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Botulinum toxin type A (BTXA) previously protected endothelial cells in free skin flaps from ischemia/reperfusion injury by inducing autophagy. Endoplasmic reticulum (ER) stress-autophagy activation may have a role in BTXA-antagonized ischemia/reperfusion damage in human dermal microvascular endothelial cells (HDMECs), however, this has yet to be proven. HDMECs were pretreated with BTXA at various concentrations for 12 h before being subjected to hypoxia and reoxygenation (H/R). Cell Count Kit 8 (CCK8) and Western blot (WB) data showed that H/R treatment significantly increased the expression of ER stress (GRP78, CHOP) and autophagy (LC3II/I, Beclin-1) proteins. The optimal BTXA pretreatment concentration was 1.6 U/mL, which resulted in the highest levels of cell survival and expression of ER stress and autophagy. Following pretreatment with 1.6 U/mL BTXA and the addition of the ER stress inducer Thapsigargin (Tg), the ER stress inhibitor 4-phenylbutyrate (4-PBA), and the inhibitor of autophagy Bafilomycin A1 (Baf A1), respectively, HDMECs were then subjected to H/R treatment. Cell survival and the percentage of ethynyldeoxyuridine-labeled cells in the BTXA pretreatment groups were reduced by the addition of Tg, 4-PBA, and Baf A1. While the expression of GRP78, CHOP, and LC3 was upregulated by Tg and Baf A1, it was downregulated by 4-PBA. The findings showed that ER stress produced by BTXA pretreatment triggers protective autophagy and protects HDMECs from H/R damage. There were no cytoprotective effects from either excessive activation or reduction of ER stress. Our results are consistent with the notion that autophagy and moderate ER stress are critical for cellular longevity and, consequently, functional integrity and may represent a potential therapeutic target.

Objective: The molecular pathways underlying hypoxemia-induced alterations in neurodevelopment of infants with congenital heart disease have not been delineated. We used transcriptome analysis to investigate differential gene expression induced by hypoxemia in an ovine artificial-womb model. Methods: Mid-gestation fetal sheep (median [interquartile range] 109 [107-112] days' gestation) were cannulated via the umbilical vessels, attached to a pumpless, low-resistance oxygenator circuit, and incubated in a sterile, fluid environment for 22 [21-23] days. Fetuses were maintained with an oxygen delivery of 20-25 mL/kg/min (normoxemia, n = 3) or 14-16 mL/kg/min (hypoxemia, n = 4). Transcriptional profiling by RNA sequencing was carried out on left frontal brains and hypoxemia-regulated genes were identified by differential gene expression analysis. Results: A total of 228 genes whose expression was up or down regulated by ≥1.5-fold (false discovery rate ≤0.05) were identified. The majority of these genes were induced in hypoxemic animals compared to normoxemic controls, and functional enrichment analysis identified respiratory electron transport as a pathway strongly upregulated in the brain during chronic hypoxemia. Further examination of hypoxemia-induced genes showed robust induction of all 7 subunits of the mitochondrial NADH:ubiquinone oxidoreductase (complex I). Other hypoxemia-induced genes included cytochrome B, a component of complex III, and ATP6, ATP8, both of which are components of complex V. Conclusions: Chronic fetal hypoxemia leads to upregulation of multiple mitochondrial respiratory complex genes critical for energy production and reactive oxygen species generation, including complex I. These data provide valuable insight into potential pathways involved in chronic hypoxemia-induced neuropathology and offers potential therapeutic targets for fetal neuroprotection in fetuses with congenital heart defects.

Recent studies suggest hypoxia can promote adipose-derived stem cells (ADSCs) to attenuate hypoxia/reoxygenation (H/R)-induced damage to human dermal microvascular endothelial cells (HDMECs). Extracellular vesicles (EVs), isolated from ADSCs, play an-important role in the fields of regenerative medicine. Here, we aimed to investigate the effect of EVs isolated from hypoxia-pretreated ADSCs (ADSC-EVs[H]) on HDMECs to attenuate ischemia/reperfusion injury of free skin flaps. First, we characterized EVs isolated from normoxia-cultured ADSCs (ADSC-EVs[N]) and ADSC-EVs(H). Experimental data indicated that EVs isolated from ADSCs consisted of lipid-bilayer vesicles that exhibited positive expression of vascular endothelial growth factor (VEGF) and marker proteins CD9, CD63 and CD81, and the mean particle size of EVs in the hypoxia-pretreated ADSCs (ADSC[H]) group was smaller (74.17 nm) than in the normoxic-cultured ADSCs (ADSC[N]) group (93.87 nm). Hypoxic pretreatment increased the number of EVs. Later, we favorably constructed the co-culture model of EVs isolated from ADSCs (ADSC-EVs) and H/R-induced HDMECs. Cell counting kit-8, Ethynyldeoxyuridine assay, western blotting and immunofluorescence staining showed that ADSC-EVs(H) promoted the survival of HDMECs and increased LC3 level. Apoptosis, reactive oxygen species (ROS) and JC-1 mitochondrial membrane potential (MMP) assays revealed that ADSC-EVs(H) reduced the apoptosis rate and ROS accumulation and increased MMP level in HDMECs, indicating that ADSC-EVs(H) effectively attenuated H/R-induced damage in HDMECs through autophagy activation and the-inhibition of apoptosis and oxidative stress. This study confirmed that ADSC-EVs(H) could effectively regulate the proliferation, apoptosis, oxidative stress, and autophagy expression of H/R-induced HDMECs in vitro, and therefore the transplantation of ADSC-EVs(H) may provide novel insights for the transplantation of free skin flaps.

Ginkgolide B (GB), a compound derived from Ginkgo biloba, exhibits significant cardioprotective properties, although its precise molecular target has yet to be identified. In this study, we synthesized a biotin-labeled GB probe (GB-biotin) to identify the molecular targets of GB. Our experiments demonstrated that treatment with GB or GB-biotin reduced mitochondrial injury, restored mitochondrial membrane potential, and decreased cell apoptosis in a concentration-dependent manner. Additionally, GB increased mitochondrial oxygen consumption rate, indicating improved mitochondrial bioenergetics. Proteomic analysis revealed that the voltage-dependent anion channel 1 (VDAC1) is a key protein that interacts with GB-biotin. This finding was further confirmed through cellular thermal shift assay (CETSA) and molecular docking, which revealed hydrogen bond formation between GB and VDAC1. Furthermore, overexpression of VDAC1 diminished the protective effects of GB, highlighting the crucial role of VDAC1 in GB-mediated cardioprotection. These findings identify VDAC1 as a therapeutic target for GB in vitro, providing valuable insights into the cardioprotective mechanisms of GB and the development of novel cardioprotective strategies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Alternative NADH dehydrogenase, also known as type II NADH dehydrogenase (NDH-2), catalyzes the same redox reaction as mitochondrial respiratory chain complex I. Specifically, it oxidizes reduced nicotinamide adenine dinucleotide (NADH) while simultaneously reducing ubiquinone to ubiquinol. However, unlike complex I, this enzyme is non-proton pumping, comprises of a single subunit, and is resistant to rotenone. Initially identified in bacteria, fungi and plants, NDH-2 was subsequently discovered in protists and certain animal taxa including sea squirts. The gene coding for NDH-2 is also present in the genomes of some annelids, tardigrades, and crustaceans. For over two decades, NDH-2 has been investigated as a potential substitute for defective complex I. In model organisms, NDH-2 has been shown to ameliorate a broad spectrum of conditions associated with complex I malfunction, including symptoms of Parkinson's disease. Recently, lifespan extension has been observed in animals expressing NDH-2 in a heterologous manner. A variety of mechanisms have been put forward by which NDH-2 may extend lifespan. Such mechanisms include the activation of pro-longevity pathways through modulation of the NAD + /NADH ratio, decreasing production of reactive oxygen species (ROS) in mitochondria, or then through moderate increases in ROS production followed by activation of defense pathways (mitohormesis). This review gives an overview of the latest research on NDH-2, including the structural peculiarities of NDH-2, its inhibitors, its role in the pathogenicity of mycobacteria and apicomplexan parasites, and its function in bacteria, fungi, and animals., Competing Interests: Declaration of competing interest The author declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Proliferation of renal tubular epithelial cells (TECs) is critical for the recovery after kidney ischemia/reperfusion (KI/R). However, there is still a lack of ideal therapies for promoting TEC proliferation. Heat shock protein A12A (HSPA12A) shows abundant expression in kidney in our previous studies. To investigate the role of HSPA12A in TEC proliferation after KI/R, an in vitro KI/R model was simulated by hypoxia (12 h) and reoxygenation (12 h) in human kidney tubular epithelial HK-2 cells. We found that, when hypoxia/reoxygenation (H/R) triggered HK-2 cell injury, HSPA12A expression was downregulated, and extracellular lactate, the readout of glycolysis, was also decreased. Loss and gain of functional studies showed that HSPA12A did not change cell viability after hypoxia but increased cell proliferation as well as glycolytic flux of HK-2 cells after H/R. When blocking glycolysis by 2-deoxy-D-glucose or oxamate, the HSPA12A promoted HK-2 cell proliferation was also abolished. Further analysis revealed that HSPA12A overexpression increased hypoxia-inducible factor 1α (Hif1α) protein expression and nuclear localization in HK-2 cells in response to H/R, whereas HSPA12A knockdown showed the opposite effects. Notably, pharmacological inhibition of Hif1α with YC-1 reversed the HSPA12A-induced increases of both glycolytic flux and proliferation of H/R HK-2 cells. Moreover, the HSPA12A increased Hif1α protein expression was not via upregulating its transcription but through increasing its protein stability in a Smurf1-dependent manner. The findings indicate that HSPA12A might serve as a promising target for TEC proliferation to help recovery after KI/R., Competing Interests: Declarations of interest The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests. Zhengnian Ding reports financial support was provided by the National Natural Science Foundation of China. Li Liu reports financial support was provided by the National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Ischemic cerebral stroke is one of the leading causes of death and disability in humans. However, molecular processes underlying the development of this pathology remain poorly understood. There are major gaps in our understanding of metabolic changes that occur in the brain tissue during the early stages of ischemia and reperfusion. In particular, it is generally accepted that both ischemia (I) and reperfusion (R) generate reactive oxygen species (ROS) that cause oxidative stress which is one of the main drivers of the pathology, although ROS generation during I/R was never demonstrated in vivo due to the lack of suitable methods. In the present study, we record for the first time the dynamics of intracellular pH and H2O2 during I/R in cultured neurons and during experimental stroke in rats using the latest generation of genetically encoded biosensors SypHer3s and HyPer7. We detect a buildup of powerful acidosis in the brain tissue that overlaps with the ischemic core from the first seconds of pathogenesis. At the same time, no significant H2O2 generation was found in the acute phase of ischemia/reperfusion. HyPer7 oxidation in the brain was detected only 24 h later. Comparison of in vivo experiments with studies on cultured neurons under I/R demonstrates that the dynamics of metabolic processes in these models significantly differ, suggesting that a cell culture is a poor predictor of metabolic events in vivo.

The Ca2+-sensing receptor (CaSR) plays an important role in regulating vascular tone. In the present study, we investigated the positive effects of the vagal neurotransmitter acetylcholine by suppressing CaSR activation in mesenteric arteries exposed to hypoxia/reoxygenation (H/R). The artery rings were exposed to a modified ‘ischemia mimetic’ solution and an anaerobic environment to simulate an H/R model. Our results showed that acetylcholine (10−6 mol/L) significantly reduced the contractions induced by KCl and phenylephrine and enhanced the endothelium-dependent relaxation induced by acetylcholine. Additionally, acetylcholine reduced CaSR mRNA expression and activity when the rings were subjected to 4 h of hypoxia and 12 h of reoxygenation. Notably, the CaSR antagonist NPS2143 significantly reduced the contractions but did not improve the endothelium-dependent relaxation. When a contractile response was achieved with extracellular Ca2+, both acetylcholine and NPS2143 reversed the H/R-induced abnormal vascular vasoconstriction, and acetylcholine reversed the calcimimetic R568-induced abnormal vascular vasoconstriction in the artery rings. In conclusion, this study suggests that acetylcholine ameliorates the dysfunctional vasoconstriction of the arteries after H/R, most likely by decreasing CaSR expression and activity, thereby inhibiting the increase in intracellular calcium concentration. Our findings may be indicative of a novel mechanism underlying ACh-induced vascular protection.

A novel discovery of two hybrid benzodioxepin-dalbergiphenol epimers, named cochindalbergiphenols A-B (1-2), and a benzofuran-dalbergiphenol hybrid, named cochindalbergiphenol C (3), were isolated and identified from the heartwood of Dalbergia cochinchinensis. The structures of all the isolated compounds were identified through NMR and HRESIMS techniques, while the absolute configurations were determined by comparing the experimental and calculated ECD spectra. Compounds 1-3 exhibited potential protective effects against hypoxia/reoxygenation (H/R) induced injury in H9c2 cells., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fructus Choerospondiatis (FC), a Mongolian medicine, was mainly used in Mongolian medical theory for the treatment of coronary heart disease (CHD). Nonetheless, the main components and mechanisms of action of FC in the treatment of coronary artery disease have not been studied clearly., Aim of the Study: The aim of this study is to identify the components of FC and analyze the pathways affected by the targets of these components to probe into the potential mechanisms of action of FC on coronary heart disease., Materials and Methods: Identification of compounds in FC employing high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (HPLC-QTOF-MS) method, then further investigate the network pharmacology and molecular docking to obtain potential targets and elucidate the potential mechanism of action of FC in the therapy of CHD. Experimental validation was established to verify the mechanism of FC in vitro., Results: 21 FC components were identified and 65 overlapping targets were gained. In addition, these ingredients regulated AMPK and PPAR signaling pathway by 65 target genes including IL6, AKT1 and PPARg, etc. Molecular docking displayed that the binding ability of the key target PPARg to FC components turned out to be better. Experimental validation proved that FC treatment decreased the expression of PPARg (p < 0.05) compare with model group, which may be involved in the PPAR signaling pathway., Conclusions: This study was the first to elucidate the mechanism of action of components of FC for the treatment of CHD using network pharmacology. It alleviated CHD by inhibiting the expression of PPARg to attenuate hypoxia/reoxygenation injury, and the results give a basis for elucidating the molecular mechanism of action of FC for the treatment of coronary heart disease., Competing Interests: Declaration of competing interest The authors report there are no competing interests to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Ischemic stroke is regarded one of the most common causes of brain vulnerability. Silibinin (SIL), extracted from the seeds of Silybinisus laborinum L., has been found to exhibit obvious therapeutic effects on neurodegenerative diseases. GAS6 has been proven to have significant neuroprotective effects; however, the role of SIL and GAS6 in ischemic stroke remains unclear. This study aimed to investigate the protective effects of SIL against cerebral ischemia-reperfusion injury in neuroblastoma N2a cells, as well as the mechanisms involved. Firstly, the toxicity of SIL was evaluated, and safe concentrations were chosen for subsequent experiments. Then, SIL exerts significant neuroprotection against hypoxia/reoxygenation (HR) injury in N2a cells, as manifested by increased cell viability, decreased apoptotic rate, LDH, and ROS generation. Additionally, SIL was found to inhibit HR-induced apoptosis, mitochondria dysfunction, and oxidative stress. However, silencing of GAS6 inhibited the neuroprotective effects of SIL. To sum up, these results suggest that SIL may be a promising therapeutic agent for the treatment of ischemic stroke., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Objective: Preeclampsia (PE) is a pregnancy-specific syndrome. Ligustrazine (LSZ) is involved in hypoxia/reoxygenation (H/R)-treated trophoblast cell regulation, but its mechanism remains elusive. This study explored the mechanism of LSZ in H/R-treated trophoblast cells to provide a theoretical basis for the new treatment method development for PE., Methods: H/R HTR8/SVneo cell model was established for PE simulation to some extent. Trophoblast cell proliferation, apoptosis rate, migration, and invasion were detected by MTT assay, flow cytometry, scratch test, and Transwell assay. miR-27a-3p expression in trophoblast cells was detected by RT-qPCR. Binding sites between miR-27a-3p and ATF3 were predicted using Starbase and verified by dual-luciferase reporter assay. Activating transcription factor 3 (ATF3), β-catenin, Cyclin D1, and c-Myc protein levels were examined using Western blot. After LSZ treatment, H/R-induced HTR8/SVneo cells were delivered with miR-27a-3p mimic or ATF3 siRNA to verify their roles in HTR8/SVneo cells., Results: LSZ facilitated the proliferation, migration, and invasion of trophoblast cells and inhibited apoptosis. miR-27a-3p was elevated in H/R-induced HTR8/SVneo cells and miR-27a-3p overexpression annulled the effect of LSZ on trophoblast cells. miR-27a-3p targeted ATF3. ATF3 silencing averted the property of LSZ on trophoblast cells. Wnt/β-catenin pathway-related proteins were repressed in H/R-induced HTR8/SVneo cells, and LSZ activated the Wnt/β-catenin pathway by promoting ATF3 expression., Conclusion: LSZ mediated the Wnt pathway by regulating the miR-27a-3p/ATF3 axis, thus promoting the proliferation and migration of trophoblast cells. The protective mechanism of LSZ showed the potential application value in the treatment of PE., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In ischemia/reperfusion (I/R) injury, both inflammation and apoptosis play a vital role, and the inhibition of excessive inflammation and apoptosis show substantial clinical potential in the treatment of I/R disease. The role of sacubitril/valsartan (SAC/VAL)-a first-in-class angiotensin receptor-neprilysin inhibitor (ARNI)-in inflammation regulation and apoptosis in the context of I/R injury needs to be further explored. In this study, we investigate the short- and long-term effects of SAC/VAL administration in treating adult murine I/R injury both in vivo and in vitro. Our results verified that the application of SAC/VAL could reduce infarct size and suppress apoptosis and the inflammatory response in the acute phase post I/R. Long-term application of SAC/VAL for four weeks significantly improved ventricular function and reversed pathological ventricular remodeling. Mechanistically, SAC/VAL treatment induces the inhibition of the GSK3β-mediated NF-κB pathway through synergistically blocking angiotensin 1 receptor (AT1R) and activating natriuretic peptide receptor (NPR). In summary, we reported the therapeutic role of SAC/VAL in regulating the GSK3β/NF-κB signaling pathway to suppress the inflammatory response and apoptosis, thereby reducing cardiac dysfunction and remodeling post I/R., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Ischemia-reperfusion (I/R) is an important risk factor for cardiovascular diseases (CVDs) and cardiac transplantation, as I/R can cause myocardial cell hypoxia/reoxygenation (H/R) injury. Recent research has shown that circular RNAs (circRNAs) may affect the progress of H/R-induced myocardial injury, but the mechanism remains unknown. Our work explored the role of circ&#95;0010729 in H2O2-induced myocardial injury., Methods: The levels of circ&#95;0010729, microRNA-1184 (miR-1184) and mRNA of receptor interacting serine/threonine kinase 1 (RIPK1) were indicated by quantitative real-time polymerase chain reaction (qRT-PCR) in human cardiac myocytes (HCMs). Meanwhile, the protein level of RIPK1 was quantified by western blot analysis. Besides, the cell functions were examined by 5-Ethynyl-29-deoxyuridine (EdU) assay, flow cytometry assay, western blot and antioxidant indexes analysis. Furthermore, the interplay between miR-1184 and circ&#95;0010729 or RIPK1 was detected by dual-luciferase reporter assay. Eventually, the in vivo experiments were applied to measure the role of circ&#95;0010729., Results: The levels of circ&#95;0010729 RNA and RIPK1 protein were increased, and the miR-1184 was decreased in HCMs exposed to H 2 O 2 . In functional analysis, circ&#95;0010729 deficiency restrained cell apoptosis and oxidative stress, whereas promoted cell proliferation in HCMs under H 2 O 2 exposure. Moreover, miR-1184 inhibited the H 2 O 2 -induced myocardial injury by targeting RIPK1. Mechanistically, circ&#95;0010729 acted as a miR-1184 sponge to regulate the level of RIPK1., Conclusion: Circ&#95;0010729 promotes H 2 O 2 -induced myocardial injury, and thus circ&#95;001729 may be targeted as a potential therapy for H/R-induced myocardial injury., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: In patients undergoing interventional or surgical coronary revascularization, subclinical hypothyroidism is common and associated with worse outcome, including the need for postoperative inotropic support. In isolated rat hearts with global ischemia/reperfusion, exogenous triiodothyronine (T3) reduces infarct size. Aim of this study was, to investigate whether or not exogenous T3 protects human myocardium from ischemia/reperfusion injury., Methods: Right atrial trabeculae from patients undergoing routine coronary artery bypass grafting were isolated and transferred to Tyrode's buffer. Electrically initiated (1 Hz) contractile stress (mN/mm 2 ) was recorded for 10 min at baseline (95% O 2 / 5% CO 2 , glucose). Sixty min hypoxia were induced by changing buffer gas and increasing stimulation rate (95% N 2 / 5% CO 2 , choline chloride, 3 Hz) before return to reoxygenation for 30 min. T3 (500 μg/l) vs. NaOH (solvent control) was administered A) throughout (n = 11 vs. n = 9) or B) only 15 min before and during reoxygenation (n = 12 vs. n = 13). Western blot analyses of established cardioprotective signaling proteins were performed., Results: At baseline, contractile stress was comparable. T3 improved the cumulative recovery of contractile stress during reoxygenation from 41 ± 16 with NaOH to 55 ± 11% of baseline with T3, when given continuously in A or from 52 ± 13 with NaOH to 63 ± 11% of baseline with T3 when given just before and during reoxygenation in B. The ratio of mitochondrial complex I matrix arm to membrane NADH:ubiquinone oxidoreductase subunits (NDUF)V2 to NDUFA9 was reduced, reflecting increased complex I activity., Conclusion: T3 increases contractile recovery of human right atrial trabeculae from hypoxia/reoxygenation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Traumatic brain injury can lead to fatal outcomes such as disability and death. Every year, it affects many patients all over the world. Not only the primary ischemic event, but also the subsequent reperfusion can cause severe brain injury. This so-called ischemia/reperfusion injury combined with mechanical forces lead to cellular disruption. Hence, this paper describes a special in-vitro model, mimicking traumatic brain injury by combining both hypoxia/reoxygenation and compression to simulate ischemia/reperfusion injury as well as the mechanical effects that occur concurrently when suffering traumatic brain injury. Through this approach, stroke, concussion, and traumatic brain injury can be studied on different cell lines in a simplified way. We used two primary mouse brain cell cultures, namely neurons and endothelial cells. Our results show that for the different cell types, different timelines of hypoxia and compression need to be explored to achieve the optimal amount of cellular damage in order to effectively mimic traumatic brain injury. Thus, this model will be useful to test potential treatments of brain injury in future in-vitro studies., (Published by Elsevier Inc.)

Myocardial ischemia/reperfusion (MI/R) may impair cardiac functions. Dexmedetomidine (DEX) is protective in various clinical cases. Therefore, this study investigated the role and mechanism of DEX in MI/R. The myocardial infarct size, apoptosis, and levels of myocardial enzymes, SOD, ROS, Ca 2+ , and inflammatory factors in DEX-treated MI/R rats were measured. Differentially expressed microRNAs (miRs) in DEX-treated MI/R rats were detected. miR-346-3p was intervened to assess the effects of DEX on MI/R rats. The targeted binding relationship between miR-346-3p and CaMKIId was predicted and verified. DEX effect on hypoxia/reoxygenation (H/R)-induced cell model was evaluated. The role of CaMKIId in DEX protection was assessed after CaMKIId overexpression in H/R cells. NF-κB pathway and NLRP3 inflammasome-related protein levels were detected. DEX alleviated the myocardial injury and Ca 2+ overload in MI/R rats, as evidenced by reduced infarct size, apoptosis and levels of myocardial enzymes, ROS, Ca 2+ , and inflammatory factors. DEX promoted miR-346-3p expression in MI/R rats, and miR-346-3p knockdown reversed DEX protection on MI/R rats. miR-346-3p targeted CaMKIId. DEX improved H/R-induced cell injury and Ca 2+ overload and inhibited NF-κB/NLRP3 inflammasome-related protein levels, which were all reversed by CaMKIId overexpression. DEX alleviated injury and Ca 2+ overload in MI/R via regulating the miR-346-3p/CaMKIId axis and inhibiting the NF-κB/NLRP3 inflammasome pathway., Competing Interests: Declaration of Competing Interest The authors declared that they have no competing interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Introduction: Attenuation of trophoblast cell dysfunction would be beneficial for retarding pre-eclampsia (PE). Vitamin D has been reported to improve trophoblast cell function in early PE, but the mechanism involved is not fully elucidated. This study is aimed to investigate whether vitamin D alleviates trophoblast cell dysfunction via regulating autophagy., Methods: Human trophoblast HTR-8 cells were cultured in hypoxia/reoxygenation (H/R) condition to simulate the oxidative stress state of early PE in vitro. MTT, Transwell and tube formation assays were respectively applied to assess cell proliferation, invasion, and angiogenesis abilities. DCFH-DA staining was performed to detect cellular reactive oxygen species levels. GFP-RFP-LC3 plasmid transfection and transmission electron microscopy were subjected to monitor autophagy. Enzyme-linked immunosorbent assay and Western blot analysis were used to detect autophagy-related and pyroptosis-associated molecules., Results: H/R led to severe impairments on the bio-function of HTR-8 cells, as evidenced by the deficiency of cell proliferation, invasion, and angiogenesis abilities, and the increase of cellular ROS production. Simultaneously, H/R inhibited autophagy and triggered pyroptosis. 1,25(OH) 2 D 3 , the hormonally active form of vitamin D, dramatically attenuated H/R-induced trophoblast dysfunction. Also, 1,25(OH) 2 D 3 activated autophagy and inhibited pyroptosis. Additionally, autophagy-enhancer rapamycin exerted similar protective effect to that of 1,25(OH) 2 D 3 , whereas autophagy-inhibitor 3-methyladenine blocked the protective effect of 1,25(OH) 2 D 3 ., Discussion: The mechanism that vitamin D alleviates trophoblast cell dysfunction is associated with autophagy induction and pyroptosis inhibition., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We have focused on the underlying role of miR-1224 in cardiomyocyte injury stimulated by hypoxia/reoxygenation (H/R). In the current study, the rat cardiomyocyte cell line H9C2 was used to construct a H/R cell model to validate the cardioprotective effects of miR-1224. Data from the dual-luciferase assay revealed that the glutathione peroxidase 4 (GPX4) was a direct target of miR-1224. Expression of miR-1224, determined using qRT-PCR, was remarkably increased while that of GPX4 protein, evaluated via western blotting, was significantly decreased in cardiomyocytes in response to H/R exposure. ROS generation, superoxide dismutase (SOD) activity, concentrations of malondialdehyde (MDA) and 4-hydroxy aldehydes (4-HNE), and H9C2 cell apoptosis were further evaluated following overexpression of miR-1224 or silencing of GPX4 in H9C2 cells. H9C2 cells under H/R conditions displayed increased synthesis of ROS, along with overexpression of miR-1224 and downregulation of GPX4. SOD activity was significantly decreased while concentrations of MDA and 4-HNE were markedly increased under H/R injury conditions. In addition, miR-1224 mimic or GPX4 siRNA plasmids dramatically enhanced H/R-mediated apoptosis, Bax expression and caspase-3 activity, with a concomitant reduction in Bcl-2 expression. Conversely, inhibition of miR-1224 exerted suppressive effects on oxidative stress and apoptosis in H9C2 cells under H/R conditions. Interestingly, silencing of GPX4 attenuated the negative effects of miR-1224 inhibition. Our results suggested that inhibition of miR-1224 caused resistance to H/R and diminished oxidative stress in vitro through targeting of GPX4., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In our previous study, we observed that donor pulmonary intravascular nonclassical monocytes play a major role in early PGF, but the specific mechanism remained unclear. In this study, we investigated the mechanistic role of monocytes in inducing pyroptosis of human pulmonary microvascular endothelial cells (HPMECs) during IRI. A murine hilar ligation model of IRI was utilized whereby left lungs underwent 1 h of ischemia and 23 h of reperfusion. Monocyte depletion by intraperitoneal clodronate-liposome treatment on pulmonary edema and pyroptosis activation were determined. In vitro experiments, we performed the co-culture experiments under hypoxia-reoxygenation (H/R) conditions to mimic the IRI environment. We monitored the expression of NLRP3, caspase-1 and IL-1β in co-cultures of monocytes (U937 cells) and HPMECs under H/R conditions. NLRP3, IL-1β and IL-1R siRNA knockdown, caspase-1 and NF-κB pathway inhibitors were employed to elucidate the mechanism modulating HPMEC pyroptosis during H/R. Treatment of mice with clodronate-liposome attenuated IR-induced pulmonary edema, cytokine production and pyroptosis activation. In vitro, NLRP3 knockdown in monocytes reduced caspase-1 and IL-1β secretion in co-cultures of monocytes and HPMECs. Reduced HPMEC pyroptosis was also observed either containing HPMECs with genetically engineered IL-1R knockdown or in co-culture treated with a Triplotide inhibitor that disrupts NF-κB signaling. Monocytes play a vital role in the development of transplant-associated ischemia-reperfusion injury. A potential role is that monocytes secrete IL-1β to induce HPMEC pyroptosis via the IL-1R/NF-κB/NLRP3 pathway., (Copyright © 2021. Published by Elsevier Inc.)

Background: Hypoxia/reoxygenation (H/R) injury of cardiomyocytes causes an irreversible damage to heart and largely results in acute myocardial infarction. Study has indicated lncRNA ROR aggravates myocardial ischemia/reperfusion (I/R) injury. Also, lncRNA ROR sponges miR-138 to promote osteogenesis. MiR-138 involves in hypoxic pulmonary vascular remodelling by targeting Mst1. However, the interaction between lncRNA ROR, miR-138 and Mst1 involved in myocardial H/R injury is still unknown., Methods: H9C2 cells were used to establish H/R injury model. The expression levels of lncRNA ROR and miR-138 were modified by transfection with the miR-138 mimics or lncRNA ROR overexpression plasmid. MTT and flow cytometry analysis were performed to detect cell proliferation and apoptosis. Dual luciferase reporter assay was used to determine interaction between lncRNA ROR and miR-138 or miR-138 and Mst1. Expression levels of lncRNA ROR, miR-138, Mst1 and apoptosis-related markers were determined by qRT-PCR or western blotting., Results: LncRNA ROR was significantly up-regulated, while miR-138 was obviously down-regulated in H/R-induced injury of H9C2 cells. Furthermore, miR-138 overexpression alleviated cardiac cell apoptosis induced by H/R injury. Mst1 was revealed to be a target of miR-138 and negatively regulated by miR-138. Mst1 overexpression reversed the protective effects of miR-138 on H/R injury of H9C2 cells. LncRNA ROR was identified as a sponge for miR-138. MiR-138 could protect H9C2 cells form H/R injury induced by lncRNA ROR overexpression., Conclusion: Our study provides that lncRNA ROR sponges miR-138 to aggravate H/R-induced myocardial cell injury by upregulating the expression of Mst1., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Shortly after joint remobilization, inflammation is induced in the joint and aggravates joint contracture via subsequent fibrosis. However, the mechanisms involved in remobilization-induced inflammation are not yet fully understood. We hypothesized that joint immobilization followed by remobilization induces hypoxia/reoxygenation, initiating inflammatory reactions through nitric oxide (NO) production via NO synthase 2 (NOS2). This study aimed to investigate whether: 1) administration of the NOS inhibitor L-NG-nitroarginine methyl ester (l-NAME) can attenuate remobilization-induced joint inflammation; and 2) hypoxia/reoxygenation is induced by joint immobilization and followed by remobilization. Unilateral knee joints of rats were immobilized using external fixators for three weeks. After removal of the fixation device, knees were allowed to move freely for one day (remobilization) with or without l-NAME administration. Without l-NAME administration, inflammatory reactions including joint swelling and inflammatory cell infiltration, edema, and upregulation of inflammatory mediator genes in the joint capsule were detected following upregulation of the NOS2 gene after remobilization. These remobilization-induced inflammatory reactions were partially attenuated by administration of l-NAME. Therefore, NOS2/NO elevation has potential as a novel treatment for remobilization-induced joint inflammation. Gene expression of the hypoxia marker hypoxia inducible factor-1α was upregulated after one day of remobilization, rather than after immobilization. These results suggest that upregulation of NOS2 by remobilization might be not due to hypoxia/reoxygenation., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Homeodomain interacting protein kinase-2 (HIPK2) has emerged as a crucial stress-responsive kinase that plays a critical role in regulating cell survival and apoptosis. However, whether HIPK2 participates in regulating cardiomyocyte survival during myocardial ischemia/reperfusion injury remains unclear. Here, we investigated the regulatory effect of HIPK2 on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its potential underlying molecular mechanism. We found that HIPK2 expression was induced in response to H/R exposure. HIPK2 depletion by small interfering RNA (siRNA)-mediated gene silencing significantly decreased the viability and exacerbated H/R-induced apoptosis and reactive oxygen species (ROS) production in cardiomyocytes. Comparatively, HIPK2 overexpression effectively rescued H/R-impaired viability and repressed H/R-induced apoptosis and ROS production in cardiomyocytes. HIPK2 overexpression significantly increased the nuclear expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and enhanced Nrf2-mediated transcriptional activity. Moreover, HIPK2 overexpression significantly increased the transcription of Nrf2/ARE target genes. Additionally, Nrf2 inhibition partially reversed the HIPK2-mediated protective effect. Overall, these results demonstrate that HIPK2 overexpression protects cardiomyocytes from H/R-induced injury by enhancing Nrf2/ARE antioxidant signaling, data that suggest HIPK2 is a potential target for cardioprotection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: To observe the effect of dexmedetomidine (DEX) on mitochondrial apoptosis of hippocampal neurons in hypoxia/reoxygenation (H/R) brain injury in developing rats, and to investigate its regulatory mechanism on HIF-1α/p53 signaling pathway., Methods: Hypoxia/reoxygenation model was used in this study. TUNEL assay was performed to detect cell apoptosis. Immunohistochemical analysis and Western-blotting analysis were conducted to detect Cytochrome-C (Cyt-c), APAF-1, Caspase-3, Neuroglobin (Ngb), HIF-1α and p53 expression. After 28 days, Morris water maze (MWM) was performed., Results: 50 μg/kg DEX improved H/R-induced brain injury and inhibited mitochondrial apoptosis in rats. Western-blotting and Immunohistochemical results demonstrated that DEX could up-regulate Ngb through α 2 receptor to inhibit H/R-induced mitochondrial apoptosis. In addition, by adding inhibitors yohimbine and 2-methoxyestradiol (2ME2), we found that DEX could activate HIF-1α/p53 signaling pathway. MWM test showed that DEX could enhance long-term learning and memory of H/R brain injury rats., Conclusion: DEX alleviates H/R-induced brain injury and mitochondrial apoptosis in developing rats through α 2 receptor, which may be related to activation of HIF-1α/p53 signaling pathway to up-regulate the expression of Ngb., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Remifentanil postconditioning (RPC) has been shown to provide potent cardioprotection against ischemia/reperfusion (I/R) injury, but the underlying mechanism has not been fully elucidated. The current study was designed to investigate whether RPC protects cardiomyocytes against I/R injury through enhancement of autophagic flux. H9c2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to mimic myocardial I/R injury in vitro. Autophagosome formation was evaluated by detecting of light chain 3 (LC3) puncta number and LC3Ⅱ levels using immunofluorescence and western blotting, respectively. Additionally, dual fluorescent staining of LC3 and lysosomal-associated membrane protein 2, a lysosomal marker protein, were used to detect autolysosome formation. Moreover, autophagic flux integrity was tracked using changes in LC3Ⅱ and p62 levels. Lastly, myocardial injury was detected by Hoechst 33342 and propidium iodide staining and MTT assay. The results showed that RPC increased autophagosome formation and promoted autophagosome-lysosome fusion, thereby improving autophagic flux in H9c2 cells. Reversal of these effect by bafilomycin A1 or chloroquine co-administration at reoxygenation onset indicated that RPC improved the impaired autophagic flux following H/R injury. Induction of autophagy was associated with increased cell viability and decreased apoptosis. Autophagy inhibition with bafilomycin A1 or chloroquine and ATG7shRNA significantly abolished RPC-induced cardioprotection. In conclusion, our finding that RPC can protect cardiomyocytes against H/R injury through enhancement of autophagic flux suggests a new mechanism for myocardial protection of opioid postconditioning., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Our previous study showed that Sphingosine-1-phosphate (S1P) could protect cardiomyocytes against hypoxia/reoxygenation (H/R) injury via the JAK-STAT pathway and maintain normal myocardial mitochondria integrity in vivo. However, it is not known yet whether S1P can relieve mitochondrial dysfunction via the mitochondrial apoptotic pathway and its detailed mechanism remains to be investigated. The aim of this study was to demonstrate the mitochondrial protective effects of S1P in a cardiomyocyte H/R injury model. In the present study, we established a H/R model in H9c2 cells. Cell viability was determined by the MTT assay, and apoptosis was evaluated by annexin V-FITC/PI staining. Mitochondrial calcium ion concentration, mitochondrial membrane potential (ΔΨm), opening of the mitochondrial permeability transition pore (mPTP), and release of cytochrome C were detected by laser confocal microscopy. The results showed that S1P inhibited the decrease in cell viability induced by H/R injury and reduced apoptosis. Confocal microscopy showed that S1P prevented loss of ΔΨm, relieved mitochondrial calcium overload, and inhibited opening of the mPTP and release of cytochrome C. The STAT3 inhibitor STATTIC can reverse the antiapoptotic effects of S1P and block the effect of S1P on mitochondria. Taken together, our results indicate that S1P protects cardiomyocytes against H/R injury by relieving mitochondrial dysfunction via the STAT3 pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hypoxia/reoxygenation (H/R) plays an important role in the pathogenesis of osteoarthritis. Fibroblast-like synoviocytes (FLS), which are highly sensitive to H/R, are thought to be associated with cartilage degradation during osteoarthritis development. In this study, we investigated the biological effects of insulin-like growth factor (IGF) system and the expression of inflammatory mediators in FLS. We also pretreated FLS with tumor necrosis factor-α (TNF-α) before H/R in order to observe the response of FLS with the background of inflammatory cytokines. H/R increased the levels of TNF-α-induced C-C chemokine ligand 5 (CCL5), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in cell-free culture supernatants; H/R also increased the expression of TNF-α-induced insulin-like growth factor binding protein 3 (IGFBP-3), downregulated the expression of insulin-like growth factor 1 (IGF-1), promoted the loss of mitochondrial membrane potential (MMP), the openness of mitochondrial permeability transition pore (MPTP), the release of intracellular reactive oxygen species (ROS), and mitochondrial matrix swelling, outer membrane rupture and decrease in cristae. Furthermore, H/R induced the expression of catabolic factors and activated the NF-κB signaling pathway in FLS. We therefore concluded that H/R may play a role in inducing inflammation and increase the TNF-α-induced inflammatory effect in FLS, contributing to osteoarthritis pathogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Fibroblast growth factor 19 (FGF19) has emerged as a crucial cytoprotective regulator that antagonizes cell apoptosis and oxidative stress under adverse conditions. However, whether FGF19 plays a cytoprotective role in preventing myocardial damage during myocardial ischemia/reperfusion injury remains unknown. In this study, we aimed to investigate the potential role of FGF19 in regulating hypoxia/reoxygenation (H/R)-induced injury of cardiomyocytes in vitro. We found that FGF19 expression was upregulated in response to H/R treatment in cardiomyocytes. Silencing of FGF19 significantly inhibited viability and increased apoptosis and reactive oxygen species (ROS) generation in cardiomyocytes with H/R treatment. In contrast, overexpression of FGF19 improved viability and inhibited apoptosis and ROS generation induced by H/R treatment, showing a cardioprotective effect. Moreover, we found that FGF19 regulated the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and the nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2). In addition, FGF19 promoted the activation of Nrf2-mediated antioxidant response element (ARE) antioxidant signaling. Notably, treatment with a GSK-3β inhibitor significantly abrogated the adverse effects of FGF19 silencing on H/R-induced injury, whereas silencing of Nrf2 partially blocked the FGF19-mediated cardioprotective effect against H/R-induced injury in cardiomyocytes. Taken together, our findings demonstrate that FGF19 alleviates H/R-induced apoptosis and oxidative stress in cardiomyocytes by inhibiting GSK-3β activity and promoting the activation of Nrf2/ARE signaling, providing a potential therapeutic target for prevention of myocardial injury., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hepatic ischemia/reperfusion (I/R) injury induces oxidative stress, hepatocyte apoptosis, and release of inflammatory cytokines, which together causes liver damage and even organ dysfunction. TNF-α-induced protein 3-interacting protein 1 (TNIP1) reportedly decreases expression of genes associated with stress response and inflammation. Thus, we investigated the effects of TNIP1 on hepatic cells injury caused by hypoxia/reoxygenation (H/R). Reduced expression of TNIP1 was determined in I/R mice compared to normal mice. Then, TNIP1 transgene mice were used to determine the effects of TNIP1 on mice after treatment for I/R. In the normal transgene (NTG) group, serum liver damage markers alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and gamma glutamyl transferase (GGT) in I/R mice significantly increased compared to the sham-operated mice. However, in the TNIP1 transgene (TNIP1-TG) group, those levels in I/R mice were reduced than that in NTG mice. Additionally, cell viability and apoptosis in the hepatic cell line L02 were detected after H/R treatment, MTT assay showed that cell viability was inhibited after H/R treatment, but reversed after ad-TNIP1 transfection. Cell apoptosis also was inhibited after ad-TNIP1 transfection, as shown by the caspase-3 and caspase-9 levels and Bcl-2 and Bax values. Furthermore, TNIP1 overexpression also attenuated the inflammatory response of L02 cells after H/R treatment. Finally, treatment with TNIP1 reduced the elevated expression of TLR2, TLR4, and Myd88 after H/R injury, but overexpression of TLR4 reversed the effects of TNIP1. In conclusion, TNIP1 may protect H/R-induced hepatic cell injury by inhibiting the TLR4/Myd88 pathway., (Copyright © 2018. Published by Elsevier Inc.)

Coptisine is a natural occurring isoquinoline alkaloid isolated from the traditional Chinese medicinal herb Rhizoma coptidis. Coptisine has been reported to have protective effects on reperfusion injury in cardiomyocytes, however, the underlying mechanism remains uncertain. Here, we used a hypoxia/reoxygenation (H/R)-treated H9c2 cell model to study the protective effects of coptisine on cardiomyocyte. The results showed that NaS 2 O 4 induced hypoxia/reoxygenation model increased apoptosis and up-regulated autophagy marker LC3-II and cleaved Caspase-3, Beclin1 and Sirt1 levels. Coptisine treatment increased cell survival, inhibited apoptosis, and reduced the protein level of LC3-II, cleaved Caspase-3, Beclin1 and Sirt1. Further, we showed that coptisine combined with chloroquine (CQ), the inhibitor of autolysosome, reduced LC3-II, suggesting that coptisine may inhibit autophagosome formation than induction of autolysosome in the autophagy events. Our results indicate that coptisine may protect cardiomyocyte damage by H/R through suppressing autophagy. Overall, our study provides a new mechanism for the treatment of coptisine on H/R-induced cardiomyocyte damage and death., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Oxidative stress and inflammation play important roles in the pathogenesis of ischemia/reperfusion (I/R)-injury. The administration of antioxidants and anti-inflammatory agents has been applied to prevent I/R-injury for several decades. Of the numerous compounds administrated therapeutically in anti-oxidative stress, nitronyl nitroxide has gained increasing attention due to its continuous ability to scavenge active oxygen radicals. However, its effect is not ideal in clinical therapy. In previous study, we linked the anti-inflammatory amino acid glycine to nitronyl nitroxide and developed a novel glycine-nitronyl nitroxide (GNN) conjugate, which showed a synergetic protection against renal ischemia/reperfusion injury. However, the underlying mechanism remains unclear. In this study, a hypoxia/reoxygenation (H/R) injury model was established in human umbilical vein endothelial cells (HUVECs) and we found that the GNN conjugate significantly elevated the cell viability via reducing the apoptosis rate in H/R-treated HUVECs. Meanwhile, GNN conjugate attenuated H/R induced mitochondrial fragmentation, mitochondrial membrane potential reduction, Cytochrome c release and autophagy. To determine the extensive applicability of GNN conjugate in different I/R models and its effect in remote organs, an in vivo hind limb I/R model was established. As expected, GNN conjugate ameliorated damages of muscle and remote organs. These results demonstrate that GNN conjugate may be an effective agent against ischemia/reperfusion injury in clinical therapy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

High-mobility group box 1 (HMGB1) translocation and release, which is involved in several tissue types of ischemia-reperfusion injuries, activate innate immunity by inducing proinflammatory cytokine production through its interaction with toll-like receptors (TLRs). Our objective was to determine the role of HMGB1 and the degree of activation of TLR-related signal transduction pathways in hypoxia/reoxygenation (H/R)-induced proinflammatory cytokine production and intra-islet graft inflammation. After islets are exposed to hypoxia-reoxygenation for 24h, TLR2/4 expression and TLR-mediated signaling was up-regulated in islets, and HMGB1 was translocated from the nucleus to the cytoplasm and released to the extracellular space. With H/R exposure, proinflammatory cytokine production (IL-1β and TNF-α) and islet injury were significantly increased, and these effects depend on TLR2/4 signaling pathways. Exogenous HMGB1 also induces islet inflammation and increases the phosphorylation of STAT3, p38 and IκBα in wild-type islets. TLR2 deficiency in TLR2-KO islets resulted in the inhibition of IL-1β production and STAT3/p38 phosphorylation after HMGB1 exposure. TLR4 deficiency in TLR4-KO islets resulted in the inhibition of TNF-α production and IκBα phosphorylation after HMGB1 exposure. Pre-incubation of the STAT3, p38, or NF-κB inhibitors significantly inhibited HMGB1-induced IL-1β or TNF-α production in islets, but the effect of HMGB1 or H/R-induced islet injury was not counteracted by a separate treatment of the STAT3 inhibitor, p38 inhibitor, or NF-κB inhibitors. HMGB1 inhibition by ethyl pyruvate or blockade by neutralizing antibodies significantly decreased the phosphorylation of STAT3, p38 and IκBα, the production of IL-1β and TNF-α, and the islet injury in wild-type islets after exposure to H/R and significantly improved early islet graft failure. Thus, our results suggest that HMGB1 released from H/R induced islets works in an autocrine manner to up-regulate STAT or p38 and augment IL-1β production via TLR2, and up-regulate NF-κB and augment TNF-α production via TLR4 in intra-islet, which are associated with H/R-induced islet injury and early graft failure., (Copyright © 2016 Elsevier B.V. All rights reserved.)

MicroRNA-122 has been reported to play a potential role in the apoptosis of myocardial cells. However, the effect of miR-122 in regulating myocardial ischemic injury has not been previously addressed. This study aimed to investigate the effect and the molecular basis of miR-122 on myocardial ischemic injury. Using the hypoxia/reoxygenation (H/R) model of rat cardiomyocytes H9C2 in vitro, we found that miR-122 was highly expressed in H9C2 cells after H/R treatment. Overexpression of miR-122 by recombinant adeno-associated viral vector infection markedly promoted the apoptosis of H9C2 cells induced by H/R treatment, whereas miR-122 inhibition significantly decreased cell apoptosis. Dual-luciferase reporter assay and western blot assay revealed that GATA-4 was a direct target gene of miR-122, and miR-122 suppressed the expression of GATA-4 via binding to its 3'-UTR. We further identified that overexpression of miR-122 inhibited the expression of GATA-4 at the mRNA and protein levels, whereas the inhibition of miR-122 upregulated the expression of GATA-4. Moreover, GATA-4 was poorly expressed in H/R H9C2 cells and the apoptosis induced by H/R was associated with the decrease in GATA-4 expression. Importantly, silencing of GATA-4 apparently abrogated the inhibitory effect of anti-miR-122 on H/R-induced cell apoptosis. In conclusion, these findings indicate that downregulation of miR-122 alleviates cardiomyocyte H/R injury through upregulation of GATA-4 expression, supplying a novel molecular target for myocardial ischemic injury., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hypoxia/reoxygenation (H/R) injury of cholangiocytes causes serious biliary complications during hepatobiliary surgeries. Molecular hydrogen (H2) has been shown to be effective in protecting various cells and organs against oxidative stress injury. Human liver cholangiocytes were used to determine the potential protective effects of hydrogen against cholangiocyte H/R injury and explore the underlying mechanisms. We found that H2 ameliorated H/R-induced cholangiocytes apoptosis. Our study revealed that H2 activated NF-E2-related factor 2 (Nrf2) and downstream cytoprotective protein expression. However, the protective function of H2 was abolished when Nrf2 was silenced. Apoptosis in cholangiocytes isolated from a rat model of liver ischemia/reperfusion injury indicated that H2 significantly attenuates ischemia/reperfusion cholangiocyte injury in vivo. In conclusion, our study shows that H2 protects intrahepatic cholangiocytes from hypoxia/reoxygenation-induced apoptosis in vitro or in vivo, and this phenomenon may depend on activating Nrf2 expression., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Aims: The α2-adrenoceptor inducer dexmedetomidine (Dex) provides renoprotection against ischemia/reperfusion (I/R) injury, but the mechanism of this effect is largely unknown. The present study investigated the effect of Dex on apoptosis induced by hypoxia/reoxygenation (H/R) and the relationship between this effect and gap junction intercellular communication (GJIC)., Main Methods: In vitro, two cell lines of normal rat kidney proximal tubular cells (NRK-52E) and HeLa cells that were transfected with a connexin 32 (Cx32) plasmid were exposed to H/R. The role of Dex in the modulation of H/R-induced apoptosis was explored by the manipulation of connexin expression, and hence gap junction (GJ) function, using a GJIC inhibitor, heptanol, and a GJIC inducer, retinoic acid. GJ function and the Cx32 protein level were determined by the parachute dye-coupling assay and Western blotting, respectively., Key Findings: Dex and heptanol significantly reduced H/R-induced apoptosis in NRK-52E cells. The anti-apoptosis effect of Dex was exhibited only in Cx32-expressing HeLa cells. One hour Dex exposure inhibited GJ function mainly via a decrease in Cx32 protein levels in NRK-52E cells., Significance: Our data suggest that Dex reduced H/R-induced apoptosis through the inhibition of GJ activity by reducing Cx32 protein levels., (Copyright © 2014 Elsevier Inc. All rights reserved.)

FTY720, sphingosine 1 phosphate (S1P) receptor agonist, is a potent immunosuppressive agent. Numerous studies have documented a relationship between S1P and cardioprotection. We therefore hypothesized that a S1P analogue FTY720 would attenuate hypoxia/reoxygenation (H/R) induced cadiomyocyte apoptosis. H9C2 cardiomyocytes were employed to establish an in vitro model of H/R. Cells were treated or not with different doses of FTY720. Cell viability was measured by flow cytometry and TUNEL staining. Western blot was used to analyze downstream signaling pathway. We observed that FTY720 inhibits the expression of cleaved caspase-3 and activates both AKT and ERK1/2 pathways. AKT pathway can be blocked by MEK kinase inhibitor PD98059. ERK1/2 pathway can be blocked by the phosphoinositide-3 kinase inhibitor wortmannin. AKT and ERK1/2 activation can also be inhibited by S1P1/3 receptor antagonist VPC23019, Gi antagonist PTX. The protein levels of TNF-α and IL1ß were upregulated during hypoxia/reoxygenation and were attenuated by FTY720. We conclude that FTY720, via its cargo of S1P, can protect cardiomyocytes against hypoxia/reoxygenation injury. This effect is achieved by inhibiting caspase-3 expression, inflammatory cytokine levels and activating AKT and ERK1/2 signaling pathways. The prosurvival signal activation is dependent on S1P1, 3 subtype receptors and Gi protein., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Alpha-lipoic acid (LA) plays an important role in the etiology of cardiovascular disease. Autophagy is widely implicated in myocardial I/R injury. We assessed the degree of autophagy by pretreatment with LA exposed to H/R in H9c2 cell based on the expression levels of Beclin-1, LC3II/LC3I, and green fluorescent protein-labeled LC3 fusion proteins. Autophagic vacuoles were confirmed in H9c2 cells exposed to H/R using transmission electron microscopy. Our findings indicated that pretreatment with LA inhibited the degree of autophagy in parallel to the enhanced cell survival and decreased total cell death in H9c2 cells exposed to H/R. We conclude that LA protects cardiomyocytes against H/R injury by inhibiting autophagy., (Copyright © 2013. Published by Elsevier Inc.)

Background: Mammalian sterile 20-like kinase 1 (Mst1) is a mammalian homolog of Hippo kinase from Drosophila and it is a critical component of the Hippo signaling pathway, which regulates a variety of biological processes ranging from cell contact inhibition, organ size control, apoptosis and tumor suppression in mammals. Mst1 plays essential roles in heart disease since its activation causes cardiomyocyte apoptosis and dilated cardiomyopathy. However, the mechanism underlying Mst1 activation in the heart is not known., Methods and Results: To identify novel cardiac proteins that may regulate Mst1 activity in the heart under pathophysiological conditions, a yeast two-hybrid screening of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait was performed. As a result, protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1) was identified as an Mst1-interacting protein. The interaction of PCMT1 with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK293 cells and native cardiomyocytes, in which PCMT1 interacted with the kinase domain of Mst1, but not with its C-terminal regulatory domain. Overexpression of PCMT1 did not affect the Mst1 expression, but significantly attenuated the Mst1 activation and its apoptotic effects in response to the hypoxia/reoxygenation induced injury in cardiomyocytes. Indeed, upregulation of PCMT1 by CGP3466B, a compound related to the anti-Parkinson's drug R-(-)-deprenyl with potent antiapoptotic effects, inhibited the hypoxia/reoxygenation induced Mst1 activation and cardiomyocyte apoptosis., Conclusions: These findings implicate PCMT1 as a novel inhibitor of Mst1 activation in cardiomyocytes and suggest that targeting PCMT1 may prevent myocardial apoptosis through inhibition of Mst1., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: The aim of this study was to investigate the cardioprotective effect of fimasartan, a newly developed angiotensin II receptor type I blocker (ARB), against myocardial ischemia/reperfusion (I/R) injury and to identify the mechanism by which it reduces mitochondrial damage., Methods: Fimasartan was administered intravenously to Sprague-Dawley rats (3mg/kg), cardiomyocytes (50 μM), and H9c2 cells (50 μM) before ischemia or hypoxia. Myocardial infarction (MI), echocardiograms, DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP in situ nick-end labeling, immunoblotting, oxygen consumption, confocal microscopic appearance, and L-type Ca(2+) current (ICa,L) were then assessed., Results: Fimasartan pretreatment remarkably reduced the rate of MI and improved cardiac performance well after I/R (n = 9/group). Fimasartan also reduced apoptotic cell death both in vivo and in hypoxia/reoxygenation (H/R)-treated H9c2 cells (n = 5~8/group). H/R-induced mitochondrial O2(-) production and collapse of membrane potential were markedly attenuated in fimasartan-treated cardiomyocytes (n = 4 ~ 6/group). Additionally, mitochondrial Ca(2+) overload during reoxygenation was suppressed by fimasartan (n = 4~6/group), and this was found to be possibly related to the inhibition of ICa,L and mitochondrial Ca(2+) uniporter. Furthermore, fimasartan pretreatment increased phosphorylations of Akt and glycogen synthase kinase-3β (n = 5 ~ 7/group), decreased pro-apoptotic p53 levels, and increased anti-apoptotic Bcl-2 levels (n = 4) during reperfusion., Conclusions: Fimasartan preconditioning has the potential to modulate Bcl-2 and suppress I/R-induced Ca(2+) overload by inhibiting ICa,L and MCU. These beneficial effects could prevent the mitochondrial dysfunction and apoptosis accompanied by I/R., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Ischemic stroke is a leading cause of death worldwide, yet therapies are limited. During periods of ischemia following reperfusion in ischemic stroke, not only loss of energy supply, but a few other factors including mitochondrial dysfunction and oxidative stress also make vital contribution to neuronal injury. Here we synthesized a steroid compound 5α-androst-3β,5,6β-triol by 3 steps starting from dehydroepiandrosterone and examined its effect on mitochondrial function and oxidative stress in primary cultured cortical neurons exposed to hypoxia followed by reoxygenation. 5α-Androst-3β,5,6β-triol dose-dependently protected cortical neurons from hypoxia/reoxygenation exposure. Rates of reduction in neuronal viability, loss of mitochondrial membrane potential, disruption of ATP production and oxidative stress were ameliorated in 5α-androst-3β,5,6β-triol pretreated cultures. In summary, these results suggest that 5α-androst-3β,5,6β-triol is neuroprotective against hypoxia/reoxygenation induced neuronal injuries through mediation of mitochondrial function and oxidative stress., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)