Hypoxia-induced Decrease Research Articles

Forskolin rescues hypoxia-induced cognitive dysfunction in zebrafish with potential involvement of O-GlcNAc cycling regulation

Repeated sublethal hypoxia exposure induces brain inflammation and affects the initiation and progression of cognitive dysfunction. Experiments from the current study showed that hypoxic exposure downregulates PKA/CREB signaling, which is restored by forskolin (FSK), an adenylate cyclase activator, in both Neuro2a (N2a) cells and zebrafish brain. FSK significantly protected N2a cells from hypoxia-induced cell death and neurite shrinkage. Intraperitoneal administration of FSK for 5 days on zebrafish additionally led to significant recovery from hypoxia-induced social interaction impairment and learning and memory (L/M) deficit. FSK suppressed hypoxia-induced neuroinflammation, as indicated by the observed decrease in NF-κB activation and GFAP expression. We further investigated the potential effect of FSK on O-GlcNAcylation changes induced by hypoxia. Intriguingly FSK induced marked upregulation of the protein level of O-GlcNAc transferase catalyzing addition of the GlcNAc group to target proteins, accompanied by elevated O-GlcNAcylation of nucleocytoplasmic proteins. The hypoxia-induced O-GlcNAcylation decrease in the brain of zebrafish was considerably restored following FSK treatment. Based on the collective results, we propose that FSK rescues hypoxia-induced cognitive dysfunction, potentially through regulation of HBP/O-GlcNAc cycling.

Effect of miR-182-5p on apoptosis in myocardial infarction

ObjectiveThis study aimed to delineate the diagnostic significance of miR-182-5p by investigating its influence on myocardial apoptosis and function, employing both in vivo and in vitro myocardial infarction models. MethodsA rat myocardial infarction model was established. Myocardial infarction area was detected using the 2,3,5-chlorotriphenyltetrazolium (TTC) method, myocardial enzyme spectrums were measured using enzyme-linked immunosorbent assay (ELISA), myocardial structure was detected by hematoxylin and eosin (HE) staining, myocardial apoptosis was detected using the TUNEL method, and expression levels of miR-182-5p and apoptosis-related molecules were detected using real-time fluorescence quantitative PCR (qPCR) and Western blot. miR-182-5p mimics and inhibitor were transfected into rat H9C2 cardiomyocytes and mouse HL-1 cardiomyocytes to establish a hypoxia model. Cardiomyocyte viability was detected using the CCK-8 method, expression levels of apoptosis-related indicators were detected using Western blot, and caspase-3/7 activity was detected using a caspase-3/7 activity detection kit. AAV9 adeno-associated virus was used to construct an miR-182-5p overexpression virus, which was injected into mice through the tail vein to create a mouse myocardial infarction model. TTC, ELISA, HE staining, echocardiography, real-time fluorescence qPCR, and Western blot methods were used to detect the effects of AAV9-miR-182-5p on myocardial injury, myocardial function, and myocardial apoptosis levels in myocardial infarction. ResultsThe rat model displayed reduced miR-182-5p expression concurrent with an increase in apoptosis. The in vitro H9C2 and HL-1 hypoxia models revealed that miR-182-5p augmented the hypoxia-induced decrease in myocardial cell viability, suppressed Bcl-2 expression, and increased Bax, Bnip3, and caspase-3/7 activity levels. The injection of AAV9-miR-182-5p significantly exacerbated myocardial tissue damage, impaired myocardial function, and enhanced apoptosis. ConclusionmiR-182-5p escalates myocardial injury during myocardial infarction by fostering apoptosis. Interventions that aim to reduce miR-182-5p levels might be crucial in halting the progression of myocardial infarction.

Hypoxia dissociates HDAC6/FOXO1 complex and aggregates them into nucleus to regulate autophagy and osteogenic differentiation.

This research aimed to elucidate the molecular mechanisms underlying the periodontitis-associated bone loss, with particular emphasis on the contributory role of hypoxic microenvironment in this process. Periodontitis generally causes alveolar bone loss and is often associated with a hypoxic microenvironment, which affects bone homeostasis. However, the regulating mechanism between hypoxia and jaw metabolism remains unclear. Hypoxia triggers autophagy, which is closely related to osteogenic differentiation, but how hypoxia-induced autophagy regulates bone metabolism is unknown. HDAC6 and FOXO1 are closely related to bone metabolism and autophagy, respectively, but whether they are related to hypoxia-induced bone loss and their internal mechanisms is still unclear. Established rat nasal obstruction model and hypoxia cell model. Immunohistochemistry was performed to detect the expression and localization of HDAC6 and FOXO1 proteins, analysis of autophagic flux and transmission electron microscopy was used to examine the autophagy level and observe the autophagosomes, co-immunoprecipitation and chromatin immunoprecipitation were preformed to investigate the interaction of HDAC6 and FOXO1. Hypoxia causes increased autophagy and reduced osteogenic differentiation in rat mandibles and BMSCs, and blocking autophagy can attenuate hypoxia-induced osteogenic differentiation decrease. Moreover, hypoxia dissociated the FOXO1-HDAC6 complex and accumulated them in the nucleus. Knocking down of FOXO1 or HDAC6 alleviated hypoxia-induced autophagy elevation or osteogenic differentiation reduction by binding to related genes, respectively. Hypoxia causes mandibular bone loss and autophagy elevation. Mechanically, hypoxia dissociates the FOXO1-HDAC6 complex and aggregates them in the nucleus, whereas HDAC6 decreases osteogenic differentiation and FOXO1 enhances autophagy to inhibit osteogenic differentiation.

Cocoa flavanols protect cognitive function, cerebral oxygenation, and mental fatigue during severe hypoxia.

We tested the hypothesis that ingestion of cocoa flavanols would improve cognition during acute hypoxia equivalent to 5,500 m altitude (partial pressure of end-tidal oxygen = 45 mmHg). Using placebo-controlled double-blind trials, 12 participants ingested 15 mg·kg-1 of cocoa flavanols 90 min before completing cognitive tasks during normoxia and either poikilocapnic or isocapnic hypoxia (partial pressure of end-tidal carbon dioxide uncontrolled or maintained at the baseline value, respectively). Cerebral oxygenation was measured using functional near-infrared spectroscopy. Overall cognition was impaired by poikilocapnic hypoxia (main effect of hypoxia, P = 0.008). Cocoa flavanols improved a measure of overall cognitive performance by 4% compared with placebo (effect of flavanols, P = 0.033) during hypoxia, indicating a change in performance from "low average" to "average." The hypoxia-induced decrease in cerebral oxygenation was two-fold greater with placebo than with cocoa flavanols (effect of flavanols, P = 0.005). Subjective fatigue was increased by 900% with placebo compared with flavanols during poikilocapnic hypoxia (effect of flavanols, P = 0.004). Overall cognition was impaired by isocapnic hypoxia (effect of hypoxia, P = 0.001) but was not improved by cocoa flavanols (mean improvement = 1%; effect of flavanols, P = 0.72). Reaction time was impaired by 8% with flavanols during normoxia and further impaired by 11% during isocapnic hypoxia (effect of flavanols, P = 0.01). Our findings are the first to show that flavanol-mediated improvements in cognition and mood during normoxia persist during severe oxygen deprivation, conferring a neuroprotective effect.NEW & NOTEWORTHY We show for the first time that cocoa flavanols exert a neuroprotective effect during severe hypoxia. Following acute cocoa flavanol ingestion, we observed improvements in cognition, cerebral oxygenation, and subjective fatigue during normoxia and severe poikilocapnic hypoxia. Cocoa flavanols did not improve cognition during severe isocapnic hypoxia, suggesting a possible interaction with carbon dioxide.

Human leukocyte antigen F-associated transcript 10 regulates the IKs potassium channel by competing for Kv7.1 ubiquitination.

The protein expression and function changes from the slow-delayed rectifying K+ current, IKs, are tightly associated with ventricular cardiac arrhythmias. Human leukocyte antigen F-associated transcript 10 (FAT10), a member of the ubiquitin-like-modifier family, exerts a protective effect against myocardial ischaemia. However, whether or how FAT10 influences the function of IKs remains unclear. Here, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and Fat10 knockout HEK293 (Fat10-/-) cells through CRISPR-Cas9 technology were used to evaluate the novel modulation of FAT10 in IKs function. Patch-clamp studies showed that the overexpression of FAT10 significantly enhanced the current density of IKs both in hiPSC-CMs and HEK293-Fat10-/- cells. In addition, a shortened action potential duration (APD) was seen from hiPSC-CMs transfected with the ad-Fat10 virus. Then, a series of molecular approaches from neonatal rat cardiomyocytes, H9C2 cells and HEK293 cells were used to determine the regulatory mechanism of FAT10 in IKs. First, western blot assays indicated that the expression of Kv7.1, the alpha-subunit of IKs, was increased when FAT10 was overexpressed. Furthermore, immunofluorescence and co-immunoprecipitation assays demonstrated that FAT10 could interact with Kv7.1. Notably, FAT10 impedes Kv7.1 ubiquitination and degradation, thereby stabilizing its expression. Finally, a hypoxia model of hiPSC-CMs was established, and the overexpression of FAT10 showed a protective effect against hypoxia-induced decreases in the current density of IKs. Taken together, these findings revealed a novel role of FAT10 in the regulation of the IKs potassium channel by competing for Kv7.1 ubiquitination, which provides a new electrophysiological insight that FAT10 could modulate Kv7.1. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

A small molecule inhibitor of SUMO prevents the hypoxia-induced decrease in IK1 current.

The inward rectifying potassium current (IK1) shapes the initial depolarization and the final repolarization phases of the cardiac action potential and is critical for maintaining the resting membrane potential of ventricular cardiomyocytes. Kir2, the inward rectifying potassium channels are the main contributors to the IK1 current, with Kir2.1 dominating. Disruption of IK1 current activity has been implicated in many cardiac arrhythmia phenotypes. Acute hypoxia during coronary occlusion changes the electrophysiological properties of the myocardium and can precipitate disordered conduction and arrhythmias due to ion channel dysregulation. However, the mechanistic basis for the proarrhythmic effects of acute hypoxia on cardiac tissue has remained unclear, limiting the development of novel therapeutics. Using whole-cell patch-clamp recording we show that acute hypoxia inhibits IK1 in rat ventricular cardiomyocytes, as well as in heterologous expression systems. We provide evidence to show that this inhibition is due to the rapid SUMOylation of Kir2.1 at lysine 49. When SUMO binds to the channel it decreases the efficacy and potency of the membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) to activate the channel. Finally, we show that a small molecule inhibitor of the SUMO-pathway precludes the hypoxia-mediated decrease in Kir2.1. We confirm that this effect is due to the direct inhibition of Kir2.1 SUMOylation within the cell. These data provide potential insight into investigation of the SUMO pathway as a target for treating the proarrhythmic effects of acute hypoxia.

Luhong Formula and Hydroxysafflor yellow A protect cardiomyocytes by inhibiting autophagy

BackgroundHeart failure (HF) is the terminal stage of all heart diseases that is characterized by irreversible cardiomyocyte injury. Equilibrium of autophagy is essential for cardiac cell survival. The Luhong formula (LHF) has been clinically applied for decades, and has exhibited significant efficacy in improving heart function and alleviating the symptoms of angina pectoris. PurposeTo clarify the mechanism of action of LHF and one of its main constituents, hydroxysafflor yellow A (HYSA), in protecting ischemic cardiomyocytes by inhibiting autophagy. MethodsCell viability was detected by CCK-8 assay with LHF or HYSA pretreatment followed by hypoxic damage. Immunofluorescence of GFP-LC3-H9C2 and GFP-LC3-HeLa cells was used to observe autophagic flux. Beclin 1 and HIF1α protein expression were assessed using western blotting. LHF was orally administered to Wistar rats following myocardial infarcion. Echocardiography was performed before the rats were sacrificed; immunohistochemistry and western blotting were used to evaluate Beclin 1 and HIF1α expression in the myocardial tissue. Hematoxylin and eosin staining as well as Masson's trichrome staining were used to measure cardiac structure and myocardial fibrosis. ResultsLHF and HYSA reversed the hypoxia-induced decrease in cell viability in vitro. LHF and HYSA induced the aggregation of GFP-LC3 puncta and reduced the expression of Beclin 1 protein in H9C2, suggesting that LHF and HYSA may inhibit autophagy activity. Pretreatment with reactive oxygen species (ROS) inducers and inhibitors revealed that LHF and HYSA inhibited autophagy by suppressing cellular ROS. Further studies demonstrated that LHF and HYSA reduced the ROS levels by inhibiting HIF1α. LHF delayed fibrosis and protected heart function in vivo in a rat model of HF following myocardial infarction. Western blotting and immunohistochemistry revealed that LHF effectively reduced the expression of Beclin 1 and HIF1α in the infarcted area of the rat heart. ConclusionThese results demonstrate that hydroxysafflor yellow A is the representative bioactive compounent of Luhong Formula on regulating autophagy to protectect cardiomyocytes from hypoxia injury. LHF and HYSA inhibit cardiac autophagy by suppressing HIF1α-mediated ROS production. This study helps to further clarify the underlying mechanism of LHF and provide a scientific basis for its development as a novel cardiovascular therapeutic agent.

FOXO3a-dependent up-regulation of HSP90 alleviates cisplatin-induced apoptosis by activating FUNDC1-mediated mitophagy in hypoxic osteosarcoma cells

Hypoxia-induced decrease in cisplatin (CDDP) sensitivity in human osteosarcoma (OS) is a significant obstacle to effective chemotherapy. Recently, mitophagy has been shown to be associated with CDDP sensitivity. However, whether it regulates hypoxia-induced decreases in CDDP sensitivity in OS and the underlying mechanisms remain unknown. In this study, we found that hypoxia activated mitophagy and suppressed mitophagy with specific inhibitors, mitochondrial division inhibitor-1 (Mdivi-1) or lysosome inhibitor chloroquine (CQ), which inhibited CDDP-induced apoptosis in hypoxic U-2OS and MG-63 cells. In addition, hypoxia upregulated the phosphorylation level of FUN14 domain-containing protein 1 (FUNDC1), whereas the activation of mitophagy and decreased CDDP sensitivity were inhibited by transfection with FUNDC1 small interfering RNA (siRNA). Hypoxia treatment also led to the up-regulation of heat shock protein 90 (HSP90), whereas HSP90 siRNA inhibited FUNDC1-mediated activation of mitophagy and decreased CDDP sensitivity. Furthermore, activation of Unc-51 like autophagy activating kinase 1 (Ulk1) was found in U-2OS and MG-63 cells after induction of hypoxia. Overexpression of Ulk1 prevented the inhibitory effect of HSP90 siRNA on the activation of FUNDC1 and mitophagy and decreased CDDP sensitivity in hypoxic U-2OS and MG-63 cells. Finally, hypoxia induced the activation of forkhead box transcription factor 3a (FOXO3a), whereas FOXO3a siRNA inhibited hypoxia-induced HSP90 up-regulation, Ulk1 activation, and FUNDC1-mediated activation of mitophagy, and decreased CDDP sensitivity in U-2OS and MG-63 cells. Using a chromatin immunoprecipitation (ChIP) assay, we confirmed that FOXO3a binds to the HSP90 promoter region. In conclusion, our findings suggest that hypoxia alleviates CDDP-induced apoptosis by activating mitophagy through the FOXO3a/HSP90/Ulk1/FUNDC1 signaling pathway in OS cells.

Effects of Ultrafine Single-Nanometer Oxygen Bubbles on Radiation Sensitivity in a Tumor-Bearing Mouse Model.

Radiation therapy against cancer cells often causes radiation resistance via accumulation of hypoxia-inducible factor 1 subunit alpha (HIF-1α) under hypoxic conditions and severe side effects. Radiation sensitizers without side effects are required to overcome hypoxia-induced radiation resistance and decrease radiation-related side effects in patients with refractory cancer. We previously developed oxygen nanobubble water (NBO2 water) and demonstrated that it suppresses hypoxia-induced radiation resistance in cancer cell lines within the single-nanometer range. This study aimed to elucidate whether NBO2 water could act as a radiosensitizer via regulation of HIF-1α in a tumor-bearing mouse model. Six-week-old female BALB/c mice subcutaneously injected with tumor cells received control water or NBO2 water for 28 days, after which biochemical examinations and radiation treatment were performed. Hypoxic tumor regions were detected immunohistochemically. We found that NBO2 water sensitized radiation reactivity in the xenografted tumors. Notably, NBO2 water administration downregulated the accumulation of HIF-1α in xenografted tumors and did not affect the vital organs of healthy mice. The combination of radiation and single-nanometer NBO2 water without severe side effects may be a promising therapeutic option to improve radiation sensitivity in cancer patients without tolerance to invasive treatments.

MiRNA Pattern in Hypoxic Microenvironment of Kidney Cancer-Role of PTEN.

MicroRNAs are post-transcriptional regulators of gene expression, and disturbances of their expression are the basis of many pathological states, including cancers. The miRNA pattern in the context of tumor microenvironment explains mechanisms related to cancer progression and provides a potential target of modern therapies. Here we show the miRNA pattern in renal cancer focusing on hypoxia as a characteristic feature of the tumor microenvironment and dysregulation of PTEN, being a major tumor suppressor. Methods comprised the CRSPR/Cas9 mediated PTEN knockout in the Renca kidney cancer cell line and global miRNA expression analysis in both in vivo and in vitro (in normoxic and hypoxic conditions). The results were validated on human cancer models with distinct PTEN status. The increase in miR-210-3p in hypoxia was universal; however, the hypoxia-induced decrease in PTEN was associated with an increase in miR-221-3p, the loss of PTEN affected the response to hypoxia differently by decreasing miR-10b-5p and increasing miR-206-3p. In turn, the complete loss of PTEN induces miR-155-5p, miR-100-5p. Upregulation of miR-342-3p in knockout PTEN occurred in the context of the whole tumor microenvironment. Thus, effective identification of miRNA patterns in cancers must consider the specificity of the tumor microenvironment together with the mutations of key suppressors.

Hypoxia: Role of SIRT1 and the protective effect of resveratrol in ovarian function.

BackgroundOvarian function is closely related to the degree of vascular network development surrounding the ovary. Maternal aging‐related construction defects in this vascular network can cause ovarian hypoxia, which impedes oocyte nutrient supply, leading to physiological changes in the ovaries and oocytes. The anti‐aging gene Sirtuin 1 (SIRT1) senses and adapts to ambient stress and is associated with hypoxic environments and mitochondrial biogenesis.MethodsThe present study is a literature review focusing on investigations involving the changes in SIRT1 and mitochondrial expression during hypoxia and the cytoprotective effects of the SIRT1 activator, resveratrol.Main findingsHypoxia suppresses SIRT1 and mitochondrial expression. Resveratrol can reverse the hypoxia‐induced decrease in mitochondrial and SIRT1 activity. Resveratrol suppresses the production of hypoxia‐inducible factor‐1α and vascular endothelial growth factor proteins.ConclusionResveratrol exhibits protective activity against hypoxic stress and may prevent hypoxia‐ or aging‐related mitochondrial dysfunction. Resveratrol treatment may be a potential option for infertility therapy.

A Retinoic Acid Receptor β 2 Agonist Improves Cardiac Function in a Heart Failure Model.

We previously demonstrated that the selective retinoic acid receptor (RAR) β 2 agonist AC261066 reduces oxidative stress in an ex vivo murine model of ischemia/reperfusion. We hypothesized that by decreasing oxidative stress and consequent fibrogenesis, AC261066 could attenuate the development of contractile dysfunction in post-ischemic heart failure (HF). We tested this hypothesis in vivo using an established murine model of myocardial infarction (MI), obtained by permanent occlusion of the left anterior descending coronary artery. Treating mice with AC261066 in drinking water significantly attenuated the post-MI deterioration of echocardiographic indices of cardiac function, diminished remodeling, and reduced oxidative stress, as evidenced by a decrease in malondialdehyde level and p38 mitogen-activated protein kinase expression in cardiomyocytes. The effects of AC261066 were also associated with a decrease in interstitial fibrosis, as shown by a marked reduction in collagen deposition and α-smooth muscle actin expression. In cardiac murine fibroblasts subjected to hypoxia, AC261066 reversed hypoxia-induced decreases in superoxide dismutase 2 and angiopoietin-like 4 transcriptional levels as well as the increase in NADPH oxidase 2 mRNA, demonstrating that the post-MI cardioprotective effects of AC261066 are associated with an action at the fibroblast level. Thus, AC261066 alleviates post-MI cardiac dysfunction by modulating a set of genes involved in the oxidant/antioxidant balance. These AC261066 responsive genes diminish interstitial fibrogenesis and remodeling. Since MI is a recognized major cause of HF, our data identify RARβ 2 as a potential pharmacological target in the treatment of HF. SIGNIFICANCE STATEMENT: A previous report showed that the selective retinoic acid receptor (RAR) β 2 agonist AC261066 reduces oxidative stress in an ex vivo murine model of ischemia/reperfusion. This study shows that AC261066 attenuates the development of contractile dysfunction and maladaptive remodeling in post-ischemic heart failure (HF) by modulating a set of genes involved in oxidant/antioxidant balance. Since myocardial infarction is a recognized major cause of HF, these data identify RARβ 2 as a potential pharmacological target in the treatment of HF.

Protective effect of Salidroside on hypoxia-related liver oxidative stress and inflammation via Nrf2 and JAK2/STAT3 signaling pathways.

High‐altitude hypoxia‐induced oxidative stress and inflammation played an essential role in the incidence and development of liver injury. Salidroside (Sal), a phenylpropanoid glycoside extracted from the plant Rhodiola rosea, has recently demonstrated antioxidant, anti‐inflammatory, and antihypoxia properties. Herein, we hypothesized that salidroside may alleviate hypoxia‐induced liver injury via antioxidant and antiinflammatory‐related pathways. A high‐altitude hypoxia animal model was established using hypobaric chamber. Male SD rats were randomly divided into the control group, hypoxia group, control +Sal group, and hypoxia +Sal group. Salidroside treatment significantly inhibited hypoxia‐induced increases of serum and hepatic pro‐inflammatory cytokines release, hepatic ROS production and MDA contents; attenuated hypoxia‐induced decrease of hepatic SOD, CAT, and GSH‐Px activities. Furthermore, salidroside treatment also potentiated the activation of Nrf2‐mediated anti‐oxidant pathway, as indicated by upregulation of n‐Nrf2 and its downstream HO‐1 and NQO‐1. In vitro study found that blocking the Nrf2 pathway using specific inhibitor ML385 significantly reversed the protective effect of salidroside on hypoxia‐induced liver oxidative stress. In addition, salidroside treatment significantly inhibited hepatic pro‐inflammatory cytokines release via JAK2/STAT3‐mediated pathway. Taken together, our findings suggested that salidroside protected against hypoxia‐induced hepatic oxidative stress and inflammation via Nrf2 and JAK2/STAT3 signaling pathways.

Specific protein 1 inhibitor mithramycin A protects cardiomyocytes from myocardial infarction via interacting with PARP.

Specific protein 1 (SP1) might act as a critical transcription regulator in myocardial infarction (MI), but little evidence about its function in regulating cardiac apoptosis, a major cause of MI development, has been revealed. This study tried to investigate the role of SP1 in MI and its interaction with poly-ADP-ribose polymerase (PARP)-1 by using SP1 inhibitor, mithramycin A (mithA). Primary mouse cardiomyocytes and commercial mouse cardiomyocytes were subjected to mithA treatment under hypoxia conditions, while cell viability, Nix promoter activity, and its expression were detected correspondingly. PARP overexpression and knockdown were conducted, respectively, in mithA-treated and SP1-overexpressing cells. Co-immunoprecipitation was used to verify the interaction between PARP and SP1. For in vivo experiments, mithA administration was performed after the injections of adenovirus for PARP overexpression, and then, MI introduction was carried out. Infarct size and lactate dehydrogenase level were measured to assess MI injury. SP1 inhibitor mithA attenuated hypoxia-induced decrease of cell viability and Nix transcriptional activation, which could be inhibited by PARP overexpression. Knockdown of PARP prevented SP1-induced transcription of Nix and cell viability change, and PARP showed direct interaction with SP1. Furthermore, mithA administration reduced MI injuries, while PARP overexpression could suppress the improvement. The cardioprotective role of SP1 inhibitor mithA was demonstrated here expanding the role of SP1 in MI development involving hypoxia-induced cardiac apoptosis. Moreover, PARP acted as a transcriptional coactivator in Nix transcription involving its interaction with SP1.

MiR-152-3p aggravates vascular endothelial cell dysfunction by targeting DEAD-box helicase 6 (DDX6) under hypoxia

ABSTRACT Stroke is a main cause of disability and death worldwide, and ischemic stroke accounts for most stroke cases. Recently, microRNAs (miRNAs) have been verified to play critical roles in the development of stroke. Herein, we explored effects of miR-152-3p on vascular endothelial cell functions under hypoxia. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia to mimic cell injury in vitro. Reverse transcription quantitative polymerase chain reaction revealed that miR-152-3p exhibited high expression in HUVECs treated with hypoxia. The inhibition of miR-152-3p reversed hypoxia-induced decrease in cell viability and the increase in angiogenesis, according to the results of cell counting kit-8 assays and tube formation assays. miR-152-3p inhibition reversed the increase in endothelial cell permeability mediated by hypoxia, as shown by endothelial cell permeability in vitro assays. In addition, the increase in protein levels of angiogenetic markers and the decrease in levels of tight junction proteins induced by hypoxia were reversed by miR-152-3p inhibition. Mechanistically, miR-152-3p directly targets 3ʹ-untranslated region of DEAD-box helicase 6 (DDX6), which was confirmed by luciferase reporter assays. DDX6 is lowly expressed in HUVECs under hypoxic condition, and mRNA expression and protein level of DDX6 were upregulated in HUVECs due to miR-152-3p inhibition. Rescue assays showed that DDX6 knockdown reversed effects of miR-152-3p on cell viability, angiogenesis and endothelial permeability. The results demonstrated that miR-152-3p aggravates vascular endothelial cell dysfunction by targeting DDX6 under hypoxia.

Keratin 1 attenuates hypoxic pulmonary artery hypertension by suppressing pulmonary artery media smooth muscle expansion.

Abnormally activated vascular smooth muscle cells are key factors in pulmonary artery remodelling (PAR) and pulmonary artery hypertension (PAH). Keratin 1 is involved in inflammatory diseases; however, its role in PAH is unknown. We speculated that keratin 1 could regulate PASMCs and prevent PAH. Rats were exposed to hypoxia (10% O2 ) or MCT (50mg/kg, intraperitoneal injection) or treated with AAV6 virus. PAR was measured through HE and Masson staining. PASMC activities were measured using MTS assay, EdU and Western blot analyses after cell knockdown with siRNAs or overexpression with Krt1 vectors. 1. Hypoxic PAR was associated with a decrease in keratin 1, especially in PASMCs. 2. Keratin 1 knockdown led to cell proliferation, migration and contraction to synthetic transformation, while keratin 1 overexpression attenuated hypoxia-induced changes in PASMCs. 3. Decreased keratin 1 induced TLR7 upregulation and mediated increases in the inflammatory factors S100a8 and S100a9. 4. Keratin 1 overexpression reduced the inflammatory factor expression induced by TLR7 activation. 5. Further studies demonstrated that keratin 1 expression was negatively correlated with pulmonary vascular pressure following prolonged hypoxia. 6. Pre-treatment with keratin 1 decreased pulmonary artery pressure and the right heart hypertrophy index and alleviated PAR in two model rats. 7. Keratin 1 exhibited a hypermethylation status in hypoxic pulmonary arteries in the sequencing. Hypoxia-induced decrease in keratin 1 expression was associated with Dnmt1 upregulation induced by YY1 downregulation in PASMCs. This study suggests that keratin 1 regulates PASMC expansion and has a preventive effect on PAH.

The role of hypoxia-induced modulation of alveolar epithelial Na+- transport in hypoxemia at high altitude.

Reabsorption of excess alveolar fluid is driven by vectorial Na+-transport across alveolar epithelium, which protects from alveolar flooding and facilitates gas exchange. Hypoxia inhibits Na+-reabsorption in cultured cells and in-vivo by decreasing activity of epithelial Na+-channels (ENaC), which impairs alveolar fluid clearance. Inhibition also occurs during in-vivo hypoxia in humans and laboratory animals. Signaling mechanisms that inhibit alveolar reabsorption are poorly understood. Because cellular adaptation to hypoxia is regulated by hypoxia-inducible transcription factors (HIF), we tested whether HIFs are involved in decreasing Na+-transport in hypoxic alveolar epithelium. Expression of HIFs was suppressed in cultured rat primary alveolar epithelial cells (AEC) with shRNAs. Hypoxia (1.5% O2, 24 h) decreased amiloride-sensitive transepithelial Na+-transport, decreased the mRNA expression of α-, β-, and γ-ENaC subunits, and reduced the amount of αβγ-ENaC subunits in the apical plasma membrane. Silencing HIF-2α partially prevented impaired fluid reabsorption in hypoxic rats and prevented the hypoxia-induced decrease in α- but not the βγ-subunits of ENaC protein expression resulting in a less active form of ENaC in hypoxic AEC. Inhibition of alveolar reabsorption also caused pulmonary vasoconstriction in ventilated rats. These results indicate that a HIF-2α-dependent decrease in Na+-transport in hypoxic alveolar epithelium decreases alveolar reabsorption. Because susceptibles to high-altitude pulmonary edema (HAPE) have decreased Na+-transport even in normoxia, inhibition of alveolar reabsorption by hypoxia at high altitude might further impair alveolar gas exchange. Thus, aggravated hypoxemia might further enhance hypoxic pulmonary vasoconstriction and might subsequently cause HAPE.

Hypoxic stress enhances extension and branching of dorsal root ganglion neuronal outgrowth.

It has been shown that painful intervertebral discs (IVDs) were associated with a deeper innervation. However, the effect of the disc's degenerative microenvironment on neuronal outgrowth remains largely unknown. The focus of this study was to determine the influence of hypoxia on dorsal root ganglion (DRG) neurite outgrowth. Toward this aim, the DRG‐derived cell line ND7/23 was either directly subjected to 2% or 20% oxygen conditions or exposed to conditioned medium (CM) collected from IVDs cultured under 2% or 20% oxygen. Viability and outgrowth analysis were performed following 3 days of exposure. Results obtained with the cell line were further validated on cultures of rabbit spinal DRG explants and dissociated DRG neurons. Results showed that hypoxia significantly increased neurite outgrowth length in ND7/23 cells, which was also validated in DRG explant and primary cell culture, although hypoxia conditioned IVD did not significantly increase ND7/23 neurite outgrowth. While hypoxia dramatically decreased the outgrowth frequency in explant cultures, it significantly increased collateral sprouting of dissociated neurons. Importantly, the hypoxia‐induced decrease of outgrowth frequency at the explant level was not due to inhibition of outgrowth branching but rather to neuronal necrosis. In summary, hypoxia in DRG promoted neurite sprouting, while neuronal necrosis may reduce the density of neuronal outgrowth at the tissue level. These findings may help to explain the deeper neo‐innervation found in the painful disc tissue.HighlightsHypoxia promoted elongation and branching of neurite outgrowth at single cell level, but reduced outgrowth density at tissue level, possibly due to hypoxia‐induced neuronal necrosis; these findings may help to explain the deeper neo‐innervation found in clinically painful tissues.

Propofol Attenuates Hypoxia-Induced Inflammation in BV2 Microglia by Inhibiting Oxidative Stress and NF-κB/Hif-1α Signaling.

Hypoxia-induced neuroinflammation typically causes neurological damage and can occur during stroke, neonatal hypoxic-ischemic encephalopathy, and other diseases. Propofol is widely used as an intravenous anesthetic. Studies have shown that propofol has antineuroinflammatory effect. However, the underlying mechanism remains to be fully elucidated. Thus, we aimed to investigate the beneficial effects of propofol against hypoxia-induced neuroinflammation and elucidated its potential cellular and biochemical mechanisms of action. In this study, we chose cobalt chloride (CoCl2) to establish a hypoxic model. We found that propofol decreased hypoxia-induced proinflammatory cytokines (TNFα, IL-1β, and IL-6) in BV2 microglia, significantly suppressed the excessive production of reactive oxygen species, and increased the total antioxidant capacity and superoxide dismutase activity. Furthermore, propofol attenuated the hypoxia-induced decrease in mitochondrial membrane potential andy 2 strongly inhibited protein expression of nuclear factor-kappa B (NF-κB) subunit p65 and hypoxia inducible factor-1α (Hif-1α) in hypoxic BV2 cells. To investigate the role of NF-κB p65, specific small interfering RNA (siRNA) against NF-κB p65 were transfected into BV2 cells, followed by exposure to hypoxia for 24 h. Hypoxia-induced Hif-1α production was downregulated after NF-κB p65 silencing. Further, propofol suppressed Hif-1α expression by inhibiting the upregulation of NF-κB p65 after exposure to hypoxia in BV2 microglia. In summary, propofol attenuates hypoxia-induced neuroinflammation, at least in part by inhibiting oxidative stress and NF-κB/Hif-1α signaling.

Ginsenoside Rb1 pretreatment attenuates myocardial ischemia by reducing calcium/calmodulin-dependent protein kinase II-medicated calcium release

Objective: The aim of this study was to investigate the protective effects of ginsenoside Rb1 and assess whether these protective effects are related to calcium/calmodulin-dependent protein kinase II (CaMKII).Methods: A myocardial ischemia (IS) rat. model and a myocardial H9C2 cell hypoxia model were established. MI was induced by occluding the left anterior descending artery for 120 min. Ginsenoside Rb1 (10 mg/kg) was administered 30 min before ischemia induction, and the treatment continued for 7 days. Results: In the rat IS injury model, ginsenoside Rb1 reduced myocardial infarct size, mean left ventricular diastolic pressure, incidence of arrhythmia, and levels of serum creatine kinase, lactate dehydrogenase, and malondialdehyde. However, the mean left ventricular systolic pressure, and maximal rising and falling rates of ventricular pressure (±dp/dtmax) increased. In the myocardial H9C2 cell hypoxia model, ginsenoside Rb1 reduced intracellular calcium concentrations ([Ca2+ ]i) during hypoxia, and markedly reversed the hypoxia-induced decrease in cell survival. Ginsenoside Rb1 was involved in the downregulation of CaMKII and the ryanodine receptor, as well as hypoxia-induced H9C2 cell survival. Conclusion: The findings of the present study suggest that ginsenoside Rb1 attenuates MI injury in rats, partially through the downregulation of CaMKII expression.