Hypoxic Cell Model Research Articles

Repair Effects of KGF on Ischemia-Reperfusion–Induced Flap Injury via Activating Nrf2 Signaling

BackgroundIschemia-reperfusion (IR) injury is a main cause to and the mechanism of necrosis after flap transplantation. Researches were hardly conducted on the role and possible mechanism of keratinocyte growth factor (KGF) in association with IR flap injury. Materials and methodsA CoCl2-stimulated hypoxia cell model was established to investigate the effects of KGF on cell viability, apoptosis, cell cycle, and reactive oxygen species level. The experiments were performed by cell counting kit-8 and flow cytometry as required. Meanwhile, the expressions of cell cycle-related and nuclear factor E2–related factor 2 (Nrf2) signaling–related genes were determined using quantitative real-time PCR and Western blot. The right dorsolateral areas of Institute of Cancer Research mice were marked as flaps, the pedicle of which formed an IR process through clamping and loosening. Tissue morphologies were observed using hematoxylin and eosin staining 24 h after the surgery. The effects of KGF on cell apoptosis and associated genes expressions were studied by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling, immunohistochemistry, and Western blot. ResultsHaCAT cells treated with 40 μM CoCl2 could not only reduce cell viability, promote cell apoptosis, arrest G1 phase of cell cycle and increase the activity of reactive oxygen species but also downregulate the expressions of c-myc, c-fos, transforming growth factor-α, Nrf2, heme oxygenase-1, and gamma-glutamyl cysteine synthetase. Additional recombinant human KGF, on one hand, could protect the cells from hypoxia injury. On the other hand, recombinant human KGF could significantly inhibit cell apoptosis, increase KGF activity, and increase the Nrf2, heme oxygenase-1, and gamma-glutamyl cysteine synthetase proteins levels in IR flap tissues. ConclusionsKGF played an important role in protecting mice flaps from IR injury, and the possible mechanism was involved in activating the Nrf2 signaling.

DL-3-n-butylphthalide protects the blood-brain barrier against ischemia/hypoxia injury via upregulation of tight junction proteins.

Background:The increased permeability of the blood-brain barrier (BBB) induced by ischemia/hypoxia is generally correlated with alteration of tight junctions (TJs). DL-3-n-butylphthalide (NBP) has been shown to exert neuroprotective effects after ischemic injury. However, few studies have assessed the correlation between NBP and TJs. This study aimed to investigate the potential effect of NBP on the TJ proteins claudin-5, zonula occludens-1 (ZO-1), and occludin during brain ischemia.Methods:A chronic cerebral hypoperfusion (CCH) Sprague-Dawley rat model was established, and NBP (20, 40, or 80 mg/kg, gavage, once a day) treatment was performed for 14 days. NBP (0.1 or 1.0 μmol/L) pre-treatment was applied to an in vitro hypoxia microvascular endothelial cell model (1% O2, 24 h). BBB permeability was assessed by performing the Evans blue assay. The expressions and localization of claudin-5, ZO-1, occludin, phosphorylated/total protein kinase B (p-Akt/Akt), phosphorylated/total glycogen synthase kinase 3β (GSK-3β)/GSK-3β, and β-catenin/β-actin were evaluated by Western blotting or immunofluorescence. Reactive oxygen species (ROS) generation was measured by flow cytometry analysis. TJ ultrastructure was observed by transmission electron microscopy.Results:In CCH rats, treatment with 40 and 80 mg/kg NBP decreased the Evans blue content in brain tissue (9.0 ± 0.9 μg/g vs. 12.3 ± 1.9 μg/g, P = 0.005; 6.7 ± 0.6 μg/g vs. 12.3 ± 1.9 μg/g, P < 0.01), increased the expression of claudin-5 (0.79 ± 0.08 vs. 0.41 ± 0.06, P < 0.01; 0.97 ± 0.07 vs. 0.41 ± 0.06, P < 0.01), and elevated the ZO-1 protein level (P < 0.05) in brain microvascular segments in a dose-dependent manner in comparison with the corresponding values in the model group. There was no significant difference in occludin expression (P > 0.05). In the hypoxia cell model, NBP pre-treatment improved TJ ultrastructure, decreased intracellular ROS level, and increased the expression of claudin-5 (P < 0.01) and ZO-1 (P < 0.01) in comparison with the corresponding values in the hypoxia group. NBP treatment also elevated the relative expression levels of p-Akt/Akt, p-GSK-3β/GSK-3β, and β-catenin/β-actin in comparison with the corresponding values in the hypoxia group (all P < 0.05).Conclusion:NBP improves the barrier function of BBB against ischemic injury by upregulating the expression of TJ proteins, possibly by reducing oxidative stress and activating the Akt/GSK-3β/β-catenin signaling pathway.

Comparison of hypoxic effects induced by chemical and physical hypoxia on cardiomyocytes

The degree and duration of chemical hypoxia induced by sodium dithionite (Na2S2O4) have not been reported. It is not yet clear how much reduction in the O2 concentration (physical hypoxia) can lead to hypoxia in cultured cardiomyocytes. In this study, oxygen microelectrodes were used to measure changes in the O2 concentration in media containing different concentrations of Na2S2O4. Then, hypoxic effects of 0.8, 1.0, and 2.0 mM Na2S2O4 or 1%, 3%, and 5% O2 in cultured cardiomyocytes from neonatal rats were observed and compared. The results showed that the O2 concentration failed to remain constant by Na2S2O4 treatment during the 180-minute observation period. Only the 2.0 mM Na2S2O4 group significantly increased the expression of hypoxia-inducible factor 1α (HIF-1α) and hypoxic responses. Notably, 3% O2 only significantly increased the expression of HIF-1α in cardiomyocytes, while 1% O2 not only increased the expression of HIF-1α but also increased the apoptotic rate in cardiomyocytes. These results suggest that Na2S2O4 is not suitable for establishing a hypoxic model in cultured neonatal rat cardiomyocytes, and neonatal rat cardiomyocytes cultured at or below 1% O2 induced significant hypoxic effects, which can be used as a starting O2 concentration for establishing a hypoxic cell model.

MALAT1 promoted cell proliferation and migration via MALAT1/miR-155/MEF2A pathway in hypoxia of cardiac stem cells.

Accumulating evidence revealed that hypoxia contributed to many human diseases, including ischemic myocardium and heart failure (HF). In recent years, the roles of hypoxia in stem cell survival and cardiac biology have been studied extensively. However, the underlying molecular mechanisms remain to be elucidated. As a leading cause of HF, ischemic heart disease was correlated with hypoxia. In this study, we firstly constructed the hypoxia cell model by CoCl2 in cardiac stem cells (CSCs) and found that hypoxia induced the cell proliferation and migration potential in CSCs. Then, we demonstrated that the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was promoted in CoCl2 -induced CSCs hypoxia model. Furthermore, we found that knockdown of MALAT1 inhibited the cell proliferation and migration in CoCl2 -induced CSCs hypoxia model. In addition, we revealed that MALAT1 regulated the microRNA-155 (miR-155) expression in CSCs under both the normal and hypoxia conditions and further, manipulation of the miR-155 expression affected the role of MALAT1 in CoCl2 -induced CSCs hypoxia cell model. We then illustrated that miR-155 regulated the myocyte enhancer factor 2A(MEF2A) expression in CSCs under both the normal and hypoxia conditions and further, changing the expression of MEF2A affected the role of miR-155. Finally, we demonstrated that MALAT1 regulated the MEF2A expression and exerted its role via modulation of the MALAT1/miR-155/MEF2A pathway. Taken together, our study illustrated that MALAT1 promoted the cell proliferation and migration in CoCl2 -induced CSCs hypoxia model, acting mechanistically by promoting MEF2A expression via "sponging" miR-155.

Effects of transfected heat shock protein 70 on cysteinyl aspartate-specific protease-12, C/EBP homologous protein and c-Jun amino-terminal kinase signal pathway in endoplasmic reticulum stress

Objective To study the effect of heat shock protein 70 (HSP70) on cysteinyl aspartate-specific protease (Caspase)-12, C/EBP homologous protein (CHOP) and c-Jun N-terminal kinase (JNK) signal pathway in the endoplasmic reticulum stress. Methods HSP70 gene was transfected into SD neonatal rat cardiomyocytes by lipofectin. We used SD neonatal rat cardiomyocytes to establish a hypoxia cell model, and then we further detected the role of HSP70 in cell apoptosis, in expression of Casepase-12, CHOP, JNK pathway in ER caused by ER stress, by overexpression or downexpression of HSP70. Results The early apoptosis rate of the blank group (9.28±0.83)%, late (28.60±1.83)%, HSP70 gene transfection group early apoptosis rate (4.30±0.72)%, late (11.6±1.66)%, HSP70 treated with antisense oligonucleotides early apoptosis rate (29.3±2.54)%, late (34.4±2.43)%. Hypoxia-induced cell apoptosis was inhibited by HSP70, but was promoted by HSP70 anti-sense oligonucleotide. Hypoxia-induced ROS production was also inhibited by HSP70, but was not inhibited by HSP70 anti-sense oligonucleotide. HSP70 signifiantly inhibited the expression of Caspase-12 and CHOP. HSP70 increased the expression of JNK. HSP70 anti-sense oligonucleotide significantly increased the expression of CHOP, but did not signifiantly inflent other tested molecules. Conclusion This study demonstrated that HSP70 reduce myocardial cell apoptosis by regulating the Caspase-12, C/EBP homologous protein and c-Jun aminoterminal kinase signal pathway of endoplasmic reticulum. Key words: Cardiomyocyte; Heat shock proteins 70; Endoplasmic reticulum stress; Cysteinyl aspartate-specific protease-12; C/EBP homologous protein; c-Jun N-terminal kinase; Apoptosis

HIF-1α promotes inflammatory response of chronic obstructive pulmonary disease by activating EGFR/PI3K/AKT pathway.

Chronic obstructive pulmonary disease (COPD) is an incomplete, reversible disease with progressive inflammation obstruction in airways. This study aims to explore the regulatory mechanism of hypoxia-inducible factor-1α (HIF-1α) in inflammatory response and progression of COPD. 71 bronchoalveolar lavage fluid (BALF) samples were collected, including 59 samples from COPD patients (COPD group) and 12 from patients with normal pulmonary function (control group). The mRNA and protein levels of HIF-1α and epidermal growth factor receptor (EGFR) in BALF were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Serum levels of interleukin-13 (IL-13), IL-9, IL-1, and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA). Hypoxia cell model was constructed by COCl2 induction in human embryonic lung cells. Expression levels of HIF-1α, EGFR and p-AKT in NCI-H1563 cells treated with 740Y-P, the phosphoinositide 3-kinase (PI3K) agonist were detected. Finally, we detected proliferation and apoptosis in NCI-H1563 cells with HIF-1α overexpression by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. The mRNA and protein levels of HIF-1α and EGFR were higher in COPD groups compared with those of control group. Serum levels of IL-13, IL-9, IL-1, and TNF-α in COPD patients were elevated. CoCl2 induction in NCI-H1563 cells led to upregulated levels of IL-13, IL-9, IL-1, and TNF-α. 740Y-P treatment remarkably activated EGFR/PI3K/AKT pathway. Overexpressed HIF-1α inhibited proliferation but induced apoptosis of NCI-H1563 cells. HIF-1α was overexpressed in COPD, which upregulated expressions of inflammatory factors via activating the EGFR/PI3K/AKT pathway. The activated EGFR/PI3K/AKT pathway induced by pulmonary inflammation further upregulated HIF-1α expression in a feedback loop, thus aggravating COPD pathological changes.

Effect of emodin on the proliferation and migration of hypoxia pulmonary artery smooth muscle cells

Objective To observe the inhibitory effect of emodin on the proliferation and migration of pulmonary artery smooth muscle cells. Methods Primary cultured human pulmonary arterial smooth muscle cells (HPASMC) were cultured in a routine manner to establish an hypoxic cell model. Emodin at different concentrations (0, 5, 10, 15 and 20 μmol/L) 48 and 72 h later, the proliferation and migration of hypoxic pulmonary artery were detected by methyl thiazol tetrazolium (MTT) assay and Transwell invasion assay. Western blotting assay was used to determine the expression of p38 protein in hypoxia HPASMC induced by emodin. Results Emodin inhibited the proliferation and migration of hypoxic HPASMC in a time-and concentration-dependent manner. When the concentration was 20 μmol/L, the effect was the most significant, and the inhibitory rates at 12, 24, 48 and 72 h were (53.46±1.81)%, (61.32±2.69)%, (76.34±3.05)% and (84.43±3.18)%, respectively. (68.90±8.49), (46.10±5.63), (27.56±5.38) in the Transwell experimental group (5, 10, 15 and 20 μmol/[(12.61±3.68)] compared with the control group [(88.59±8.56)]. Compared with the control group, the experimental group p38 protein expression was reduced (P=0.001). Conclusion Emodin can significantly inhibit the proliferation and migration of human pulmonary artery smooth muscle cells under hypoxic conditions and down-regulate the expression of p38 protein. Key words: Emodin; Pulmonary artery smooth muscle cells; Proliferation; Migration

The expression of major histocompatibility complex class I in endometrial epithelial cells from dairy cow under a simulating hypoxic environment

During the first trimester of pregnancy in dairy cows, the fetus is mainly recognized by the expression of major histocompatibility complex class I (MHC-I) in the maternal immune system. Before the embryo begins to implant in the maternal uterus, it requires a long time to remodel and develop vessels in the placenta. During this stage, the embryos are exposed to a hypoxic environment. However, the expression of bovine MHC-I has not been determined in hypoxic bovine endometrial epithelial cells (bEECs). Hypoxia-inducible factor-1α (HIF-1α) is a marker for hypoxic conditions in cells. In the present study, we used cobalt chloride (CoCl2) to establish a hypoxic cell model and then determined the expression of the classical gene BoLA-A and the non-classical gene MICB. qRT-PCR and western blot assays were applied to determine the expression of HIF-1α. qRT-PCR was performed to detect the levels of BoLA-A and MICB mRNAs. The results showed that HIF-1α expression was increased in the hypoxic cell model. The expression of BoLA-A was increased, but MICB levels were decreased in bEECs. Our study provides a basis for exploring the cattle intrauterine environment during pregnancy.

Effects of heat shock protein 70 regulating endoplasmic reticulum calcium channel related protein on cardiomyocyte apoptosis

Objective To observe the role of heat shock protein 70 (HSP70) in endoplasmic reticulum (ER) stress-induced cell apoptosis, and to investigate the molecular mechanism underlying the HSP70 regulating ER stress induced-apoptosis, secretion and calcium signal transduction. The fidings provide new intervention targets and theoretical basis of subcellular molecular application for myocardial protection. Methods We used SD neonatal rat cardiomyocytes to establish a hypoxia cell model, and then we further detected the role of HSP70 in cell apoptosis, production of reactive oxygen species (ROS), expression of calcium sensing receptor (CaSR) and sarco endoplasmic reticulum Ca2+ -ATPase (SERCA) in ER caused by ER stress, by overexpression or downexpression of HSP70. Results The early apoptosis rate of the blank group [(9.28±0.83)%], late apoptosis rate [(28.60±1.83)%], ROS formation rate is 80.0%, HSP70 gene transfection group early apoptosis rate [(4.30±0.72)%], late apoptosis rate [(11.6±1.66)%], ROS formation rate is 38.5%, HSP70 treated with antisense oligonucleotides early apoptosis rate [(29.3±2.54)%], late apoptosis rate [(34.4±2.43)%], ROS formation rate of 77.0%, respectively, suggesting that HSP70 significantly inhibited the cell apoptosis and the production of ROS. The expression of CaSR in HSP70 gene transfection group was significantly lower than that in HSP70 antisense oligonucleotide group, but SERCA showed no significant difference, indicating that HSP70 significantly inhibited the expression of CaSR, but it had no influence on the expression of SERCA. Conclusion HSP70 inhibited the Ca2+ current via inhibiting the expression of CaSR, and the JNK pathway might play important roles in cell apoptosis and ROS-induced Ca2+ current. Key words: Heat shock protein 70; Endoplasmic reticulum stress; Myocardial protection; Calmodulin; Calcium sensitive receptors; Apoptosis

Regulation of COX-2 expression and epithelial-to-mesenchymal transition by hypoxia-inducible factor-1α is associated with poor prognosis in hepatocellular carcinoma patients post TACE surgery.

Currently, it is not entirely clear whether hypoxia-inducible factor-1α (HIF-1α) is involved in the regulation of COX-2 expression and epithelial-to-mesenchymal transition (EMT), and whether these events affect the prognosis of hepatocellular carcinoma (HCC) patients treated with transcatheter arterial chemoembolization (TACE). In this report the relationship between HIF-1α and COX-2 protein expression, EMT in tumor specimens from HCC patients after TACE surgery and the clinical significance of HIF-1α and COX-2 expression were analyzed using statistical approaches. HepG2 cells treated with CoCl2 was employed as a hypoxia cell model in vitro to study hypoxia-induced HIF-1α, COX-2 expression, and EMT alteration. The results showed that HIF-1α and COX-2 protein expression increased in HCC tissues after TACE surgery. Moreover, there was positive correlation between upregulation of HIF-1α and COX-2. Elevated expression of HIF-1α increased both Snail and Vimentin protein expression, while it reduced E-cadherin protein expression. It was further verified that hypoxia enhanced protein expression of HIF-1α and COX-2 in HepG2 cells treated with CoCl2. Upregulation of HIF-1α and COX-2, together with EMT alteration resulted in increased migration and invasion of HepG2 cells under hypoxia. In conclusion, TACE surgery results in aggravated hypoxia status, leading to increased HIF-1α protein expression in HCC tissue. To adapt to hypoxic environment, HIF-1α stimulates COX-2 protein expression and promotes EMT process in hepatocellular cancer cells, which enhances HCC invasion and metastasis, and might contribute to poor prognosis in HCC patients post TACE treatment.

Establishment of CoCl2 induced hypoxic cell model of oral squamous cell carcinoma and a preliminary study on the biological behaviour change

Establishment of CoCl2 induced hypoxic cell model of oral squamous cell carcinoma and a preliminary study on the biological behaviour change

Establishment of CoCl2 induced hypoxic cell model of oral squamous cell carcinoma and a preliminary study on the biological behaviour change

To mimic oral squamous cell carcinoma (OSCC) cell hypoxia by using chemical agent CoCl2 and to investigate its biological behaviour. Oral squamous cell carcinoma cell lines HSC-3 and SCC-4 were exposed to different concentration of CoCl2. HSC-3 and SCC-4 cells were treated with 50, 100, 150, 200 µmol/L CoCl2. Expression of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and B-cell lymphoma-2 (BCL-2) were measured by real time polymerase chain reaction (PCR) and Western blotting in both mRNA and protein level. Cell proliferation, cell apoptosis and cell cycle were detected to analyze its biological behaviour. Both wound healing and Transwell assay were applied to test the ability of cell igration. The result showed that after treatment of 150 µmol/L CoCl2 for 24 h, mRNA level of HIF-1α, VEGF and Bcl-2 was increased by 6.00 ± 0.20, 5.40 ± 0.40, 5.40 ± 0.30 (SCC-4); 5.60 ± 0.30, 5.20 ± 0.60, 5.80 ± 0.40(HSC-3). OSCC cells treated with 150 µmol/L CoCl2 for 24 h were collected. Compared with control group, the growth rate of cells was significantly decreased, P value was less than 0.05 (when HSC-3, SCC-4 cultured for 2 and 3 days). The apoptosis of OSCC cells was increased when treated with 150 µmol/L CoCl2 for 24 h:HSC-3 2.25% (control group) and 5.82% (treatment group); SCC-4 2.58% (control group) and 10.27% (treatment group). The migration ablility of OSCC cells was decreased when using 150 µmol/L CoCl2 for 24 h. The migration area ratio was (31.5 ± 2.3) % (HSC-3), (29.1 ± 1.5) % (SCC-4) in control group and (18.3 ± 1.9) % (HSC-3), (13.2 ± 0.8)% (SCC-4) in treatment group (P < 0.05). The hypoxic cell model of OSCC could be induced by CoCl2. The expression level of hypoxic markers was up regulated significantly and the cells biological behaviour changed including decreased cell proliferation, increased apoptosis and decreased migration.

Effects of A2BR on the biological behavior of mouse renal fibroblasts during hypoxia.

Fibroblasts are the effector cells of collagen secretion in renal interstitial fibrosis (RIF), and their proliferation and activation are essential for the development of RIF. Hypoxic ischemia in local tissues has been identified in chronic kidney diseases (CKDs), with adenosine (ADO) as a key signaling molecule. The current study investigated the association between ADO and the biological behavior of renal fibroblasts by establishing an invitro hypoxia cell model. This aimed to provide experimental evidence for the prevention and treatment of RIF. NIH3T3 fibroblasts were exposed to hypoxia, and the subtypes of the ADO receptor (AR) on the cell surface were identified by a TaqMan probe‑based assay. Cells were divided into the following four groups: i)Control; ii)5'‑N‑ethylcarboxamidoadenosine (NECA); iii)PT, NECA+8‑phenyltheophylline (PT); and iv)MRS, NECA+N‑(4‑cyanophenyl)‑2‑[4‑(2,3,6,7‑tetrahydro‑2,6‑dioxo‑1,3‑dipropyl‑1H‑purin‑8‑yl)phenoxy]‑acetamide (MRS1754). The mRNA levels of transforming growth factor‑β1 (TGF‑β1), procollagenα1(I) and α‑smooth muscle actin (α‑SMA) were measured following 24, 48, and 72h of hypoxia. Cell proliferation was evaluated by a 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay at 0, 12, 24, 48 and 72h. The results demonstrated that A2BR was the predominant AR subtype present in hypoxia‑stimulated fibroblasts. NECA significantly induced fibroblast proliferation and upregulated the expression of TGF‑β1, procollagenα1(I) and α‑SMA mRNA, while 8‑PT and MRS1754 inhibited fibroblast proliferation and downregulated the expression of TGF‑β1, procollagenα1(I) and α‑SMA mRNA. The blockage of A2BR in hypoxia significantly inhibited the proliferation and activation of fibroblasts, and reduced the production of profibrotic cytokines, thus preventing the generation and development of fibrosis.

Nerve Protective Effect of Asiaticoside against Ischemia-Hypoxia in Cultured Rat Cortex Neurons.

BackgroundAsiaticoside is one of the main functional components of the natural plant Centella asiatica urban. Studies have reported it has several functions such as anti-depression and nerve cell protection. Asiaticoside can reduce the cerebral infarct size in acute focal cerebral ischemia in a mouse model and asiatic acid glycosides can significantly improve neurobehavioral scores. Currently, there is a lack of understanding of asiaticoside in regard to its neural protective mechanism in cerebral ischemia. This study aimed to solve this problem by using an ischemia-hypoxia cell model in vitro.Material/MethodsAn in vitro ischemia hypoxia cell model was successfully established by primary cultured newborn rat cortical neurons. After being treated by asiaticoside for 24 h, cell survival rate, lactate dehydrogenase release quantity, and B-cell lymphoma gene-2 (BCL-2), Bax, and caspase-3 protein expressions was detected.ResultsAfter 10 nmol/L or 100 nmol/L of asiaticoside were given to the cells, cell survival rate increased significantly and presented concentration dependence. Asiaticoside can reduce lactate dehydrogenase release. Lactate dehydrogenase release in model cells is gradually reduced with the increase of asiaticoside concentration. The lactate dehydrogenase release in asiaticoside 10 nmol/L group, asiaticoside 100 nmol/L group and ischemia hypoxia group were 26.75±1.05, 22.36±2.87 and 52.35±5.46%, respectively (p<0.05). It was also found that asiaticoside could modulate the expression of apoptotic factors, including bcl-2, Bax, and caspase-3.ConclusionsAsiaticoside helps to protect in vitro ischemia hypoxia neurons. This nerve cell protection may be mediated by the BCL-2 protein.

Effect of intermittent hypoxia on lipid metabolism in liver cells and the underlying mechanism

To determine the effect of intermittent hypoxia on lipid metabolism in liver cells and to explore the possible molecular pathways involved in this process. An intermittent hypoxia cell model system was established by incubating the human hepatic cell lines L02 and HepG2 in an atmosphere of 2% O₂, 5% CO₂ and 93% N₂ for 8 hours per day over a period of 1, 2, 3, 4 or 5 days. Cells cultured in normoxia conditions (21% O₂) served as controls. Changes in intracellular lipid droplets and triglyceride (TG) levels were assessed by biochemical assays and oil red staining. Changes in intracellular reactive oxygen species (ROS) were assessed by inverted fluorescence microscopy and flow cytometry. Changes in expression of hypoxia-inducible factor (HIF)-1a and HIF-2a proteins, and the downstream ADFP, SREBP-1c and FAS proteins, were detected by western blotting. For both L02 and HepG2 cell lines, the cells grown under hypoxic conditions showed significantly higher lipid droplet accumulation and TG content than the cells grown under normoxic conditions (F(L02) =61.83, FHepG2 =104.19, P less than 0.01). Both oxygen concentration and time appeared to be correlated with these lipid-related changes (F(L02) =39.60, FHepG2 =76.39, P less than 0.01). The ROS fluorescence index was significantly increased after 2 days of intermittent hypoxia L02: 0.703 ± 0.129 vs. 3.310 ± 0.198, t =22.0637 and HepG2:0.617 ± 0.156 vs. 2.33 ± 0.42, t =7.2003, P < 0.05); in addition, increasing trends were observed in the ROS content and intensity of green fluorescence in conjunction with increased time of exposure to intermittent hypoxia (F(L02) =1021.84, FHepG2 =49.89, P less than 0.01). Compared with their respective control groups, the L02 and HepG2 cells both showed significantly upregulated expression of HIF-1a ADFP, SREBP-1c and FAS (L02:FHIF-1a =371.19, FsREBP-1c =204.49, FFAS =38.20, FADFP =154.31, P less than 0.05 and HepG2:FHF-1a =150.84, FSRERBP-1c =107.35, FFAs =279.71, FADFP =352.06, P less than 0.01). In contrast, the HIF-2a level was markedly decreased in the cells after 4 and 5 days of exposure to intermittent hypoxia (F(L02) =125.29, FHcpG2 =10.68, P less than 0.05). Under intermittent hypoxic conditions, ROS may regulate the expression of hypoxia-inducible factors and the adipose differentiation-related protein,as well as influence fatty acid metabolism via a HIF-1 a-SREBP-1 c-FAS signal and upregulation of the ADFP protein, in liver cells.

Regulation of transplanted mesenchymal stem cells by the lung progenitor niche in rats with chronic obstructive pulmonary disease

BackgroundStem cell transplantation is a promising method for the treatment of chronic obstructive pulmonary disease (COPD), and mesenchymal stem cells (MSCs) have clinical potential for lung repair/regeneration. However, the rates of engraftment and differentiation are generally low following MSC therapy for lung injury. In previous studies, we constructed a pulmonary surfactant-associated protein A (SPA) suicide gene system, rAAV-SPA-TK, which induced apoptosis in alveolar epithelial type II (AT II) cells and vacated the AT II cell niche. We hypothesized that this system would increase the rates of MSC engraftment and repair in COPD rats.MethodsThe MSC engraftment rate and morphometric changes in lung tissue in vivo were investigated by in situ hybridization, hematoxylin and eosin staining, Masson’s trichrome staining, immunohistochemistry, and real-time PCR. The expression of hypoxia inducible factor (HIF-1α) and stromal cell-derived factor-1 (SDF-1), and relationship between HIF-1α and SDF-1 in a hypoxic cell model were analyzed by real-time PCR, western blotting, and enzyme-linked immunosorbent assay.ResultsrAAV-SPA-TK transfection increased the recruitment of MSCs but induced pulmonary fibrosis in COPD rats. HIF-1α and SDF-1 expression were enhanced after rAAV-SPA-TK transfection. Hypoxia increased the expression of HIF-1α and SDF-1 in the hypoxic cell model, and SDF-1 expression was augmented by HIF-1α under hypoxic conditions.ConclusionsVacant AT II cell niches increase the homing and recruitment of MSCs to the lung in COPD rats. MSCs play an important role in lung repair and promote collagen fiber deposition after induction of secondary damage in AT II cells by rAAV-SPA-TK, which involves HIF-1α and SDF-1 signaling.

Hydrogen sulfide prevents hypoxia-induced apoptosis via inhibition of an H2O2-activated calcium signaling pathway in mouse hippocampal neurons

Hydrogen sulfide (H2S), an endogenous gaseous mediator, has been shown to exert protective effects against damage to different organs in the human body caused by various stimuli. However, the potential effects of H2S on hypoxia-induced neuronal apoptosis and its mechanisms remain unclear. Here, we exposed mouse hippocampal neurons to hypoxic conditions (2% O2, 5% CO2 and 93% N2 at 37°C) to establish a hypoxic cell model. We found that 4-h hypoxia treatment significantly increased intracellular reactive oxygen species (ROS) levels, and pretreatment with NaHS (a source of H2S) for 30min suppressed hypoxia-induced intracellular ROS elevation. The hypoxia treatment significantly increased cytosolic calcium ([Ca2+]i), and pretreatment with NaHS prevented the increase in [Ca2+]i. Additionally, polyethylene glycol (PEG)–catalase (a H2O2 scavenger) but not PEG–SOD (an O2- scavenger) conferred an inhibitory effect similar to H2S on the hypoxia-induced increase in [Ca2+]i. Furthermore, we found that pretreatment with NaHS could significantly inhibit hypoxia-induced neuronal apoptosis, which was also inhibited by PEG-catalase or the inositol 1,4,5-triphosphate (IP3) receptor blocker xestospongin C. Taken together, these findings suggest that H2S inhibits hypoxia-induced apoptosis through inhibition of a ROS (mainly H2O2)-activated Ca2+ signaling pathway in mouse hippocampal neurons.

Chronic hypoxia promotes an aggressive phenotype in rat prostate cancer cells

In general, tumors cells that are resistant to apoptosis and increase angiogenesis are a result of the hypoxic responses contributing to the malignant phenotype. In this study, we developed a chronic hypoxic cell model (HMLL), by incubating the prostate cancer MatLyLu cells in a hypoxic chamber (1% O2) over 3 weeks. Surviving cells were selected through each cell passage and were grown in the hypoxic condition up to 8 weeks. This strategy resulted in survival of only 5% of the cells. The surviving hypoxic cells displayed a greater stimulation on hypoxic adaptive response, including a greater expression of glucose transporter1 (Glut1) and VEGF secretion. In addition, higher invasion activity was observed in the chronic hypoxic HMLL cells as compared to MatLyLu cells exposed to acute hypoxia (1% O2, 5 h) using the matrigel assay. To further examine the role of HIF-1α in tumor progression, both MatLyLu and HMLL cells were transfected with dominant-negative form of HIF-1α (DNHIF-1α). The Matrigel invasion activity induced by chronic hypoxia was significantly attenuated by DNHIF-1α. These results suggest that signaling pathways leading to hypoxic response may be differentially regulated in chronic hypoxic cells and acute hypoxic cells. Chronic hypoxia may play a greater role than acute hypoxia in promoting the aggressive phenotype of tumor cells. This observation mimics the clinical scenario where tumor cells following treatment with radiation are subjected to hypoxic conditions. The reemergence of tumor following treatment usually results in tumor cells that are more aggressive and metastatic.