Effects of leptin on proliferation and differentiation of hypoxic rat retinal progenitor cells in vitro / 南方医科大学学报

OBJECTIVE@#To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition.@*METHODS@#SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for β-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture.@*RESULTS@#The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P < 0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P < 0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and β-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P < 0.05).@*CONCLUSION@#In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.

CoCl2 simulated hypoxia induce cell proliferation and alter the expression pattern of hypoxia associated genes involved in angiogenesis and apoptosis

BACKGROUND/AIMS: Hypoxia microenvironment plays a crucial role during tumor progression and it tends to exhibit poor prognosis and make resistant to various conventional therapies. HIF-1α acts as an important transcriptional regulator directly or indirectly associated with genes involved in cell proliferation, angiogenesis, apoptosis and energy metabolism during tumor progression in hypoxic microenvironment. This study was aimed to investigate the expression pattern of the hypoxia associated genes and their association during breast cancer progression under hypoxic microenvironment in breast cancer cells. METHODS: Cell proliferation in MCF-7 and MDA-MB-231 cell lines treated with different concentration of CoCl2 was analyzed by MTT assay. Flow cytometry was performed to check cell cycle distribution, whereas cell morphology was examined by phase contrast microscopy in both the cells during hypoxia induction. Expression of hypoxia associated genes HIF-1α, VEGF, p53 and BAX were determined by semiquantitative RT-PCR and real-time PCR. Western blotting was performed to detect the expression at protein level. RESULTS: Our study revealed that cell proliferation in CoCl2 treated breast cancer cells were concentration dependent and varies with different cell types, further increase in CoCl2 concentration leads to apoptotic cell death. Further, accumulation of p53 protein in response to hypoxia as compare to normoxia showed that induction of p53 in breast cancer cells is HIF-1α dependent. HIF-1α dependent BAX expression during hypoxia revealed that after certain extent of hypoxia induction, over expression of BAX conquers the effect of anti-apoptotic proteins and ultimately leads to apoptosis in breast cancer cells. CONCLUSION: In conclusion our results clearly indicate that CoCl2 simulated hypoxia induce the accumulation of HIF-1α protein and alter the expression of hypoxia associated genes involved in angiogenesis and apoptosis.

Polydatin protects H9c2 cells from hypoxia-induced injury via up-regulating long non-coding RNA DGCR5

Polydatin (PD), a monocrystalline polyphenolic drug mainly found in the roots of Polygonum cuspidatum, has various pharmacological activities. Long non-coding RNAs (lncRNA) DiGeorge syndrome critical region gene 5 (DGCR5) was found to participate in the suppression of multiple cancers. Here, we proposed to study the effect of PD on myocardial infarction (MI) by inducing DGCR5. CCK-8 assay was performed to detect the viability of H9c2 cells. Flow cytometry was utilized to test apoptosis of H9c2 cells. These results determined the optimal concentration and effect time of hypoxia as well as PD. Si-DGCR5 was transfected into cells and the expression level was determined by qRT-PCR. Western blot was utilized to evaluate the expression of apoptosis-related proteins, Bcl-2, Bax, and cleaved-caspase-3, as well as autophagy-associated proteins including Beclin-1, p62, and LC3-II/LC3-I. As a result, PD efficiently attenuated hypoxia-induced apoptosis and autophagy in H9c2 cells. The expression of DGCR5 was down-regulated by hypoxia and up-regulated by PD. Besides, knocking-down the expression of DGCR5 inhibited the protection of PD in H9c2 cells. In addition, PD up-regulated the accumulation of DGCR5, DGCR5 decreased the expression of Bcl-2 and p62, raised the expression of Bax and cleaved-caspase-3, and the proportion of LC3-II/LC3-I. PD stimulated the PI3K/AKT/mTOR and MEK/ERK signaling pathways via up-regulating the expression of DGCR5. Our data demonstrated that PD reduced cell apoptosis and autophagy induced by hypoxia in cardiomyocytes. Moreover, PD activated PI3K/AKT/mTOR and MEK/ERK signaling pathways by up-regulating the expression of DGCR5.

Emodin protects H9c2 cells from hypoxia-induced injury by up-regulating miR-138 expression

Myocardial infarction (MI) is a common presentation for ischemic heart disease, which is a leading cause of death. Emodin is a Chinese herbal anthraquinone used in several diseases. However, the effect of emodin in hypoxia-induced injury in cardiomyocytes has not been clearly elucidated. Our study aimed to clarify the functions of emodin in hypoxia-induced injury in rat cardiomyocytes H9c2 and explore the underlying mechanism. The effects of emodin on cell viability and apoptosis were analyzed by the Cell counting kit-8 assay and flow cytometry assay, respectively. The cell proliferation- and cell apoptosis-related proteins were detected by western blot. qRT-PCR was used to determine the relative expression of miR-138. Cell transfection was performed to alter miR-138 and MLK3 expression. miR-138 target was performed by dual luciferase activity assay. Sirt1/AKT and Wnt/β-catenin pathways-related factors phosphorylation were analyzed by western blot. Emodin inhibited hypoxia-induced injury in H9c2 cells by promoting cell viability and reducing cell apoptosis. miR-138 was down-regulated by hypoxia treatment but up-regulated by emodin. Up-regulation of miR-138 alleviated hypoxia-induced cell injury. Down-regulation of miR-138 attenuated the growth-promoting effect of emodin on hypoxia-induced injury, whereas up-regulation of miR-138 enhanced the growth-promoting effects of emodin. The underlying mechanism might be by inactivating Sirt1/AKT and Wnt/β-catenin pathways. MLK3 was negatively regulated by miR-138 expression and inactivated Sirt1/AKT and Wnt/β-catenin pathways. Emodin alleviated hypoxia-induced injury in H9c2 cells via up-regulation of miR-138 modulated by MLK3, as well as by activating Sirt1/AKT and Wnt/β-catenin pathways.

Andrographolide protects mouse astrocytes against hypoxia injury by promoting autophagy and S100B expression

Andrographolide (ANDRO) has been studied for its immunomodulation, anti-inflammatory, and neuroprotection effects. Because brain hypoxia is the most common factor of secondary brain injury after traumatic brain injury, we studied the role and possible mechanism of ANDRO in this process using hypoxia-injured astrocytes. Mouse cortical astrocytes C8-D1A (astrocyte type I clone from C57/BL6 strains) were subjected to 3 and 21% of O2 for various times (0-12 h) to establish an astrocyte hypoxia injury model in vitro. After hypoxia and ANDRO administration, the changes in cell viability and apoptosis were assessed using CCK-8 and flow cytometry. Expression changes in apoptosis-related proteins, autophagy-related proteins, main factors of JNK pathway, ATG5, and S100B were determined by western blot. Hypoxia remarkably damaged C8-D1A cells evidenced by reduction of cell viability and induction of apoptosis. Hypoxia also induced autophagy and overproduction of S100B. ANDRO reduced cell apoptosis and promoted cell autophagy and S100B expression. After ANDRO administration, autophagy-related proteins, S-100B, JNK pathway proteins, and ATG5 were all upregulated, while autophagy-related proteins and s100b were downregulated when the jnk pathway was inhibited or ATG5 was knocked down. ANDRO conferred a survival advantage to hypoxia-injured astrocytes by reducing cell apoptosis and promoting autophagy and s100b expression. Furthermore, the promotion of autophagy and s100b expression by ANDRO was via activation of jnk pathway and regulation of ATG5.

Aged garlic extract and S-allylcysteine prevent apoptotic cell death in a chemical hypoxia model

BACKGROUND: Aged garlic extract (AGE) and its main constituent S-allylcysteine (SAC) are natural antioxidants with protective effects against cerebral ischemia or cancer, events that involve hypoxia stress. Cobalt chloride (CoCl2) has been used to mimic hypoxic conditions through the stabilization of the α subunit of hypoxia inducible factor (HIF-1α) and up-regulation of HIF-1α-dependent genes as well as activation of hypoxic conditions such as reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and apoptosis. The present study was designed to assess the effect of AGE and SAC on the CoCl2-chemical hypoxia model in PC12 cells. RESULTS: We found that CoCl2 induced the stabilization of HIF-1α and its nuclear localization. CoCl2 produced ROS and apoptotic cell death that depended on hypoxia extent. The treatment with AGE and SAC decreased ROS and protected against CoCl2-induced apoptotic cell death which depended on the CoCl2 concentration and incubation time. SAC or AGE decreased the number of cells in the early and late stages of apoptosis. Interestingly, this protective effect was associated with attenuation in HIF-1α stabilization, activity not previously reported for AGE and SAC. CONCLUSIONS: Obtained results show that AGE and SAC decreased apoptotic CoCl2-induced cell death. This protection occurs by affecting the activity of HIF-1α and supports the use of these natural compounds as a therapeutic alternative for hypoxic conditions

The Effect of Brimonidine on Transepithelial Resistance in a Human Retinal Pigment Epithelial Cell Line

PURPOSE: To investigate the effects of brimonidine, an alpha-2-adrenergic agonist, on barrier function in ARPE-19 cells by measuring transepithelial resistance (TER). METHODS: ARPE-19 cells were cultured into a confluent monolayer on a microporous filter. Brimonidine was added to the apical medium, and the barrier function of the cells was evaluated by measuring TER. A subset of cells was treated under hypoxic conditions, and the TER changes observed upon administration of brimonidine were compared to those observed in cells in normoxic conditions. RESULTS: The ARPE cell membrane reached a peak resistance of 29.1+/-7.97 Omega cm2 after four weeks of culture. The TER of the cells treated under normoxic conditions increased with brimonidine treatment; however, the TER of the cells treated under hypoxic conditions did not change following the administration of brimonidine. CONCLUSIONS: Barrier function in ARPE-19 cells increased with brimonidine treatment. Understanding the exact mechanism of this barrier function change requires further investigation.

Study of rat hepatocytes in primary culture submitted to hypoxia and reoxygenation: action of the cytoprotectors prostaglandin E1, superoxide dismutase, allopurinol and verapamil / Estudo de hepatócitos de rato em cultura primária submetidos a hipóxia e reoxigenação: ação dos citoprotetores prostaglandina E1, superóxido dismutase, alopurinol e verapamil

Context: Exposure of hepatocytes to pathological conditions in a microenvironment of hypoxia and reoxygenation is very frequent in hepatic diseases. Several substances present perspectives for cytoprotective action on hepatocyte submitted to reoxygenation after hypoxia and simple hypoxia. Objective: We research therapeutic options for hepatocytes submitted to hypoxia and hypoxia + reoxygenation injury. Methods: Primary culture of rat hepatocytes was submitted to hypoxia (2 hours) plus reoxygenation (2 hours) and simple hypoxia (4 hours) in the presence or the absence of cytoprotectors. The hepatocyte lesion was evaluated by functional criteria through percentage of lactate dehydrogenase released and cell viability. The effects of the cytoprotectors prostaglandin E1 3 ηg/mL, superoxide dismutase 80 μg/mL, allopurinol 20 μM and verapamil 10-4 M were studied in this model of injury. Results: Reoxygenation after hypoxia induced more significant lesion in cultured hepatocytes compared to simple hypoxia, detected by analysis of functional criteria. There was a significant reduction of percentage of lactate dehydrogenase released and a significant increase of percentage of cell viability in the hypoxia + reoxygenation + cytoprotectors groups compared to hypoxia + reoxygenation groups. Prostaglandin E1, superoxide dismutase and verapamil also protected the group submitted to simple hypoxia, when evaluated by functional criteria. Conclusions: We conclude that reoxygenation after hypoxia significantly increased the lesion of cultured rat hepatocytes when compared to simple hypoxia. Prostaglandin E1, superoxide dismutase, allopurinol and verapamil acted as cytoprotectors to the rat cultured hepatocytes submitted to hypoxia + reoxygenation in vitro. The substances prostaglandin E1, superoxide dismutase and verapamil protected hepatocytes submitted to simple hypoxia on the basis of all the criteria studied in this experimental model.

Contexto: A exposição dos hepatócitos a condições patológicas em que ocorram microambientes de hipóxia e reoxigenação são muito frequentes em doenças hepáticas. Várias substâncias apresentam perspectivas de ação citoprotetora para hepatócitos submetidos a reoxigenação após hipóxia e hipóxia simples. Objetivo: Pesquisaram-se opções terapêuticas para o dano dos hepatócitos submetidos a hipóxia e hipóxia + reoxigenação. Métodos: Hepatócitos de rato em cultura primária foram submetidos a hipóxia (2 horas) mais reoxigenação (2 horas) e hipóxia simples (4 horas), na presença ou ausência dos citoprotetores. A lesão dos hepatócitos foi avaliada por critérios funcionais através da percentagem liberada de desidrogenase láctica e da viabilidade celular. Os efeitos dos citoprotetores prostaglandina E1 3 ηg/mL, superóxido dismutase 80 μg/mL, alopurinol 20 μM e verapamil 10-4M, foram estudados neste modelo de injúria celular. Resultados: A reoxigenação após hipóxia induziu lesão mais significativa nos hepatócitos cultivados comparado com hipóxia simples, conforme demonstrado pela análise dos critérios funcionais. Houve significativa redução da porcentagem liberada de desidrogenase láctica e aumento significativo da percentagem de viabilidade celular nos grupos hipóxia + reoxigenação + citoprotetores em comparação com o grupo hipóxia + reoxigenação. Prostaglandina E1, superóxido dismutase e verapamil também protegeram o grupo hipóxia simples, quando avaliado pelos critérios funcionais. Conclusões: Conclui-se que a reoxigenação após hipóxia aumentou significativamente a lesão dos hepatócitos de rato cultivados, em comparação com a hipóxia simples. Prostaglandina E1, superóxido dismutase, alopurinol e verapamil foram citoprotetores para os hepatócitos de rato submetidos a hipóxia + reoxigenação in vitro. As substâncias prostaglandina E1, superóxido dismutase e verapamil protegeram os hepatócitos submetidos a hipóxia simples com base em...

The blockade of cyclooxygenases-1 and -2 reduces the effects of hypoxia on endothelial cells

Hypoxia activates endothelial cells by the action of reactive oxygen species generated in part by cyclooxygenases (COX) production enhancing leukocyte transmigration. We investigated the effect of specific COX inhibition on the function of endothelial cells exposed to hypoxia. Mouse immortalized endothelial cells were subjected to 30 min of oxygen deprivation by gas exchange. Acridine orange/ethidium bromide dyes and lactate dehydrogenase activity were used to monitor cell viability. The mRNA of COX-1 and -2 was amplified and semi-quantified before and after hypoxia in cells treated or not with indomethacin, a non-selective COX inhibitor. Expression of RANTES (regulated upon activation, normal T cell expressed and secreted) protein and the protective role of heme oxygenase-1 (HO-1) were also investigated by PCR. Gas exchange decreased partial oxygen pressure (PaO2) by 45.12 ± 5.85 percent (from 162 ± 10 to 73 ± 7.4 mmHg). Thirty minutes of hypoxia decreased cell viability and enhanced lactate dehydrogenase levels compared to control (73.1 ± 2.7 vs 91.2 ± 0.9 percent, P < 0.02; 35.96 ± 11.64 vs 22.19 ± 9.65 percent, P = 0.002, respectively). COX-2 and HO-1 mRNA were up-regulated after hypoxia. Indomethacin (300 æM) decreased COX-2, HO-1, hypoxia-inducible factor-1alpha and RANTES mRNA and increased cell viability after hypoxia. We conclude that blockade of COX up-regulation can ameliorate endothelial injury, resulting in reduced production of chemokines.

A new indication of sildenafil in medicine: hypoxic cell sensitizer for penile cancer.

PURPOSE: To evaluate the feasibility and results of sildenafil used as radiosensitizer by causing penile vasodilatation and reducing tumor hypoxia in radical radiotherapy for penile cancer. MATERIALS AND METHODS: Three men with histopathologic diagnosis of squamous carcinoma of penis were treated with radical external radiotherapy with concomitant sildenafil. Acute and late adverse events were noted according to RTOG toxicity criteria. Response to radiotherapy, as well as disease-free and overall survivals, was evaluated. RESULTS: All three patients achieved tumescence, completed the treatment and achieved complete response. One patient had recurrence at 10 months and died of the disease after 23 months from starting the treatment. Other two patients are disease-free and alive at 48 and 53 months respectively. No excess acute or late adverse event was observed. CONCLUSION: Sildenafil may act as radiosensitizer by causing penile vasodilatation and reducing hypoxia. Larger controlled studies are required to validate the hypothesis.

Tackling radioresistance of hypoxic cells by metabolic modulation of bioenergetics--a 31P MRS study on perfused Ehrlich ascites tumor cells.

In an attempt to overcome resistance of hypoxic cells to radiotherapy, the combination of a hematoporphyrin derivative (Hpd) and 2-deoxy-D-glucose (2-DG), a promising radiomodifier, was evaluated by assessing the alterations in phosphorylated metabolites and bioenergetics induced in perfused Ehrlich ascites tumor (EAT) cells, using Phosphorus-31 Magnetic Resonance Spectroscopy (31P-MRS). By reducing flow rate of perfusion, a relatively hypoxic condition of tumor was simulated. Changes in bioenergetics levels induced by the combined treatment of Photosan, a Hpd, and 2-DG, under reduced perfusion conditions were more pronounced. Significantly higher uptake of 2-DG and irreversibility of the reduction in cellular bioenergetics induced by the combined treatment, observed under simulated hypoxic conditions, might have considerable implications in optimizing tumor radiotherapy using 2-DG as an adjuvant. These result also suggest the usefulness of this technique to easily simulate hypoxic conditions of tumors in vitro that could be used for rapid in vitro pharmacological evaluation of promising therapeutic strategies.

A clinical study to assess chlorpromazine as hypoxic cell sensitizer in head & neck cancer treated with conventional radiation.

A randomized prospective study was conducted to evaluate the effectiveness of chlorpromazine as a sensitizer of radiation in advanced head and neck cancers. Patients with unresectable laryngopharyngeal cancers except glottic cancers, with histologically proven squamous cell carcinoma staged III and IV were accrued for the study. Patients received radiation to a total dose of 6000 cGy in six weeks in both the groups except that patients in the study group received 50 mgs Chlorpromazine (CPZ) in divided doses. Fourteen of 20 patients showed complete response in the control group whereas 34 of 38 patients in chlorpromazine treated group had complete regression of the tumour (p = 0.016). The survival was (p = 0.08) better in patients receiving CPZ. This preliminary study shows beneficial effects of chlorpromazine. No adverse effects due to chlorpromazine in conjunction with radiation were documented.

Protective effects of diltiazem on the mechanical performance of the hypoxic myocardium

1. To determine whether diltiazem protects the hypoxic myocardium by reducing contractile work, we have compared the effects of diltiazem and quiescence on left ventricular (LV) papillary muscle subjected to hypoxia. Papillary muscles were obtained from male Charles River CD rats weighing 150-250 g. 2. Four groups of muscles were studied: control (N = 6), non-stimulation (N = 10), diltiazem 10(-4) M (N = 6) and diltiazem 10(-4) M plus non-stimulation (N = 10). 3. Isolated rat LV papillary muscles were studied in Krebs-Henseleit solution with a calcium concentration of 2.52 mM at 28 degrees C while contracting isometrically at a stimulation rate of 0.2 Hz. Resting tension and active isometric tension were measured. 4. Both diltiazem and quiescence significantly attenuated contracture tension during hypoxia (0.91 +/- 0.10 vs 2.26 +/- 0.49 g/mm2 for diltiazem vs control, and 0.55 +/- 0.18 vs 2.26 +/- 0.49 g/mm2 for quiescence vs control). Recovery of active tension was improved in the diltiazem groups during reoxygenation (4.16 +/- 0.42 vs 3.75 +/- 0.51, 3.53 +/- 0.15 vs 2.90 +/- 0.13, 5.84 +/- 0.33 vs 6.48 +/- 0.29 and 5.98 +/- 0.90 vs 7.67 +/- 0.68 g/mm2 for diltiazem, diltiazem non-stimulation, non-stimulation and control groups). 5. The results suggest that the protective effect of diltiazem during hypoxia was due to the reduction in energy demand of the myocardium