptosis-related protein, namely cleaved caspase-3, indicating that THP could reduce apoptosis by inhibiting autophagy. In-depth studies have found that THP could inhibit the activation of the ULK1/FUNDC1 pathway of mitophagy and the occurrence of mitophagy by reducing the phosphorylation degree of ULK1 at Ser555 and FUNDC1 at Ser17. The application of ULK1 agonist BL-918 reversely verified the effect of THP on reducing the phosphorylation of ULK1 and FUNDC1. In summary, THP inhibited mitophagy through the ULK1/FUNDC1 pathway to reduce H/R injury in H9c2 cells.