Effects of tanshinone IIA on proliferation,apoptosis and expression of HIF-1α, VEGF and wild-type P53 in human hepatoma HepG2 cells under hypoxia

AIM:To investigate the effects of tanshinone IIA (Tan IIA) on proliferation, apoptosis and its molecular mechanism in human hepatoma HepG2 cells under hypoxic condition. METHODS:Hypoxia model was established by treatment with cobalt chloride (CoCl2). The cells were divided into normoxia control group, hypoxia control group and hypoxia combined at different concentrations of Tan IIA groups. After HepG2 cells were incubated with different concentrations of Tan IIA (0.5, 1.0, 2.0, 5.0 and 10.0 mg/L) for 24 h, 48 h and 72 h under hypoxic condition, the cell proliferation was determined by MTT assay. After Tan IIA was added to the media at different concentrations for 24 h and 48 h, the apoptotic cells were observed by Hoechst 33258 staining. The protein levels of hypoxia-inducible factor 1 alpha (HIF-1α), vascular endothelial growth factor (VEGF) and wild-type P53 were detected by Western blotting after cultured with different concentrations of Tan IIA for 48 h. RESULTS:Tan IIA inhibited the proliferation of HepG2 cells in a dose- and time-dependent manner. Tan IIA induced the typical morphology of apoptotic cells and increased the apoptotic rate in a dose- and time-dependent manner after treatment with 1.0 mg/L~5.0 mg/L for 24 h and 48 h under hypoxic condition. The protein levels of HIF-1α and VEGF were weakly expressed in HepG2 cells under normoxia but up-regulated after incubated under hypoxia for 48 h. The protein expression of HIF-1α and VEGF were decreased with the increase in the concentration of Tan IIA under hypoxia. The protein expression of wild-type P53 was increased with the increase in the concentrations of Tan IIA under hypoxia. CONCLUSION: Tan IIA significantly inhibits the proliferation and induces the apoptosis of human hepatoma HepG2 cells under hypoxia, which may be related to the down-regulation of HIF-1α and VEGF and up-regulation of wild-type P53.     

MicroRNA-210 negatively regulates hypoxic hPASMC proliferation by targeting MKP-1

AIM:To investigate the possible interactions between microRNA-210 (miR-210) and mitogen-activated protein kinase phosphatase 1 (MKP-1) and the effect on the proliferation of hypoxic human pulmonary artery smooth muscle cells (hPASMCs). METHODS:hPASMCs were cultured in 21% O2 and 5% CO2 (normoxia) or 1% O2 and 5% CO2 (hypoxia) for 48 h, and then transfected with mimic or inhibitor of miR-210 or MKP-1 small interfering RNA (si-RNA). The levels of RNA, miRNA and protein were isolated separately. RESULTS:The level of miR-210 was significantly increased in cultured hPASMCs exposed to 1% O2 for 48 h, and the expression of MKP-1 at mRNA and protein levels was also increased. Furthermore, inhibition of miR-210 expression increased the mRNA and protein levels of MKP-1 in the hPASMCs and decreased the cell proliferation under hypoxia. Conversely, over-expression of miR-210 prevented hypoxia-induced MKP-1 expression with no effect on the cell proliferation. Knockdown of MKP-1by siRNA abolished the preventive effect of miR-210 inhibitor on the cell proliferation under hypoxia. CONCLUSION: MKP-1 is a target of miR-210 and mediates the negative regulation of miR-210 inhibitor in hypoxic hPASMCs.     

In vitro killing effect of adenovirus-mediated herpes simplex virus thymidine kinase gene under regulation of hypoxic response element on hepatoma cell line HepG2

AIM: To investigate the in vitro killing effect of adenovirus-mediated herpes simplex virus thymidine kinase gene (HSV-TK) driven by hypoxic response element (HRE) on hepatoma cell line HepG2. METHODS: Recombinant adenoviral vector Ad-HRE-TK was constructed with HSV-TK under the control of HRE using AdEasy system. Then Ad-HRE-TK was transfected into hepatoma cell line HepG2 and the cells were cultured under normoxic or hypoxic conditions. After treated with GCV for 3 d, the sensitivity to GCV of HepG2 was measured by MTT method. RESULTS: Over 95% HepG2 cells infected with Ad-HRE-TK cultured under hypoxic condition were killed when the MOI was 100 and the concentration of GCV was 50 mg/L. On the contrary, no killing effect of GCV was observed in cells cultured under normoxic condition. CONCLUSION: HRE promotes the expression of HSV-TK specifically under hypoxic condition and induces the specific killing effect of GCV.     

Sevoflurane preconditioning inhibits brain injury in hypoxic mice

AIM: To observe the effects of sevoflurane preconditioning on brain injury in hypoxic mice and its possible mechanism. METHODS: Male C57BL/6J mice were randomly divided into control (C) group, hypoxia (H) group, 2% sevoflurane preconditioning for 30 min + hypoxia (S1+H) group, 2% sevoflurane preconditioning for 60 min+hypoxia (S2+H) group and 4% sevoflurane preconditioning for 30 min + hypoxia (S3+H) group. The hypoxia model was established by continuous inhalation of (6.5±0.1)% O₂ for 24 h. The sevoflurane preconditioning treatments, S1, S2 and S3, were conducted by inhalation of 2% sevoflurane for 30 min, 2% sevoflurane for 60 min and 4% sevoflurane for 30 min, respectively, with the carrier of (21.0±0.5)% O₂, followed by washout for 15 min and then hypoxia treatment. The histological changes of the hippocampal CA1 area were observed under light microscope and transmission electron microscope (TEM), and serum lactate dehydrogenase (LDH) activity was measured by colorimetric method. Furthermore, the protein levels of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in brain tissue homogenate were examined by ELISA, and the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured by microplate reader. RESULTS: After hypoxia for 24 h, cell edema or pyknosis in the hippocampal CA1 area was observed in H group. Sevoflurane preconditioning reduced hypoxic injury, and the cell ultrastructure under TEM was significantly improved in S2+H group. Compared with C group, the serum LDH activity and the levels of EPO, VEGF and MDA in brain tissues were significantly increased in H group, while the activity of SOD and GPx decreased. After sevoflurane pretreatment, the serum LDH activity and the levels of EPO and VEGF in brain tissues were lower than those in H group, and the most significant difference was observed in S2+H group. Moreover, the MDA content and SOD activity decreased, and the GPx activity increased in the sevoflurane preconditioning groups. CONCLUSION: Sevoflurane preconditioning attenuates brain injury in hypoxic mice by regulating antihypoxic protein synthesis and reducing oxidative stress.     

Regulation of hypoxic response elements to expression of hVEGF165 gene transferred to cardiomyocytes under anoxic and normoxic conditions

AIM:To investigate effects of hypoxic response elements (HRE) on expression of hVEGF165 gene transferred to myocardiocyte under anoxic and normoxic conditions in vitro.METHODS:Myocardiocytes from neonatal SD rats were isolated and cultured in serum-free medium.The r-AAV vector packaged with 293T cells was used to transfer cultured myocardiocytes.Myocardiocytes were divided into eight groups: Group Ⅰ: myocardiocytes were cultured for 24 hours under normoxic conditions as control;Group Ⅱ: cells was cultured under hypoxia for 8 hours;Group Ⅲ: infected cells were cultured for 24 hours under normoxic conditions;Group Ⅳ: infected cells were cultured under hypoxia for 8 hours;GroupⅤ: infected cells were cultured under hypoxia for 8 hours and normoxia for 4 hours;GroupⅥ: infected cells were cultured under hypoxia for 8 hours and normoxia for 8 hours;GroupⅦ: infected cells were cultured under hypoxia for 8 hours and normoxia for 12 hours;Group Ⅷ: infected cells were cultured under hypoxia only for 20 hours.hVEGF165 protein was quantified from culture medium using ELISA,hVEGF165 protein in myocardiocytes was detected with immunofluorescence and expression of hVEGF165 mRNA was also determined by RT-PCR.RESULTS:95% myocardiocytes showed beating rhythmically with 86% of cTn-I positive staining.87% virual transferred efficiency was achieved.The contents of hVEGF165 protein in group Ⅳ,Ⅴ and Ⅷ were higher than those in other groups (P<0.01).Immunofluorescence positive cells were observed in group Ⅳ,Ⅴ and group Ⅷ.RT-PCR revealed that a 484 bp strip was also found in the same groups.CONCLUSION:rAAV-HRE9-hVEGF165 vector infected cultured myocardiocyte successfully.Under hypoxia,expression of hVEGF165mRNA and hVEGF165 protein were regulated by HRE,whereas under normoxic conditions the expression of hVEGF165 mRNA and hVEGF165 protein were ceased.     

An improved method to develop cell culture model of intermittent hypoxia

AIM: To establish and validate a novel model of cultured cells for imitating intermittent hypoxia. METHODS: In a chamber with experiment cabin and simulated air control cabin, the frequency and duration of the intermittent hypoxia model according to the time of hypoxia and reoxygenation were evaluated. The A549 cells were randomly divided into 7 groups, named as control (Con) group, 6 h intermittent hypoxia (6IH) group, 9 h intermittent hypoxia (9IH) group, 6 h simulated air control (6AC) group, 9 h simulated air control (9AC) group, 4 h sustained hypoxia (4SH) group, 6 h sustained hypoxia (6SH) group, respectively. When the model was established, the cellular morphology was observed under inverted microscope. The mRNA expression of hypoxia-inducible factor (HIF)-1α was detected by real-time PCR. The protein expression of HIF-1α was analyzed by immunohistochemistry. RESULTS: The intermittent hypoxia cycle (5% O₂ 60 min-20% O₂ 30 min for 6 cycles) was established. The damaged A549 cells were observed in 6IH group, 9IH group and 6SH group. Compared with 6IH group, the expression of HIF-1α at mRNA and protein levels was significantly increased in 9IH group (P<0.05). The expression of HIF-1α at mRNA and protein levels in 6IH group and 9IH group was higher than that in 4SH group and 6SH group, respectively (P<0.05). No significant difference among the control group, 6AC group and 9AC group was found. CONCLUSION: The model (5% O₂ 60 min-20% O₂ 30 min for 6 cycles) can simulate the pathological process of obstructive sleep apnea hypopnea syndrome. This model is suitable for studying intermittent hypoxia in adherent cells.     

Hypoxia induces myofibroblast formation and stimulates production of collagen I in myofibroblasts through ERK1/2 pathway

AIM: To investigate the effect of hypoxia on the myofibroblast transdifferentiation from fibroblasts,and associated signaling of hypoxia on the production of collagen I in cultured rat renal cortical myofibroblasts.METHODS: The study is composed of two relevant parts.In the first part,a normal rat renal interstitial fibroblast cell line NRK-49F was treated with hypoxia (1% O2) or normoxia (21% O2) for 6 h,12 h and 24 h.The expression of hypoxia inducible factor-1α (HIF-1α) was examined by Western blotting in order to make sure the hypoxic condition is reliable.The myofibroblast transformation from fibroblasts induced by hypoxia was assayed by detecting the protein levels of α-smooth muscle actin (α-SMA).In the second part,the object was done on the primary cultured rat renal cortical myofibroblasts.Myofibroblasts were subjected to hypoxic or normoxic conditions for variety of times.The levels of HIF-1α in cell lysates and collagen I protein in supernatant culture medium and the activation of extracellular signal-regulated kinase (ERK)1/2 MAPK pathway were analyzed by Western blotting.RT-PCR was carried out to measure the levels of collagen I mRNA at different time points (2 h,4 h and 6 h).The distribution of HIF-1α in myofibroblasts was demonstrated by immunocytochemistry.The changes of collagen I production were detected after PD98059,a specific inhibitor of ERK1/2 activation pretreatment and during the hypoxia incubation.The activity of gelatinase matrix metalloproteinase-2(MMP-2) and MMP-9 in the supernatant medium from the cultured cells were assayed by gelatin zymography.RESULTS: Significant increased levels of HIF-1α protein appeared in cell lysates under hypoxia for 6 h.Furthermore,HIF-1α was translocated into nuclei of myofibroblasts after 6 h exposure of myofibroblasts to hypoxia.The levels of α-SMA protein increased in NRK-49F under hypoxia for 12 h (187%±32%,P<0.05).The level of collagen I protein in culture medium was increased in hypoxia treated myofibroblasts at 6 h (171%±27%,P<0.05) and 12 h (256%±61%,P<0.05).Collagen I mRNA expression was increased in cells under hypoxia condition for 4 h (189%±28%,P<0.05) and 6 h (221%±44%,P<0.05).The activities of MMP-2 and MMP-9 in the supernatant medium were not significantly changed at different experimental time points between the normoxic and hypoxic conditions.Activation of ERK1 /2 occurred as early as 15 min,sustained the high level at 30 min and 60 min and was back to the baseline level at 2 h.Blockade of ERK activation with PD98059 abolished hypoxia-induced expressions of collagen I protein.CONCLUSION: Hypoxia contributes to the renal interstitial fibrosis through inducing formation of myofibroblasts and stimulating the production of collagen I in myofibroblasts.     

Effect of diltiazem on heme oxygenase-1 and nitric oxide synthase in rats with pulmonary hypertension

AIM: To investigate the action of diltiazem (a calcium antagonist) on the expression of heme oxygenase (HO) -1 and nitric oxide synthase (NOS) in the small pulmonary arteries (SPA) of rat in chronic hypoxia. METHODS: Chronic pulmonary arterial hypertension models were established by treating the rats in hypoxic environment for 6 weeks. After 2 weeks of hypoxia, rats were treated with diltiazem (15 mg/kg/day). Right ventricular systolic pressure (RVSP) and right ventricular hypertrophy index (RVHI) were measured. Pathological changes in the lungs were observed under the light microscope and transmission electron microscope. The expression and distribution of heme oxygenase (HO) -1, endothelial NOS (eNOS) and inducible NOS (iNOS) were tested by immunohistochemistry and Western blot. Guanosine-3', 5'-cyclic monophosphate (cGMP) of lung tissues were detected with radioimmunoassay. RESULTS: Diltiazem significantly decreased abnormal RVSP, and RVHI in model rats, attenuated the SPA media thickeness, and recovered abnormal eNOS and iNOS expression in SPA. Whereas diltiazem had little effect on the increased HO-1 expression in SPA caused by hypoxia and ultrastructure injury in endothelium. cGMP levels were corresponded with HO-1. CONCLUSION: Diltiazem has a significant effect on inhibiting hypoxic pulmonary hypertension structural remodeling. These effects might be partly attributed to the suppression of iNOS, promotion of eNOS, and not attenuation HO-1 expression in the lung of hypoxic rats.     

Inmbition of nodosin on cell proliferation and expression of Bcl-2 and Bax in HepG2 cells

AIM:To investigate the effects of nodosin extracted from Chinese traditional medicine on the proliferation of HepG2 cells cultured in vitroand to detect the protein expression of Bcl-2 and Bax in HepG2 cells. METHODS:HepG2 cells were treated with different concentrations (1.25, 2.5, 5, 10 and 20 μmol/L) of nodosin for 24 h. The morphological changes of HepG2 cells were observed under inverted microscope. The inhibitory rates of HepG2 cell growth were detected by MTT assay. The apoptotic rates and the protein expression of Bcl-2 and Bax were analyzed by flow cytometry. RESULTS:Shrunken and suspended HepG2 cells increased with the increases in the concentrations of nodosin. The apoptotic rates and the expression of Bax increased with the increases in the doses of nodosin, while the expression of Bcl-2 decreased. CONCLUSION: Nodosin inhibits the growth of HepG2 cells in a dose-dependent manner. The inhibition of HepG2 cell growth is induced by decreasing Bcl-2 and increasing Bax, thus promoting cell apoptosis.     

Effect of hypoxia on invasion and migration of lung carcinoma cells and its molecular basis

AIM: To investigate the effect of hypoxia on the invasion and migration of lung carcinoma cells. METHODS: Lung carcinoma cell H128 was exposed to normoxia (air, 5% CO₂), hypoxia (5% O₂,5% CO₂,90% N₂) or anoxia (95% N₂,5% CO₂) conditions for 48 hours. The migration ability of the cells was assayed by wound healing methods. The invasiveness ability was determined with HABM-HEM model. The cells exposed to hypoxia were inoculated under skin in nude mice, and then the growth of the tumor and the rate of metastasis to lymph node or lung were observed. The expression of E-cadherin and β1-integrin on the cells were also assayed by flow cytometry. RESULTS: Compared with normoxic group, the invasiveness and migration in hypoxic group were increased. The rates of tumorgenesis and metastasis to lung in anoxic group were evidently decreased. The expression of E-cadherin was decreased, and the expression of β1-integrin was increased in hypoxic group. In anoxia group, the invasiveness, migration, the expression of E-cadherin and β1-integrin were all decreased. CONCLUSIONS: Moderate hypoxia down-regulated the expression of E-cadherin, up-regulated the expression of β1-integrin, and increased the invasiveness and metastasis of carcinoma cells. Serious hypoxia decreased the expression of adhesive molecules, and the proliferation, invasiveness and migration were also decreased.     

H IF-1αm ediates effect of interm ittent hypoxia on biological behavior of A 549 cells in vitro

ATM: To investigate whether hypoxia-inducible factor 1α (HIF-1α) mediates the effect of intermittent hypoxia on A549 cell viability, apoptosis and invasive ability METHODS: A549 cells were transfected with HIF-1α-siRNA and cultured under intermittent hypoxia. The expression of HIF-1α and its downstream genes, such as Bcl-2, Bax, P53, P21 and VEGF at mRNA and protein levels was determined by real-time PCR and Western blot. The viability of the A549 cells was measured by MTT assay. The apoptosis and cell cycle distribution of the A549 cells were examined by flow cytometry. The invasive ability of the A549 cells was detected by transwell test. RESULTS: The expression levels of HIF-1α, Bcl-2 and VEGF in non-HIF-1α-siRNA transfected A549 cells cultured in intermittent hypoxia environment[blank controlgroup(IH C),empty vector control group (IH E) and negative control group (IH N)] were higher than those in the A549 cells in normoxia group (RA), but the expression levels of Bax and P21 were lower than those in RA group (P<0.05). The siRNA-mediated HIF-1α gene silencing[intermittent hypoxia silenced group (IHS)] resulted in obvious down-regulation of HIF-1α, Bcl-2 and VEGF, and significant increase in the protein expression of P21 and Bax(P<0.05). The expression level of P53 in intermittent hypoxia groups was significantly higher than that in RA group, and no significant difference of P53 expression in different intermittent hypoxia groups was observed. Compared with normoxia, intermittent hypoxia resulted in significantly enhanced cell viability, decreased apoptosis, and enhanced invasive ability of non-HIF-1α-siRNA transfected A549 cells (P<0.05). The siRNA-mediated HIF-1α gene silencing resulted in significant cell viability inhibition, elevated apoptotic rate and decreased invasive ability under hypoxic condition (P<0.05).CONCLUSION: Intermittent hypoxia promotes the viability and invasion of A549 cells by HIF-1α-mediated downstream gene expression. HIF-1α gene silencing inhibits A549 cell growth and invasion under intermittent hypoxia by inhibition of HIF-1α signal pathways in vitro.     

Construction of human anti-HBc ScFv eukaryotic expression vector and expression of anti-HBc ScFv in HepG2 cells

AIM: To construct an eukaryotic expression vector of human single-chain variable fragment against hepatitis B virus core protein (anti-HBc ScFv) and detect its expression in HepG2 cells. METHODS: Anti-HBc ScFv genes were amplified from the plasmids abstracted from positive clone and inserted into pEGFP-c1 vector that contained green fluorescent protein gene. The recombinant plasmids were transfected into HepG2 cells, and resistant clones were obtained by G418 selection. The expression of the gene of fusion protein was determined by fluorescent invert microscope and ELISA. RESULTS: Recombinant plasmids were successfully constructed. The plasmid transfected HepG2 cells were obtained by G418 selection. Specific fluorescence was observed in HepG2 cells 48 hours after transfection. ELISA analysis confirmed the expression of anti-HBc ScFv in the cells. CONCLUSION: The construction of human anti-HBc ScFv eukaryotic expression vector and its expression in HepG2 cells lay the foundation for advanced research of intracellular anti-HBc ScFv.     

Response of mitochondrial reactive oxygen species in pulmonary arterial smooth muscle cells under acute hypoxic condition

AIM:To observe the response of mitochondrial reactive oxygen species (ROS) in rat pulmonary artery smooth muscle cells (PASMCs) under acute hypoxic condition. METHODS:The cultured PASMCs were under normoxic (35 ℃, 5% CO2, 21% O2, 74% N2) or acute hypoxic (35℃, 5% CO2, 1% O2, 94% N2) condition. The cells were incubated with molecular probes chloromethyl dichlorodihydrofluorescein diacetate (CM-H2DCF/DA) and RedoxSensor Red CC-1 to detect the ROS generation by laser scanning confocal microscopy. The mitochondria were isolated and mitochondrial inhibitors were used to detect the ROS generation functional unit sites by spectrophotometry under acute hypoxic condition. RESULTS:Under acute hypoxic condition, the intracellular ROS was significantly increased in hypoxia group with 3.35 folds higher of H2O2 than that in normoxia group. The contents of H2O2 and O-·2 in hypoxia group were 1.61 folds higher than those in normoxia group. Compare with hypoxia goup, pretreatment with the mitochondrial electron transport chain (ETC) complex I inhibitor MPP, the complex II inhibitors NPA and TTFA as well as the complex III pre-ubisemiquinone site inhibitor myxothiazol all remarkably reduced hypoxia-induced increase in ROS generation in PASMCs (reduced by 60%, 73%, 75% and 61%, respectively, P<0.01), whereas the complex III postubisemiquinone site inhibitor antimycin A and the complex IV inhibitor NaN3 had no effect on hypoxia-induced increase in ROS generation (increased by 13% and 9.1%, respectively, P>0.05). Direct detection of mitochondrial ROS showed the same results as the intracellular ROS. CONCLUSION: The intracellular ROS increases significantly in rat PASMCs under acute hypoxic condition. The mitochondrial ETC complex I, complex II and complex III pre-ubisemiquinone sites increase ROS generation, whereas the complex III postubisemiquinone site and complex IV do not produce this effect under acute hypoxic condition.     

Effects of EGLN1 siRNA on growth of rat pulmonary artery smooth muscle cells under hypoxic condition

AIM: To observe whether EGLN1 gene is involved in the growth of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EGLN1 gene expression was interference by siRNA. METHODS: The rat primary pulmonary arterial smooth muscle cells were cultured, and the specific lipidosome of EGLN1 siRNA was constructed and transfected into the PASMCs. The transfected PASMCs were cultured under hypoxia or normoxia conditions, respectively. The viability of the PASMCs was detected by CCK-8 assay. The protein expression of EGLN1 and vascular endothelial growth factor (VEGF) was determined by Western blot. RESULTS: The viability of the PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition in a time-dependent manner. In hypoxia or normoxia condition, the viability and VEGF protein expression of the PASMCs were suppressed by EGLN1 siRNA. CONCLUSION: EGLN1 gene may involve in the growth of rat PASMCs by regulating VEGF protein level under hypoxic condition.     

Effects of levcromakalim on pulmonary arterial endothelial cells and smooth muscle cells exposed to hypoxia

AIM:To explore the effects of levcromakalim(Lev) on pulmonary arterial endothelial cells (PAEC) and smooth muscle cells (PASMC) exposed to hypoxia and the mechanisms involved.METHODS:The effects of Lev on [Ca²⁺]_i, and expression of PKC_α, eNOS, iNOS and PDGF-B mRNA and protein levels were observed. The nitrite (NO₂^-) and entothelin-1(ET-1) concentrations in supernatant in cultured PAEC and PASMC were measured. The proliferation and apoptosis of PASMC was also detected.RESULTS:When PASMC were exposed to hypoxia, Lev reduced concentration of ET-1 in cultured cell supernatant, lowed the expression of PKC_α, iNOS and PDGF-B both at mRNA and protein levels, decreased [Ca²⁺]_i concentration, increased proliferation and promoted the apoptosis in PASMC. However, in the presence of Lev, the [Ca²⁺]_i concentration was not changed in the hypoxic PAEC. The NO₂^- concentration in cultured cell supernant and expression of eNOS at mRNA and protein levels in hypoxic PASMC and PAEC were also unchanged. The downregulated ET-1 activity in PASMC and PAEC and proliferation in PASMC involved in the inhibition of PKC_α signaling pathway.CONCLUSIONS:Lev reduce some disadvatage effect of hypoxia on PASMC and PAEC. The mechanism of Lev action may partly involve in the downregulation of PKC_α signaling functions.     

Role of α1 adrenoceptor in proliferation of hypoxic pulmonary artery smooth muscle cells

AIM: To investigate the role of α₁ and β₂ adrenoceptors(α₁AR and β₂AR) in the proliferation of hypoxic pulmonary artery smooth muscle cells (PASMCs).METHODS: PASMCs were isolated by an explant method from neonatal bovine pulmonary arteries. The cultured PASMCs were exposed to 6.6% O₂ for 6 h, 12 h and 24 h. The method of -TdR incorporation was used to measure the proliferation of PASMCs. _i was assayed with Fura-2/AM. The mRNA expression of α₁AR, β₂AR, c-fos and c-myc was determined by Northern blotting. The effects of activation of α₁AR and β₂AR, and inhibition of α₁AR on the above indexes were observed by treating PASMCs with different AR agonists and antagonists under hypoxic condition.RESULTS: Significant increase in TdR incorporation in hypoxic PASMCs with α₁AR activation was observed, and marked decrease in that was induced by α₁AR inhibition. However, no significant change was found after β₂AR activation. _i , the mRNA expression of c-fos, c-myc, α₁AR and β₂AR in PASMCs were increased after hypoxia.CONCLUSION: Hypoxia induces the increase in _i and mRNA expression of c-fos and c-myc, leading to the proliferation of PASMCs. The hypoxic proliferation of PASMCs is intervened by α₁AR, but not β₂AR. The remodeling of pulmonary arteriole and pulmonary hypertension may be involved in the processes of pulmonary arteriole constriction and proliferation induced by hypoxia through up-regulation of α₁AR.     

Effects of G6PD silencing by siRNA on expression of HIF-1α in human colon cancer cells

AIM: To study the effects of glucose-6-phosphate dehydrogenase (G6PD) silencing by small interference RNA(siRNA) on the levels of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) and hypoxia-inducible factor 1α(HIF-1α) under hypoxia in human colon cancer cell line LoVo.METHODS: Specific siRNA expression vector targeting G6PD gene was constructed. The recombinant plasmid was identified by restriction endonuclease and DNA sequencing, and then transfected into LoVo cells. The effects of G6PD silencing were evaluated by detecting the activity and mRNA expression of G6PD. LoVo cells were cultured in vitro under hypoxic condition. NADPH levels were determined.The mRNA and protein levels of HIF-1α were analyzed by RT-PCR and Western blotting,respectively. RESULTS: The recombinant plasmid for G6PD silencing by siRNA was successfully constructed and transfected into LoVo cells. Compared with untransfected cells,the mRNA expression of G6PD in transfected cells was decreased by 43% and G6PD activity was decreased by 63.5%. Under hypoxic condition, the level of NADPH in transfected cells was significantly decreased (41% vs 100%, P<0.05).HIF-1α protein was also decreased significantly but its mRNA expression had no change as compared with the control cells. CONCLUSION: G6PD silencing by siRNA decreases NADPH level, resulting in the decline of HIF-1α stability in cancer cells under hypoxic condition. By this mechanism, G6PD silencing can influence the hypoxic responses in cancer.