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.     

Effect of hypoxia-inducible factor-1α knockdown by siRNA on viability and CEACAM1 expression in oral squamous cell carcinoma

AIM: To investigate the relationship between hypoxia-inducible factor-1α (HIF-1α) and viability and apoptosis of oral squamous cell carcinoma and its mechanism. METHODS: The expression of HIF-1α and carcinoembryonic antigen-related cell adhesion molecular 1 (CEACAM1) at mRNA and protein levels in oral squamous cell carcinoma cell lines Tca8113 and CAL27 and normal epithelial cell line NOK was determined by RT-PCR and Western blot. The expression of HIF-1α in CAL27 cells was silenced by RNA interference (RNAi) technique. The cells were divided into blank control group, non-sense control group and siRNA-HIF-1α group. The viability of CAL27 cells was measured by MTT assay and the apoptotic rate was analyzed by flow cytometry. The protein levels of HIF-1α, P21, vascular endothelial growth factor (VEGF), Bcl-2 and Bax were examined by Western blot. RESULTS: The expression of HIF-1α and CEACAM1 in oral squamous cell carcinoma cells was significantly higher than that in normal cells (P<0.05), and the expression of HIF-1α and CEACAM1 was positively correlated. The protein expression of HIF-1α in siRNA-HIF-1α group was significantly lower than that in blank control group (P<0.05). Knockdown of HIF-1α significantly inhibited CAL27 cell viability (P<0.05), promoted apoptosis (P<0.05), increased the protein levels of P21 and Bax (P<0.05), and significantly decreased the levels of VEGF and Bcl-2 (P<0.05). CONCLUSION: HIF-1α is over-expressed in oral squamous cell carcinoma. Knockdown of HIF-1α significantly inhibits cell viability and promotes apoptosis possibly through regulating the expression of HIF-1α downstream target genes and tumor angiogenesis.     

Effects of HIF-1α silencing on proliferation of rat hepatoma cells

AIM：To study the effect of hypoxia-inducible factor 1α (HIF-1α) silencing on the proliferation of hepatoma cells under hypoxia. METHODS：Rat hepatoma cell line CBRH-7919 was used in this study. Hypoxia model was established by treating the cells with cobalt chloride (CoCl2). The expression of HIF-1α was silenced by small interfe-rence RNA. Real-time RT-PCR and Western blotting were used to detect the mRNA and/or protein expression of HIF-1α, vascular endothelial growth factor (VEGF), p21 and cyclin D1 in CBRH-7919 cells under hypoxia. The proliferation of CBRH-7919 cells was measured by the technique of 5-bromo-2’-deoxyuridine (BrdU) incorporation. RESULTS：The expression of HIF-1α and VEGF at mRNA and protein levels was significantly increased under hypoxia (P<0.05). Silencing of HIF-1α significantly inhibited the expression of HIF-1α, VEGF and cyclin D1 at mRNA and/or protein levels, while increased the protein expression of p21 (P<0.05). The BrdU-positive cells in HIF-1α siRNA transfection group were significantly less than those in control group. CONCLUSION：HIF-1α silencing significantly inhibits the proliferation of hepatoma cells under hypoxia.     

Role of HIF-1α/VEGF pathway in treatment of cerebral ischemia-reperfusion injury by hyperbaric oxygen pretreatment

AIM: To investigate the role of hypoxia-inducible factor-1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway in hyperbaric oxygen (HO) pretreatment in rats with cerebral ischemia-reperfusion (IR) injury. METHODS: Healthy male SD rats (n=32) were randomly divided into control group IR group, HO-IR group and HO-IR-HIF-1α inhibitor group (HO-IR-I group). The IR model was established by middle cerebral artery occlusion. The corresponding blood vessels of the rats in control group were only exposed. The rats in HO-IR group and HO-IR-I group were treated with HO for 4 weeks before the animal modeling. The rats in HO-IR-I group received 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazol (YC-1) by intraperitoneal injection at 4 mg/kg before HO preconditioning every day. At 1 d and 7 d after modeling, the neurological assessment was evaluated.At the end of the 7 th day, after observation, the rats were sacrificed by anesthesia to measure the infarct volume of the brain tissue. The protein levels of HIF-1α, VEGF and apoptosis-related proteins Bax and Bcl-2 were determined by Western blot. The number of apoptotic cells was detected by TUNEL. RESULTS: Compared with control group, the neurological function score was decreased, while the cerebral infarction volume ratio, the protein levels of HIF-1α, VEGF, Bcl-2 and Bax, and the apoptotic cells were increased in IR group, HO-IR group and HO-IR-I group (P<0.05). Compared with IR group, the neurological function score, and the protein levels of HIF-1α,VEGF and Bcl-2 were increased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were decreased in HO-IR group and HO-IR-I group (P<0.05). Compared with HO-IR group, the neurological function score, and the protein levels of HIF-1α, VEGF and Bcl-2 were decreased, while the cerebral infarction volume ratio, the protein level of Bax and apoptotic cells were increased in HO-IR-I group (P<0.05). CONCLUSION: The mechanism of HO preconditioning attenuating cerebral IR injury may be related to the regulation of apoptosis by inducing HIF-1α/VEGF signaling pathway activation.     

Effects of CARHSP1 gene expression on viability,apoptosis and immune factors of vascular endothe-lial cells induced by hypoxia in atherosclerosis

AIM: To investigate the effect of calcium-regulated heat stable protein 1 (CARHSP1) gene expression on the viability, apoptosis and expression of interleukin-6 (IL-6) and C-reactive protein (CRP) in vascular endothe-lial cells induced by hypoxia.METHODS: The protein expression of CARHSP1 was detected by Western blot in atherosclerotic plaques. Human umbilical vein endothelial cells (HUVECs) were treated with hypoxia, and the cells were divided into normal culture group, hypoxia group, hypoxia+CARHSP1-siRNA group and hypoxia+pcDNA3.1-CARHSP1 group. The viability and apoptotic rate of the HUVECs were measured by CCK-8 assay and flow cytometry, respectively. The mRNA expression of IL-6 and CRP was detected by RT-PCR. The protein levels of caspase-3, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot.RESULTS: The protein expression of CARHSP1 in atherosclerotic plaques was significantly higher than that in control group (P<0.05). Hypoxia significantly increased the expression of CARHSP1. The cell viability and the protein expression of Bcl-2 were significantly lower in hypoxia group than those in normal culture group (P<0.05). The apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were significantly higher than those in normal culture group (P<0.05). Compared with hypoxia group, the cell viability and protein expression of Bcl-2 were significantly increased in hypoxia+CARHSP1-siRNA group, while the apoptotic rate and the protein levels of IL-6, CRP, cleaved caspase-3 and Bax were decreased significantly (P<0.05). The cell viability and protein expression of Bcl-2 were decreased significantly in hypoxia+pcDNA3.1-CARHSP1 group, while the apoptotic rate and the protein le-vels of IL-6, CRP, cleaved caspase-3 and Bax were increased significantly (P<0.05).CONCLUSION: The expression of CARHSP1 is increased in atherosclerotic plaques, and inhibition of CARHSP1 expression improves the viability, reduces the apoptosis, and down-regulates the expression of IL-6 and CRP in the HUVECs. Over-expression of CARHSP1 exerts the opposite effect.     

Hypoxia-induced caveolin-1 up-regulation is involved in migration and invasion of lung adenocarcinoma A549 cells

AIM:To investigate the regulatory role of hypoxia mimic reagent cobalt chloride (CoCl2) on caveolin-1 (Cav-1) generation and the influence of Cav-1 on the abilities of migration and invasion of human lung adenocarcinoma A549 cells. METHODS:The concentrations of Cav-1 and hypoxia-inducible factor (HIF)-1α in pleural effusion of the patients with lung cancer (MPE) or tuberculous pleurisy (TBPE) were detected, and the correlation was also compared. A549 cells were treated with CoCl2 at different concentrations and time in the presence or absence of HIF-1α inhibitor YC-1. The concentrations of Cav-1 and HIF-1α in the cell supernatants were measured by ELISA. The effects of Cav-1 induced by CoCl2 on the migration and invasion of A549 cells were determined by scratch test and Transwell invasion trial, respectively. RESULTS:The levels of Cav-1 and HIF-1α in MPE were significantly higher than those in TBPE. There was a highly positive correlation between Cav-1 and HIF-1α levels in the pleural effusion. CoCl2 induced the generation of Cav-1 and HIF-1α in A549 cells in a concentration- and time-dependent manner, the peak occurred at 200 μmol/L or 24 h, while the concentration over 200 μmol/L or after treated over 24 h, a concentration- or time-dependent inhibition was observed. HIF-1α inhibitor YC-1 concentration-dependently inhibited the generation of HIF-1α and Cav-1 induced by CoCl2 in A549 cells. CoCl2 enhanced A549 cells migration and invasion, with 200 μmol/L played the strongest role, which were down-regulated significantly in the presence of YC-1. CONCLUSION: The alteration of hypoxia-induced Cav-1 generation might be involved in the migration and invasion of A549 cells. A possible role for HIF-1α is indicated in Cav-1 generation.     

Angiogenic effect of HIF-1α on extranodal nasal-type NK/T-cell lymphoma

AIM: To study the angiogenic effect of hypoxia inducible factor 1α(HIF-1α) and its significance on human extranodal nasal-type NK/T-cell lymphoma. METHODS: The protein levels of HIF-1α, vascular endothelial growth factor(VEGF) and VEGF receptor 2(VEGFR2) in human extranodal nasal-type NK/T-cell lymphoma were detected by immunohistochemistry. Microvessel density (MVD) of the tumor tissues was determined by labeling of microvessel endothelium with CD34 antibody. The correlation between the expression of HIF-1α, VEGF and VEGFR2 and MVD was analyzed with SPSS 13.0 statistical software. RESULTS: The positive expression of HIF-1α was observed in 39 cases (39/50, 78%) and the positive expression of VEGFR2 was 27 cases (27/50, 54%) of human extranodal nasal-type NK/T-cell lymphoma. A statistical difference of HIF-1α and VEGFR2 expression between tumor tissues and normal lymphocytes in lymph node was observed (P<0.05). In the tumor tissues, the co-expression of VEGF or VEGFR2 with HIF-1α was 72% and 64%, respectively, significantly higher than that without HIF-1α co-expression (P<0.05). The expression of HIF-1α, VEGFR and VEGFR2 was positively correlated with MVD of the tumor tissues (P<0.01). HIF-1α was expressed in all 15 cases of extranodal nasal-type NK/T-cell lymphoma with angiocentric infiltration.CONCLUSION: HIF-1α may promote angiogenesis of extranodal nasal-type NK/T-cell lymphoma through VEGF/VEGFR2 signaling pathway.     

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.     

Effect of bim silencing by siRNA on hypoxia-induced apoptosis of rat cardiomyocytes

AIM：To investigate the effect of BH3-only protein Bim (Bcl-2 interacting mediator of cell death) on apoptosis of rat cardiomyocytes induced by hypoxia. METHODS：Rat cardiomyocytes were isolated from infant rats aged 1~3 days and then primarily cultured. The antibody targeting α-actin of striated muscle was used to identify the cardiomyocytes. The siRNAs of bim were transfected into the cardiomyocytes with liposome, and the expression of Bim was determined by Western blotting. The cardiomyocytes were divided into blank control group, hypoxia group, hypoxia+liposome group, hypoxia+negative control siRNA group and hypoxia+bim-siRNA group.The frequency and rhythm of cardiomyocyte beating were observed and recorded under inverted microscope. The activity of lactate dehydrogenase (LDH) in the culture medium was assessed by automatic biochemical analyzer. The viability of the cells was analyzed by MTT assay. The cell apoptotic rate was measured by flow cytometry. The protein expression of Bim, Bax, Bcl-2, p-p38 MAPK and p38 MAPK was detected by Western blotting. RESULTS：Immunohistochemical identification confirmed that the rat cardiomyocytes were successfully cultured. The expression of Bim was obviously inhibited after transfected with bim-siRNAs and the silencing efficiency of bim-siRNA-2 was the highest (86.73%). The frequency of cardiomyocyte beating was slowed down after hypoxia and the rhythm was disordered, while the frequency of beating was obviously increased after silencting the expression of bim. Compared with control group, the LDH in the culture medium was increased (P<0.01), and the viability of the cardiomyocytes was reduced in hypoxia group (P<0.05). The apoptotic rate was increased (P<0.01). After transfection with bim-siRNA, the release of LDH was decreased, and the viability of the cardiomyocytes was increased. The apoptotic rate was decreased. The results of Western blotting showed that hypoxia increased the expression of Bax and p-p38 MAPK (P<0.05), and decreased the expression of Bcl-2 (P<0.01), while transfection with bim-siRNA reduced the effects caused by hypoxia (P<005). These were greatly related to the decrease of apoptosis. However, the expression of p38 MAPK was not changed. CONCLUSION：The apoptosis of cardiomyocytes induced by hypoxia can be inhibited by silencing the expression of bim gene by down-regulation of p-p38 MAPK and Bax expression and up-regulation of Bcl-2 expression.     

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.     

Influence of hypoxia-inducible factor-1 alpha on lung cancer cell A549 growth in vitro and in vivo

AIM: To study the role of hypoxia-inducible factor-1alpha(HIF-1α) on lung cancer cells A549 growth in vitro and in vivo. METHODS: To observe the growth rate of A549 cells after HIF-1α transfected, A549 cells (1×10⁶/mouse) were inoculated subcutaneously into 20 nude mice, which were randomly divided into two groups: the control group (group A, n=10), the HIF-1α transfected group(group B, n=10). The weights of subcutaneous tumor were detected. The resected specimens were made into paraffin-embedded sections. The proliferating cell nuclear antigen (PCNA) was identified by immunohistochemistry(ISH). The expressions of HIF-1α、 apoptosis-related protein survivin and bcl-2 were analyzed by Western blot. RESULTS: The growth rates of the HIF-1α transfected lung cancer cells A549 were significantly increased, and more importantly, the HIF-1α transfected lung cancer cells A549 was able to enhance lung cancer growth in nude mice(P＜0.05). The PCNA were increased significantly in group B, compared with group A. The expressions of HIF-1α, survivin and bcl-2 in group B were increased significantly than that of group A. CONCLUSIONS: HIF-1α increases lung cancer cells A549 growth in vitro and in vivo and its mechanism may be due to promotion of proliferation and inhibition of apoptosis.     

Expression of hypoxia-inducible factor 1α in human gingival tissues with chronic periodontitis

AIM：To observe the expression of hypoxia-inducible factor 1α (HIF-1α) in human gingival tissues with chronic periodontitis. METHODS：A total of 55 volunteers, including 15 healthy controls, 20 cases of moderate chronic periodontitis and 20 cases of severe chronic periodontitis, were involved in this study, and their gingival specimens were taken and fixed in 4% neutral formalin. The histological changes of gingival tissues were observed by HE staining, and the expression of HIF-1α in gingival tissues was detected by immunohistochemical staining. RESULTS： The proportion of HIF-1α positive cells in gingival tissues was significantly higher in chronic periodontitis groups than that in healthy control group (P<0.01), and that in severe chronic periodontitis group was significantly higher than that in moderate chronic periodontitis group (P<0.05). There was a significantly positive correlation between the severity of chronic periodontitis and the proportion of HIF-1α positive cells in gingival tissues. CONCLUSION：The expression of HIF-1α in human gingival tissues is increased with the severity of chronic periodontitis, suggesting that hypoxia may play an important role in chronic periodontitis.     

Tongxinluo improves anti-hypoxia ability of vascular endothelial cells via PI-3K/Akt/HIF pathway

AIM: To investigate the effects of PI-3K/Akt/HIF pathway on anti-hypoxia ability of vascular endothelial cells influenced by Tongxinluo under hypoxic condition. METHODS: Human umbilical vein endothelial cells (HUVECs) were divided into the following groups: control group, Tongxinluo (100 mg/L) group, hypoxia group and hypoxia+Tongxinluo (100 mg/L) group. The CCK-8 assay were used to detect the viability and proliferation rate of the cells in each group. The protein levels of HIF-1α, Bcl-2, Mcl-1, Bax and phosphorylated Akt were studied by immunoblotting analysis. The HUVECs were transiently transfected with the dominant negative mutant of HIF-1α (DN-HIF). The apoptotic rates were analyzed by flow cytometry (FCM). The HUVECs were transiently transfected with the dominant negative mutant of PI-3K (Δp85) or Akt (DN-Akt) to investigate the role of PI-3K/Akt signal pathway in the anti-hypoxia ability of Tongxinluo on endothelial cells. RESULTS: Under hypoxic condition, although the proliferation rate increased significantly in Tongxinluo group compared with hypoxia group, the degree was notably weak compared with control group. The protein levels of HIF-1α, Bcl-2, Mcl-1 and phosphorylated Akt were up-regulated by Tongxinluo. Meanwhile, the expression of Bax was down-regulated. Inhibition of HIF-1α activation by DN-HIF and inhibition of the PI-3K/Akt pathway by Δp85 or DN-Akt attenuated the increase in HIF-1α expression and HUVEC viability induced by Tongxinluo. The percentage of apoptotic HUVECs was down-regulated to a certain extent by Tongxinluo. CONCLUSION: Tongxinluo improves the anti-hypoxia ability of vascular endothelial cells by up-regulating the protein level of HIF-1α, promoting the expression of anti-apoptotic factors, improving the cell viability and eventually reducing the apoptotic rate.These effects of Tongxinluo depend on PI-3K/Akt signal pathway.     

Effect of down-regulation of X-box binding protein 1 gene expression on viability and apoptosis of glioma cells

AIM: To investigate the effect of down-regulation of X-box binding protein 1 (XBP1) expression on the viability and apoptosis of glioma cells. METHODS: The mRNA expression of XBP1 in the glioma tissues was detected by qPCR. Small interfering RNA (siRNA) interfering with XBP1 expression (XBP1-siRNA) was transfected into human brain glioma U251 cells. At the same time, control group (the cells without special treatment) and negative control (NC-siRNA) group (transfected with siRNA without any interference) were set up. The mRNA expression of XBP1 in the 3 groups 48 h after transfection was detected by qPCR. The protein levels of XBP1, proliferating cell nuclear antigen (PCNA), B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated X protein (Bax), cyclin D1 (cyclin D1), phosphatidylinositol 3-kinase (PI3K) and phosphorylated Akt (p-Akt) were determined by Western blot. The cell viability was measured by CCK-8 assay. The cell cycle distribution and apoptosis were analyzed by flow cytometry. RESULTS: The expression level of XBP1 in the glioma tissues was significantly higher than that in the tumor adjacent tissues (P<0.05). The XBP1 expression at mRNA and protein levels was significantly decreased in the cells transfected with XBP1-siRNA (P<0.05). No statistically significant difference of the cell viability, cell cycle, apoptotic rate and the protein levels of PCNA, Bcl-2, Bax, cyclin D1, PI3K and p-Akt between NC-siRNA group and control group was observed. Compared with control group, the cell viability, S-phase cells and the protein levels of PCNA, Bcl-2, cyclin D1, PI3K, and p-Akt in XBP1-siRNA group were decreased significantly, and the apoptotic rate, G₀/G₁-phase cells and Bax protein expression were significantly increased (P<0.05). CONCLUSION: Down-regulation of XBP1 gene expression in brain glioma cells reduces the viability of cancer cells, blocks the cells in G₁ phase and promote apoptosis. The mechanism is related to the inhibition of PI3K/Akt signaling pathway.     

Effects of several harmful factors on expression of glycine receptor α1 subunit in neonatal rat myocardial cells

AIM: To study the expression of glycine receptor α₁ subunit in neonatal rat myocardial cells and to investigate the effect of lipopolysaccharide (LPS), hypoxia/reoxygenation, isoproterenol (ISO) and high concentration of glucose (HG) on the expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells. METHODS: Neonatal rat myocardial cells were cultured in vitro. The expression of glycine receptor α₁ subunit was detected by Western blotting. The neonatal rat myocardial cells were treated with LPS (20 mg/L), ISO (100μmol/L) or high concentration of glucose (25 mmol/L) for 24 h, or were exposed to hypoxia for 3 h followed by reoxygenation for 3 h. Subsequently, the cell viability was measured by CCK-8 assay, and the expression of glycine receptor α₁ subunit was determined by Western blotting. RESULTS: The expression of glycine receptor α₁ subunit in the neonatal rat myocardial cells was positively detectable by Western blotting. Compared with control group, no significant difference of the cell viability (P>0.05) in LPS group, ISO group, hypoxia/reoxygenation group and HG group was observed. The expression of glycine receptor α₁ subunit was increased (P<0.01) in LPS group, ISO group and hypoxia/reoxygenatio group, but decreased (P<0.01) in HG group. CONCLUSION: Glycine receptor α₁ subunit exists in the neonatal rat myocardial cells. A certain concentration of LPS or ISO, or hypoxia/reoxygenation for a certain period upregulate the expression of glycine receptor α₁ subunit, but HG downregulates the expression of glycine receptor α₁ subunit in cultured neonatal rat myocardial cells.     

Relationship between peritubular capillary injury and renal interstitial fibrosis in mice with unilateral ureteral obstruction

AIM: To observe the injury of peritubular capillary (PTC), hypoxia and interstitial fibrosis after unilateral ureteral obstruction (UUO), and to explore the effects of PTC injury and hypoxia on interstitial fibrosis in mouse model of UUO. METHODS: Forty-eight male KM mice were randomly divided into control group and UUO group. On the 1st, 3rd, 7th and 14th days, 6 mice in each group were sacrificed. The changes of pathomorphism in the kidney were observed by HE and Masson staining. The expression levels of thrombospondin-1 (TSP-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α), and PTC density were detected by immunohistochemistry. The protein expression of VEGF was also determined by Western blotting. RESULTS: No histological abnormalities of the kidneys were observed in sham-operated mice. The expression of TSP-1 was increased 1 day after UUO, and significantly increased on the 3rd, 7th and 14th days(P<0.05). The expression of VEGF was obviously decreased(P<0.05). PTC density was gradually decreased. The expression of HIF-1α was gradually increased, and renal interstitial area was gradually expanded. PTC density was negatively correlated with the expression of TSP-1 and HIF-1α (r=-0.874 and r=-0.930, respectively). VEGF expression was positively correlated with PTC density (r=0.745). PTC density was negatively correlated with the area of renal fibrosis (r=-0.787). HIF-1α expression was positively correlated with the area of renal fibrosis (r=0.835, P<0.05). CONCLUSION: In mouse UUO model, the expression of TSP-1 is increased. The expression of VEGF is reduced. The peritubular capillary injury and tissue hypoxia are aggravated, and renal interstitial fibrosis area is expanded. Ischemia and hypoxia may play an important role in the progression of UUO.     

Regulatory effects of DIS3 expression on colony formation ability in human myeloma cells and tube structure formation of endothelial cells

AIM:To investigate the effects of DIS3 expression on the colony formation ability of 3 kinds of human myeloma cells and tube structure formation of the endothelial cells. METHODS:Human myeloma cell lines NCI-H929, RPMI-8226 and U266 were selected as the study objects, and DIS3 gene over-expression vector and DIS3-siRNA were designed and constructed respectively. The cell experiments were divided into 5 groups:control group, siRNA negative control (siRNA-NC) group, siRNA-DIS3 group, empty vector group and DIS3 over-expression group. The colony formation ability was tested by the plate colony formation assay. Western blot was used to detect the protein expression of hypoxia inducible factor-1α (HIF-1α) and HIF-3α. The expression of angiogenesis-related molecules angiogenin 1 (Ang1), Ang2 and vascubar enelothelial growth factor-A(VEGF-A) at the mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. Matrigel method was used to detect the effect of supernatant from each group of the cells on the tube structure formation of HUVECs. RESULTS:The trends of the following indexes in NCI-H929 cells, RPMI-8226 cells and U266 cells were similar. Compared with empty vector group, the colony formation ability of the cells in DIS3 over-expression group was significantly inhibited (P<0.05). Compared with siRNA-NC group, siRNA-DIS3 significantly enhanced the colony formation ability of the cells (P<0.05). DIS3 over-expression significantly reduced the expression of HIF-1α and HIF-3α (P<0.05), while knock-down of DIS3 expression significantly increased the protein levels of HIF-1α and HIF-3α (P<0.05). In addition, DIS3 over-expression significantly reduced the expression of Ang1, Ang2 and VEGF-A at mRNA and protein levels (P<0.05), while siRNA-DIS3 significantly promoted the expression of Ang1, Ang2 and VEGF-A (P<0.05). Compared with empty vector group, the supernatant from DIS3 over-expression group significantly inhibited the tube structure formation of HUVECs (P<0.01). Compared with siRNA-NC group, the supernatant from siRNA-DIS3 group significantly promoted the tube structure formation of HUVECs (P<0.05). CONCLUSION:DIS3 over-expression significantly inhibits the colony formation ability of human myeloma cells and tube structure formation of HUVECs, which may be closely related to the regulation of the expression of HIF-1α and HIF-3α.