Cobalt Chloride-induced Hypoxia Research Articles

Effect of exercise during pregnancy on offspring development through ameliorating high glucose and hypoxia in gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) women require prenatal care to minimize short- and long-term complications. The mechanism by which exercise during pregnancy affects organ development and whether glucose transporter (GLUT) 1 plays a role in GDM offspring organ development remains unknown. To determine the effect of exercise during pregnancy on the cardiac, hepatic and renal development of GDM mother's offspring. Placenta samples were collected from humans and mice. GDM mouse models were created using streptozotocin along with a GDM with exercise group. The hearts, livers and kidneys of 3- and 8-week-old offspring were collected for body composition analysis and staining. The effects of high glucose levels and hypoxia were investigated using HTR8/SVneo. Transwell and wound-healing assays were performed to assess cell migration. Immunofluorescence accompanied with TUNEL and Ki67 staining was used to explore apoptosis and proliferation. Exercise during pregnancy downregulated the GLUT1 and hypoxia inducible factor-1α expression in placenta from individuals with GDM. Cobalt chloride-induced hypoxia and high glucose levels also significantly decreased migration and apoptosis of HTR8/SVneo cells. In addition, exercise reduced inflammatory cell infiltration in the liver and decreased the tubular vacuolar area in the kidneys of offspring. GDM affects the growth and development of organs in offspring. Exercise during pregnancy can reverse adverse effects of GDM on the development of the heart, liver, and kidney in offspring.

Tibetan-Origin Edible Chinese Herbal Prescription C18 Protects H9C2 Cardiomyocytes from Cobalt Chloride-induced Hypoxia Injury through the PI3K/AKT Signaling Pathway

Background Altitude sickness is often prone to occur during tourism or work in high-altitude areas. In China, traditional Tibetan medicines have a long history of preventing or treating altitude sickness, especially altitude hypoxia, which may lead to myocardial cell apoptosis and myocardial hypoxia-reoxygenation injury. Purpose This study investigated the effect of a Tibetan-origin edible Chinese herbal prescription (named C18) on protecting H9C2 cardiomyocytes from cobalt chloride-induced hypoxia injury and its potential mechanism. Materials and Methods In this study, a hypoxic injury model of H9C2 cardiomyocytes induced by cobalt chloride was established first. Then the cell viability, relevant antioxidant indicators malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and protein expression (hypoxia-inducible factor 1 alpha (HIF-1α), phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (p-AKT)) were measured after pretreatment with or without C18. At last, the specific PI3K/AKT inhibitor LY294002 was applied to verify the antihypoxia signaling pathway. Results C18 could significantly promote normal H9C2 cardiomyocyte proliferation and inhibit apoptosis of hypoxic H9C2 cardiomyocytes, reduce the release of lactate dehydrogenase and MDA, and increase the levels of SOD and GSH-Px antioxidant enzymes. In addition, C18 could significantly downregulate the expression of HIF-1α protein and upregulate the expression of intracellular p-AKT. Moreover, these effects of C18 can be blocked by the specific PI3K/AKT inhibitor LY294002. Conclusion C18 protects H9C2 cardiomyocytes from cobalt chloride-induced hypoxia injury through the PI3K/AKT signaling pathway.

Differential effects of coverslip-induced hypoxia and cobalt chloride mimetic hypoxia on cellular stress, metabolism, and nuclear structure

Hypoxia has profound effects on cell physiology, both in normal or pathological settings like cancer. In this study, we asked whether a variant of coverslip-induced hypoxia that recapitulates the conditions found in the tumor microenvironment would elicit similar cellular responses compared to the well established model of cobalt chloride-induced hypoxia. Comparable levels of nuclear HIF-1α were observed after 24 h of coverslip-induced hypoxia or cobalt chloride treatment in CAL-27 oral squamous carcinoma cells. However, cellular stress levels assessed by reactive oxygen species production and lipid droplet accumulation were markedly increased in coverslip-induced hypoxia compared to cobalt chloride treatment. Conversely, mitochondrial ATP production sharply decreased after coverslip-induced hypoxia but was preserved in the presence of cobalt chloride. Coverslip-induced hypoxia also had profound effects in nuclear organization, assessed by changes in nuclear dry mass distribution, whereas these effects were much less marked after cobalt chloride treatment. Taken together, our results show that coverslip-induced hypoxia effects on cell physiology and structure are more pronounced than mimetic hypoxia induced by cobalt chloride treatment. Considering also the simplicity of coverslip-induced hypoxia, our results therefore underscore the usefulness of this method to recapitulate in vitro the effects of hypoxic microenvironments encountered by cells in vivo.

A Hypoxia-Regulated Retinal Pigment Epithelium-Specific Gene Therapy Vector Reduces Choroidal Neovascularization in a Mouse Model.

Wet age-related macular degeneration (wAMD) is characterized by the presence of choroidal neovascularization (CNV). Although there are some clinical drugs targeting vascular endothelial growth factor (VEGF) and inhibiting CNV, two major side effects limit their application, including the excessive activity of anti-VEGF and frequent intraocular injections. To explore better treatment strategies, researchers developed a hypoxic modulator retinal pigment epithelium (RPE)- specific adeno-associated virus (AAV) vector expressing endostatin to inhibit CNV. However, the mechanism of endostatin is complex. Instead, soluble fms-like tyrosine kinase-1 (sFlt-1) can inhibit VEGF-induced angiogenesis through two simple and clear mechanisms, giving rise to sequestration of VEGF and forming an inactive heterodimer with the membrane-spanning isoforms of the VEGF receptor Flt-1 and kinase insert domain-containing receptor. In this study, we chose sFlt-1 as a safer substitute to treat wAMD by inhibiting VEGFinduced angiogenesis. The AAV2/8-Y733F-REG-RPE-sFlt-1 vector was delivered by intravitreal injection to the eyes of mice. AAV2/8-Y733F vector is a mutant of the AAV2/8 vector, and the REG-RPE promoter is a hypoxia-regulated RPE-specific promoter. Two animal models were used to evaluate the function of the vector. In the cobalt chloride-induced hypoxia model, the results demonstrated that the AAV2/8- Y733F-REG-RPE-sFlt-1 vector induced the expression of the sFlt-1 gene in RPE cells through hypoxia. In the laser-induced CNV model, the results demonstrated that the AAV2/8-Y733F-REG-RPE-sFlt- 1 vector reduced laser-induced CNV. Hypoxia regulated, RPE-specific AAV vector-mediated sFlt-1 gene is a hypoxiaregulated antiangiogenic vector for wAMD.

Cobalt Chloride-induced Hypoxia Can Lead SKBR3 and HEK293T Cell Lines toward Epithelial-mesenchymal Transition.

Hypoxia is a common characteristic of the tumor microenvironment. In response to hypoxia, expression of the hypoxia-inducible factor (HIF) can lead to activation of downstream molecular events such as epithelial-mesenchymal transition (EMT), invasion, and angiogenesis. In this study, CoCl2 was used to simulate hypoxia in SKBR3 and HEK293T cell lines to investigate whether this treatment can induce hypoxia-associated EMT and invasion in the studied cells. SKBR3 and HEK293T cells were treated with different concentrations of CoCl2 at different exposure times and their viability was analyzed. To confirm successful hypoxia induction, the expression levels of HIF1α and vascular endothelial growth factor A (VEGFA) mRNA were assessed. Additionally, the expression of EMT-associated markers including snail, E-cadherin, N-cadherin, and vimentin, as well as invasion-related genes including matrix metalloproteinase-2 (MMP2) and MMP9 was measured. We found that cell viability in CoCl2-treated cells was concentration-dependent and was not affected at low doses. While the expression of HIF and VEGFA genes was upregulated following hypoxia induction. E-cadherin expression was significantly downregulated in HEK293T cells; while, N-cadherin and snail were upregulated in both cell lines. Moreover, an increment of MMP expression was only observed in SKBR3 cells. Taken together, the findings indicated that CoCl2 can mimic hypoxia in both cell lines, but EMT was triggered in SKBR3 cells more effectively than in HEK293T cells, and invasion was only stimulated in SKBR3 cells. In conclusion, SKBR3 cancer cells can be used as an EMT model to better understand its control and manipulation mechanisms and to investigate new therapeutic targets for the suppression of tumor metastasis.

Autophagy promotes GSDME-mediated pyroptosis via intrinsic and extrinsic apoptotic pathways in cobalt chloride-induced hypoxia reoxygenation-acute kidney injury

Cobalt is a transition element that abundantly exists in the environment. Besides direct hypoxia stress, cobalt ions indirectly induce hypoxia-reoxygenation injury (HRI), the main cause of acute kidney injury (AKI), a life-threatening clinical syndrome characterized by the necrosis of the proximal tubular epithelial cells (PTECs) and inflammation. Pyroptosis, a type of inflammatory programmed cell death, might play an essential role in HRI-AKI. However, whether pyroptosis is involved in cobalt chloride (CoCl2)-induced HRI-AKI remains unknown. Autophagy is a cellular biological process maintaining cell homeostasis that is involved in cell damage in AKI, yet the underlying regulatory mechanism of autophagy on pyroptosis has not been fully understood. In this study, the in vitro and in vivo models of CoCl2-induced HRI-AKI were established with HK-2 cell line and C57BL/6J mouse. Pyroptosis-related markers were detected with western blotting and immunofluorescence assays, and results showed that gasdermin E (GSDME)-mediated pyroptosis was involved in the cell damage in HRI-AKI. Specific chemical inhibitors of caspase 3, caspase 8, and caspase 9 significantly inhibited GSDME-mediated pyroptosis, verifying that GSDME-mediated pyroptosis was induced via the activation of caspase 3/8/9. The western blotting and immunofluorescence assays were adopted to detect the accumulation of the autophagosomes, and results suggested that HRI increased the autophagic level. The effects of autophagy on apoptosis and pyroptosis were evaluated using lentivirus transfection assays to knock down autophagy-specific genes atg5 and fip200, and results demonstrated that autophagy induced GSDME-mediated pyroptosis via apoptotic pathways in HRI-AKI. Our results revealed the involvement of GSDME-mediated pyroptosis in CoCl2-induced HRI-AKI and promoted the understanding of the regulatory mechanism of GSDME cleavage. Our study might provide a potential therapeutic target for HRI-AKI, and will be helpful for the risk evaluation of cobalt exposure.

Role of vasorin, an anti-apoptotic, anti-TGF-β and hypoxia-induced glycoprotein in the trabecular meshwork cellsand glaucoma.

Glaucoma, one of the leading causes of irreversible blindness, is commonly associated with elevated intraocular pressure due to impaired aqueous humour (AH) drainage through the trabecular meshwork. The aetiological mechanisms contributing to impaired AH outflow, however, are poorly understood. Here, we identified the secreted form of vasorin, a transmembrane glycoprotein, as a common constituent of human AH by mass spectrometry and immunoblotting analysis. ELISA assay revealed a significant but marginal decrease in vasorin levels in the AH of primary open‐angle glaucoma patients compared to non‐glaucoma cataract patients. Human trabecular meshwork (HTM) cells were confirmed to express vasorin, which has been shown to possess anti‐apoptotic and anti‐TGF‐β activities. Treatment of HTM cells with vasorin induced actin stress fibres and focal adhesions and suppressed TGF‐β2‐induced SMAD2/3 activation in HTM cells. Additionally, cobalt chloride‐induced hypoxia stimulated a robust elevation in vasorin expression, and vasorin suppressed TNF‐α‐induced cell death in HTM cells. Taken together, these findings reveal the importance of vasorin in maintenance of cell survival, inhibition of TGF‐β induced biological responses in TM cells, and the decreasing trend in vasorin levels in the AH of glaucoma patients suggests a plausible role for vasorin in the pathobiology of ocular hypertension and glaucoma.

Ameliorative Effect of Ananas comosus on Cobalt Chloride-Induced Hypoxia in Caco2 cells via HIF-1α, GLUT 1, VEGF, ANG and FGF.

In the present study, protective effects of Ananas comosus i.e., pineapple pulp (PA) against cobalt chloride (CoCl2)‑induced hypoxia in Caco-2cells were evaluated. PA reduces levels of lipid peroxidation, reactive oxygen and nitrogen species. It was proved to be cytoprotective and increased anti-oxidant activity against CoCl2-induced hypoxia. The inference drawn from this experiment was CoCl2-induced hypoxia that regulates hypoxia-inducible factor-1 (HIF-1), a transcription factor. It was also confirmed that PA pre-treatment inhibited the expression of HIF-1α, thereby downregulating the hypoxia-associated gene/protein expressions such as GLUT-1, VEGF, ANG and FGF. Finally, supplementation of PA could help in snow-balling the digestive hormones like leptin and CCK in hypoxic conditions. Therefore, this report provides substantial proof of reducing the hypoxia-induced loss of appetite at high-altitude environments. Graphical Abstract.

β-Escin alleviates cobalt chloride-induced hypoxia-mediated apoptotic resistance and invasion via ROS-dependent HIF-1α/TGF-β/MMPs in A549 cells.

Hypoxia is contributed in various pathophysiological conditions including obesity, cardiovascular diseases, and cancer. In cancer, hypoxia is a salient phenomenon and has been correlated with tumor progression, metastasis, and provoke resistance to therapies in cancer patients, which exert with stabilization of main effector, hypoxia inducible factor-1 alpha (HIF-1α). Therefore, therapeutic targeting of hypoxic responses in cancer is the potential approach to improve the better treatment efficacy. In the present study, we evaluated the effect of β-Escin (β-Es) on hypoxia-induced resistance to apoptosis and metastasis in human non-small-cell lung cancer cells. The MTT assay revealed that β-Es treatment decreased the A549 cells viability under cobalt chloride-induced hypoxia. Apoptotic proteins were analyzed by western blot that showed cancer cells treated with β-Es induced cell death in hypoxia condition as proteins compared with normoxia. Moreover, we observed that cobalt chloride induced hypoxia through the generation of intracellular reactive oxygen species and stabilized the transcriptional factor HIF-1α, which leads to cancer metastasis. This notion was supported by the migration, invasion, and adhesion assays. Furthermore, hypoxia increased the expression of transforming growth factor-β, and the activation of matrix metalloproteinases were suppressed by the treatment of β-Es as well as pretreatment with N-acetylcysteine (NAC). Therefore, we demonstrate that a concurrent activation of HIF-1α, transforming growth factor-β, and matrix metalloproteinases participate in hypoxia-induced metastasis and that β-Es prevent A549 cells metastasis by inhibition of reactive oxygen species.

Long non-coding RNA ROR mitigates cobalt chloride-induced hypoxia injury through regulation of miR-145

Obstructive sleep apnoea-hypopnoea syndrome (OSAHS) is a condition causing apnea and hypopnea. LncRNA-ROR revealed properties in regulating hypoxia response. Our study explored the roles of ROR in CoCl2-induced hypoxia injury in HK-2 cells. HK-2 cells were treated with CoCl2 to induce hypoxia injury. Cell viability, cell apoptosis and apoptotic proteins were detected using CCK-8, flow cytometry and western blot, respectively. The alter expression of ROR and miR-145 was achieved through transfection. Moreover, the expressions of HIF-α and ERK and MAPK related factors were examined using western blot. We found that CoCl2 decreased cell viability and increased apoptosis as well as increased the expression of ROR. ROR overexpression increased cell viability, decreased cell apoptosis. ROR overexpression upregulated anti-apoptotic proteins Bcl-2 and decreased p53, Bax and cleaved-Caspase-3. ROR overexpression also increased the expression of HIF-α. On the opposite, ROR silence led to the opposite results as relative to ROR overexpression. ROR overexpression decreased expression of miR-145. Co-transfection with ROR overexpression and miR-145 impaired the promoting effects of ROR in CoCl2 treated cells. ROR increased phosphorylation of ERK while decreased phosphorylation of MAPK. In conclusion, lncRNA ROR alleviated CoCl2-induced hypoxia injury through regulation of miR-145 as well as modulating ERK and MAPK signalling. Highlights CoCl2 induces ROR upregulation; Overexpression of ROR reduces CoCl2-induced HK-2 cell injury; Silence of ROR promotes CoCl2-induced HK-2 cell injury; Overexpression of ROR decreases miR-145 expression; ROR overexpression modulates ERK and MAPK signalling pathways through regulation of miR-145.

A derivative of betulinic acid protects human Retinal Pigment Epithelial (RPE) cells from cobalt chloride-induced acute hypoxic stress

The Retinal Pigment Epithelium (RPE) is a monolayer of cells located above the choroid. It mediates human visual cycle and nourishes photoreceptors. Hypoxia-induced oxidative stress to RPE is a vital cause of retinal degeneration such as the Age-related Macular Degeneration. Most of these retinal diseases are irreversible with no efficient treatment, therefore protecting RPE cells from hypoxia stress is an important way to prevent or slow down the progression of retinal degeneration. Betulinic acid (BA) and betulin (BE) are pentacyclic triterpenoids with anti-oxidative property, but little is known about their effect on RPE cells. We investigated the protective effect of BA, BE and their derivatives against cobalt chloride-induced hypoxia stress in RPE cells. Human ARPE-19 cells were exposed to BA, BE and their eighteen derivatives (named as H3H20) that we customized through replacing moieties at C3 and C28 positions. We found that cobalt chloride reduced cell viability, increased Reactive Oxygen Species (ROS) production as well as induced apoptosis and necrosis in ARPE-19 cells. Interestingly, the pretreatment of 3-O-acetyl-glycyl- 28-O-glycyl-betulinic acid effectively protected cells from acute hypoxia stress induced by cobalt chloride. Our immunoblotting results suggested that this derivative attenuated the cobalt chloride-induced activation of Akt, Erk and JNK pathways. All findings were further validated in human primary RPE cells. In summary, this BA derivate has protective effect against the acute hypoxic stress in human RPE cells and may be developed into a candidate agent effective in the prevention of prevalent retinal diseases.

Effect of cobalt chloride-induced hypoxia on proliferation of human umbilical cord-derived mesenchymal stem cells and related gene and protein expressions

Effect of cobalt chloride-induced hypoxia on proliferation of human umbilical cord-derived mesenchymal stem cells and related gene and protein expressions

Melatonin attenuates apoptosis and mitochondrial depolarization levels in hypoxic conditions of SH-SY5Y neuronal cells induced by cobalt chloride (CoCl2)

Melatonin (MEL) and its metabolites serve as endogenous reactive oxygen species (ROS) scavengers and have a wide spectrum of antioxidant activity. Cobalt chloride is one of the commonly used hypoxia-mimetic agents, due to blocking the degradation of and triggering the accumulation of hypoxia-inducible factor-1 alpha (HIF-1α ) protein, which is very well known as a critical regulator of the cellular response against hypoxia. In the current study we aimed to determine the possible protective effects of melatonin on a cobalt chloride-induced hypoxia model of SH-SY5Y neuronal cells. Group I was the control group and SH-SY5Y cells were incubated in normal culture media without any chemical administration. In Group II, SH-SY5Y cells were incubated with 1 μM MEL for 24 h. In Group III cells were incubated with 200 μM cobalt chloride for 24 h. The last group, Group 4, was a combination group of cobalt chloride and MEL. Cells were preincubated with 1 μM for 12 h and then 200 μM cobalt chloride for 24 h. We performed the cell viability test (MTT) and checked the caspase-3 and -9 activities and the lipid peroxidation (LP), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), mitochondrial depolarization, and intracellular ROS levels. We observed that cobalt chloride increased intracellular ROS, caspase-3 and -9, lipid peroxidation, and mitochondrial membrane depolarization values, while decreasing GSH levels and GSH-Px activity. However, GSH and GSH-Px values were increased by melatonin treatment although lipid peroxidation level, intracellular ROS production, and caspase-3 and -9 activities were decreased by the treatment. In conclusion, we observed that melatonin incubation protects neuronal cells against hypoxia-induced oxidative stress.

Preferential cytotoxicity of the type I ribosome inactivating protein alpha-momorcharin on human nasopharyngeal carcinoma cells under normoxia and hypoxia

All primary nasopharyngeal carcinoma (NPC) tumors contain hypoxic regions which are implicated in decreased local control and increased distant metastases, as well as resistance to chemotherapy in advanced NPC patients. One of the promising therapeutic approaches for NPC is to use drugs that can target hypoxic factors in tumors. In the present investigation, the type I ribosome inactivating protein α-momorcharin (α-MMC), isolated from seeds of the bitter gourd Momordica charantia, reduced cell viability and inhibited clonogenic formation of human NPC CNE2 and HONE1 cells under normoxia and cobalt chloride-induced hypoxia. By comparison, α-MMC exhibited only slight cytotoxicity on human nasopharyngeal epithelial NP69 cells under normoxia. Interestingly, α-MMC suppressed the expression levels of hypoxia-inducible factor 1-alpha (HIF1α) and vascular endothelial growth factor (VEGF) in hypoxic NPC, as well as the growth of human umbilical vein endothelial cells. Further study disclosed that α-MMC targeted endoplasmic reticulum and down-regulated unfolded protein response (UPR) in NPC cells. Moreover, α-MMC induced apoptosis in NPC cells in a dose- and time-dependent manner. It initiated mitochondrial- and death receptor-mediated apoptotic signaling in CNE2 cells, but there was hardly any effect on HONE1 cells. In addition, α-MMC brought about G0/G1 phase cell cycle arrest in CNE2 cells and S phase arrest in HONE1 cells. Collectively, α-MMC preferentially exhibited inhibitory effect on normoxic and hypoxic NPC cells partly by blocking survival signaling (e.g. HIF1α, VEGF and UPR), and triggering apoptotic pathways mediated by mitochondria or death receptor. These observations indicate the potential utility of α-MMC for prophylaxis and therapy of NPC.

Effects of Salidroside on cobalt chloride-induced hypoxia damage and mTOR signaling repression in PC12 cells.

Salidroside (SA), a phenylpropanoid glycoside isolated from Rhodiola rosea L., has been documented to exert a broad spectrum of pharmacological properties, including protective effects against neuronal death induced by various stresses. To provide further insights into the neuroprotective functions of SA, this study examined whether SA can attenuate cobalt chloride (CoCl2)-induced hypoxia damage and mammalian target of rapamycin (mTOR) signaling repression in PC12 differentiated cells. Differentiated PC12 cells were exposed to CoCl2 for 12 h to mimic hypoxic/ischemic conditions and treated with SA at the same time, followed by electron microscopy and analysis of cell viability, intracellular reactive oxygen species (ROS) level, hypoxia-inducible factor-1α (HIF-1α) level, and the regulated in development and DNA damage responses (REDD1)/mTOR/ p70 ribosomal S6 kinase (p70S6K) signaling pathway. Our data indicated that SA can dramatically attenuate the ultrastructural damage of mitochondria induced by CoCl2 and significantly decrease CoCl2-induced ROS production. Moreover, phosphorylated mammalian target of rapamycin (p-mTOR) was significantly reduced by CoCl2, and this inhibition was relieved by the treatment of SA in PC12 cells, as evidenced by immunoblot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The SA effects were blocked by pretreatment of RAD001. The results indicate that SA can rescue CoCl2-induced repression of REDD1/mTOR/ p70S6K signal transduction in PC12 cells. Our data demonstrate that SA is able to attenuate CoCl2-induced hypoxia damage and mTOR signaling repression, suggesting that SA may protect brain neurons from ischemic injury through mTOR signaling, and provide new insights into the prevention and treatment of cerebral ischemic.

Overexpression of cytoglobin gene inhibits hypoxic injury to SH-SY5Y neuroblastoma cells.

A plasmid for cytoglobin expression, pAcGFP1-C1-cytoglobin, was transfected into SH-SY5Y cells. Cobalt chloride was used to establish a model of hypoxia. Western blotting indicated that cytoglobin was overexpressed and there was low expression of hypoxia-inducible factor-1α in SH-SY5Y cells after transfection. Following cobalt chloride-induced hypoxia, cytoglobin and hypoxia-inducible factor-1α expression gradually increased in SH-SY5Y cells. Flow cytometry showed that with increasing duration of hypoxia, the proportion of normal cells significantly diminished in the transfected and non-transfected groups. The proportion of cells in the early stages of apoptosis increased. However, the proportion of apoptotic cells was significantly lower in the transfected group compared with the non-transfected group. These results demonstrate that cytoglobin and hypoxia-inducible factor-1α are strongly up-regulated by hypoxia, and that there is a strong relationship between hypoxia-inducible factor-1α and cytoglobin during hypoxic injury.

Cobalt Chloride-induced Hypoxia Ameliorates NLRP3-Mediated Caspase-1 Activation in Mixed Glial Cultures

Hypoxia has been shown to promote inflammation, including the release of proinflammatory cytokines, but it is poorly investigated how hypoxia directly affects inflammasome signaling pathways. To explore whether hypoxic stress modulates inflammasome activity, we examined the effect of cobalt chloride (CoCl2)-induced hypoxia on caspase-1 activation in primary mixed glial cultures of the neonatal mouse brain. Unexpectedly, hypoxia induced by oxygen-glucose deprivation or CoCl2 treatment failed to activate caspase-1 in microglial BV-2 cells and primary mixed glial cultures. Of particular interest, CoCl2-induced hypoxic condition considerably inhibited NLRP3-dependent caspase-1 activation in mixed glial cells, but not in bone marrow-derived macrophages. CoCl2-mediated inhibition of NLRP3 inflammasome activity was also observed in the isolated brain microglial cells, but CoCl2 did not affect poly dA:dT-triggered AIM2 inflammasome activity in mixed glial cells. Our results collectively demonstrate that CoCl2-induced hypoxia may negatively regulate NLRP3 inflammasome signaling in brain glial cells, but its physiological significance remains to be determined.

Anti-Inflammatory Effects of Lutein in Retinal Ischemic/Hypoxic Injury: In Vivo and In Vitro Studies

Lutein protects retinal neurons by its anti-oxidative and anti-apoptotic properties in ischemia/reperfusion (I/R) injury while its anti-inflammatory effects remain unknown. As Müller cells play a critical role in retinal inflammation, the effect of lutein on Müller cells was investigated in a murine model of I/R injury and a culture model of hypoxic damage. Unilateral retinal I/R was induced by a blockade of internal carotid artery using the intraluminal method in mice. Ischemia was maintained for 2 hours followed by 22 hours of reperfusion, during which either lutein (0.2 mg/kg) or vehicle was administered. Flash electroretinogram (flash ERG) and glial fibrillary acidic protein (GFAP) activation were assessed. Lutein's effect on Müller cells was further evaluated in immortalized rat Müller cells (rMC-1) challenged with cobalt chloride-induced hypoxia. Levels of IL-1β, cyclooxygenase-2 (Cox-2), TNFα, and nuclear factor-NF-kappa-B (NF-κB) were examined by Western blot analysis. Lutein treatment minimized deterioration of b-wave/a-wave ratio and oscillatory potentials as well as inhibited up-regulation of GFAP in retinal I/R injury. In cultured Müller cells, lutein treatment increased cell viability and reduced level of nuclear NF-κB, IL-1β, and Cox-2, but not TNFα after hypoxic injury. Reduced gliosis in I/R retina was observed with lutein treatment, which may contribute to preserved retinal function. Less production of pro-inflammatory factors from Müller cells suggested an anti-inflammatory role of lutein in retinal ischemic/hypoxic injury. Together with our previous studies, our results suggest that lutein protected the retina from ischemic/hypoxic damage by its anti-oxidative, anti-apoptotic, and anti-inflammatory properties.

Hypoxia regulates cell proliferation and steroidogenesis through protein kinase A signaling in bovine corpus luteum

Hypoxia is an important physiological process which ensures corpus luteum (CL) formation and development, thus playing an important role in steroidogenesis. Recent studies have shown that CL develops in an analogous to tumorigenesis by accumulation of hypoxia-inducible factor-1 alpha subunit (HIF1A) in response to hypoxia. To investigate the relationship among hypoxia, steroidogenesis, and cell proliferation during CL lifespan, histological and steroidogenic analyses of CL were performed at various CL stages in non-pregnant Holstein. Also, the hypoxia-mediated steroidogenesis and cell proliferation were studied in vitro with both primary luteal and luteinized granulosa cells. Our results showed that progesterone (P 4) concentration increased with the upregulation of steroidogenic protein including steroidogenic acute regulatory protein (STAR) and CYP11A1 (P450scc) in the middle luteal stage. On the other hand, the cell proliferation- or hypoxia-associated proteins were upregulated in the early stage, including the proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor A (VEGFA), HIF1A, and aryl hydrocarbon receptor nuclear translocator (ARNT). In primary culture, phospho-protein kinase A (p-PKA) was downregulated, as were P 4 secretion and steroidogenic proteins both under oxygen-conditioned hypoxia in luteal cells and cobalt chloride-induced hypoxia in luteinized granulosa cells. However, under the treatment of hypoxia, PCNA, which was downregulated in luteal cells, was upregulated together with HIF1A and VEGFA in luteinized granulosa cells. Taken together, present study suggested that hypoxia downregulated steroidogenesis through PKA signaling and that the hypoxia-regulated cell proliferation could be activated during CL formation.

Cobalt chloride-induced hypoxia stimulates inflammatory response in adipocytes

Objective To explore the relationship between hypoxia induced by cobalt chloride( CoCl2 ) and inflammation in 3T3-LI adipocytes, and the underlying molecular mechanism. Methods 3T3-L1 pre-adipocytes were induced under standard differentiation process. Differentiated 3T3-L1 adipocytes were incubated with CoCl2 to induce hypoxia. The mRNA and protein expressions of hypoxia inducible factor-1 α ( HIF-1 α ), glucose transporter 1 ( Glut1 ), adipocytokines, and inflammatory cytokines, as well as the related signaling pathways were determined with quantitative realtime-PCR and Western-blot in this cell model. Results CoCl2 at the concentration of 200 μmol/L significantly up-regulated HIF-1 α and Glut1 expressions. After adipocytes were treated with CoCl2, the mRNA levels of endoplasmic reticulum stress marker genes, such as C/EBP homologous protein (CHOP) and glucose-regulated protein78 (GRP78) were markedly increased, adiponectin mRNA expression was significantly decreased, while resistin and leptin mRNA expressions were significantly increased. In addition, the mRNA expressions of cyclooxygenase 2, interleukin-6, and other inflammatory factors were also increased. The phosphorylation of IκBα,which could inhibit the activation of NF-κB, was stimulated by CoCl2. ConclusionsThe results indicate that hypoxia may induce inflammatory response via NF-κB activiation in adipocytes. Key words: Hypoxia; Adipocyte; Endoplasmic reticulumstress; Inflammation