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1.
Journal of Zhejiang University. Science. B ; (12): 50-63, 2023.
Article in English | WPRIM | ID: wpr-971468

ABSTRACT

Accumulating evidence has confirmed the links between transfer RNA (tRNA) modifications and tumor progression. The present study is the first to explore the role of tRNA methyltransferase 5 (TRMT5), which catalyzes the m1G37 modification of mitochondrial tRNAs in hepatocellular carcinoma (HCC) progression. Here, based on bioinformatics and clinical analyses, we identified that TRMT5 expression was upregulated in HCC, which correlated with poor prognosis. Silencing TRMT5 attenuated HCC proliferation and metastasis both in vivo and in vitro, which may be partially explained by declined extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). Mechanistically, we discovered that knockdown of TRMT5 inactivated the hypoxia-inducible factor-1 (HIF-1) signaling pathway by preventing HIF-1α stability through the enhancement of cellular oxygen content. Moreover, our data indicated that inhibition of TRMT5 sensitized HCC to doxorubicin by adjusting HIF-‍1α. In conclusion, our study revealed that targeting TRMT5 could inhibit HCC progression and increase the susceptibility of tumor cells to chemotherapy drugs. Thus, TRMT5 might be a carcinogenesis candidate gene that could serve as a potential target for HCC therapy.


Subject(s)
Humans , Carcinoma, Hepatocellular/pathology , Cell Hypoxia , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Liver Neoplasms/pathology , Signal Transduction/genetics , tRNA Methyltransferases/metabolism
2.
Journal of Zhejiang University. Science. B ; (12): 32-49, 2023.
Article in English | WPRIM | ID: wpr-971467

ABSTRACT

Hypoxia, as an important hallmark of the tumor microenvironment, is a major cause of oxidative stress and plays a central role in various malignant tumors, including glioblastoma. Elevated reactive oxygen species (ROS) in a hypoxic microenvironment promote glioblastoma progression; however, the underlying mechanism has not been clarified. Herein, we found that hypoxia promoted ROS production, and the proliferation, migration, and invasion of glioblastoma cells, while this promotion was restrained by ROS scavengers N-acetyl-L-cysteine (NAC) and diphenyleneiodonium chloride (DPI). Hypoxia-induced ROS activated hypoxia-inducible factor-1α (HIF-1α) signaling, which enhanced cell migration and invasion by epithelial-mesenchymal transition (EMT). Furthermore, the induction of serine protease inhibitor family E member 1 (SERPINE1) was ROS-dependent under hypoxia, and HIF-1α mediated SERPINE1 increase induced by ROS via binding to the SERPINE1 promoter region, thereby facilitating glioblastoma migration and invasion. Taken together, our data revealed that hypoxia-induced ROS reinforce the hypoxic adaptation of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway, and that targeting ROS may be a promising therapeutic strategy for glioblastoma.


Subject(s)
Humans , Cell Hypoxia , Cell Line, Tumor , Glioblastoma/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Plasminogen Activator Inhibitor 1/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Tumor Microenvironment , Brain Neoplasms/pathology
3.
Journal of Southern Medical University ; (12): 807-814, 2023.
Article in Chinese | WPRIM | ID: wpr-986992

ABSTRACT

OBJECTIVE@#To investigate the regulatory role of the long non-coding RNA LINC00926 in pyroptosis of hypoxia-induced human umbilical vein vascular endothelial cells (HUVECs) and explore the molecular mechanism.@*METHODS@#HUVECs were transfected with a LINC00926-overexpressing plasmid (OE-LINC00926), a siRNA targeting ELAVL1, or both, followed by exposure to hypoxia (5% O2) or normoxia. The expression of LINC00926 and ELAVL1 in hypoxia-treated HUVECs was detected using real-time quantitative PCR (RT-qPCR) and Western blotting. Cell proliferation was detected using Cell Counting Kit-8 (CCK-8), and the levels of IL-1β in the cell cultures was determined with ELISA. The protein expression levels of pyroptosis-related proteins (caspase-1, cleaved caspase-1 and NLRP3) in the treated cells were analyzed using Western blotting, and the binding between LINC00926 and ELAVL1 was verified with RNA immunoprecipitation (RIP) assay.@*RESULTS@#Exposure to hypoxia obviously up-regulated the mRNA expression of LINC00926 and the protein expression of ELAVL1 in HUVECs, but did not affect the mRNA expression of ELAVL1. LINC00926 overexpression in the cells significantly inhibited cell proliferation, increased IL-1β level and enhanced the expressions of pyroptosis-related proteins (all P < 0.05). LINC00926 overexpression further up-regulated the protein expression of ELAVL1 in hypoxia-exposed HUVECs. The results of RIP assay confirmed the binding between LINC00926 and ELAVL1. ELAVL1 knockdown significantly decreased IL-1β level and the expressions of pyroptosis-related proteins in hypoxia-exposed HUVECs (P < 0.05), while LINC00926 overexpression partially reversed the effects of ELAVL1 knockdown.@*CONCLUSION@#LINC00926 promotes pyroptosis of hypoxia-induced HUVECs by recruiting ELAVL1.


Subject(s)
Humans , Caspase 1 , ELAV-Like Protein 1 , Human Umbilical Vein Endothelial Cells , Pyroptosis , RNA, Messenger , RNA, Long Noncoding/genetics , Cell Hypoxia
4.
Chinese Critical Care Medicine ; (12): 627-632, 2023.
Article in Chinese | WPRIM | ID: wpr-982644

ABSTRACT

OBJECTIVE@#To investigate the effects of tanshinone IIA on apoptosis and autophagy induced by hypoxia/reoxygenation in H9C2 cardiomyocytes and its mechanism.@*METHODS@#H9C2 cardiomyocytes in logarithmic growth phase were divided into control group, hypoxia/reoxygenation model group and tanshinone IIA low-dose, medium-dose and high-dose groups (50, 100, 200 mg/L tanshinone IIA were treated after hypoxia/reoxygenation respectively). The dose with good therapeutic effect was selected for follow-up study. The cells were divided into control group, hypoxia/reoxygenation model group, tanshinone IIA+pcDNA3.1-NC group and tanshinone IIA+pcDNA3.1-ABCE1 group. The cells were transfected with the overexpressed plasmids pcDNA3.1-ABCE1 and pcDNA3.1-NC and then treated accordingly. Cell counting kit-8 (CCK-8) was used to detect H9C2 cell activity in each group. The apoptosis rate of cardiomyocytes was detected by flow cytometry. The ATP-binding cassette transporter E1 (ABCE1), apoptosis-related proteins Bcl-2 and Bax, caspase-3, autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3II/I) and p62 mRNA expression level of H9C2 cells in each group were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The protein expression levels of the above indexes in H9C2 cells were detected by Western blotting.@*RESULTS@#(1) Cell activity and ABCE1 expression: tanshinone IIA inhibited the activity of H9C2 cells induced by hypoxia/reoxygenation, and the effect was significant at medium-dose [(0.95±0.05)% vs. (0.37±0.10)%, P < 0.01], mRNA and protein expression of ABCE1 were significantly reduced [ABCE1 mRNA (2-ΔΔCt): 2.02±0.13 vs. 3.74±0.17, ABCE1 protein (ABCE1/GAPDH): 0.46±0.04 vs. 0.68±0.07, both P < 0.05]. (2) Expression of apoptosis-related proteins: medium-dose of tanshinone IIA inhibited the apoptosis of H9C2 cells induced by hypoxia/reoxygenation [apoptosis rate: (28.26±2.52)% vs. (45.27±3.07)%, P < 0.05]. Compared with the hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated the protein expression of Bax and caspase-3 in H9C2 cells induced by hypoxia/reoxygenation, and significantly up-regulated the protein expression of Bcl-2 [Bax (Bax/GAPDH): 0.28±0.03 vs. 0.47±0.03, caspase-3 (caspase-3/GAPDH): 0.31±0.02 vs. 0.44±0.03, Bcl-2 (Bcl-2/GAPDH): 0.53±0.02 vs. 0.37±0.05, all P < 0.05]. (3) Expression of autophagy-related proteins: compared with the control group, the positive rate of LC3 in the hypoxia/reoxygenation model group was significantly increased, while the positive rate of LC3 in the medium-dose of tanshinone IIA group was significantly decreased [(20.67±3.09)% vs. (42.67±3.86)%, P < 0.01]. Compared with hypoxia/reoxygenation model group, medium-dose of tanshinone IIA significantly down-regulated Beclin-1, LC3II/I and p62 protein expressions [Beclin-1 (Beclin-1/GAPDH): 0.27±0.05 vs. 0.47±0.03, LC3II/I ratio: 0.24±0.05 vs. 0.47±0.04, p62 (p62/GAPDH): 0.21±0.03 vs. 0.48±0.02, all P < 0.05]. (4) Expression of apoptosis and autophagy related proteins after transfection with overexpressed ABCE1 plasmid: compared with tanshinone IIA+pcDNA3.1-NC group, the protein expression levels of Bax, caspase-3, Beclin-1, LC3II/I and p62 in tanshinone IIA+pcDNA3.1-ABCE1 group were significantly up-regulated, while the protein expression level of Bcl-2 was significantly down-regulated.@*CONCLUSIONS@#100 mg/L tanshinone IIA could inhibit autophagy and apoptosis of cardiomyocytes by regulating the expression level of ABCE1. So, it protects H9C2 cardiomyocytes injury induced by hypoxia/reoxygenation.


Subject(s)
Humans , Apoptosis , ATP-Binding Cassette Transporters/metabolism , Autophagy , bcl-2-Associated X Protein/metabolism , Beclin-1/metabolism , Caspase 3/metabolism , Follow-Up Studies , Myocytes, Cardiac , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/metabolism , Cell Hypoxia
5.
Journal of Zhejiang University. Medical sciences ; (6): 750-757, 2023.
Article in English | WPRIM | ID: wpr-971092

ABSTRACT

Phenotypic transformation of pulmonary artery smooth muscle cells (PASMCs) is a key factor in pulmonary vascular remodeling. Inhibiting or reversing phenotypic transformation can inhibit pulmonary vascular remodeling and control the progression of hypoxic pulmonary hypertension. Recent studies have shown that hypoxia causes intracellular peroxide metabolism to induce oxidative stress, induces multi-pathway signal transduction, including those related to autophagy, endoplasmic reticulum stress and mitochondrial dysfunction, and also induces non-coding RNA regulation of cell marker protein expression, resulting in PASMCs phenotypic transformation. This article reviews recent research progress on mechanisms of hypoxia-induced phenotypic transformation of PASMCs, which may be helpful for finding targets to inhibit phenotypic transformation and to improve pulmonary vascular remodeling diseases such as hypoxia-induced pulmonary hypertension.


Subject(s)
Humans , Pulmonary Artery , Hypertension, Pulmonary , Vascular Remodeling/genetics , Hypoxia/genetics , Myocytes, Smooth Muscle , Cell Proliferation/physiology , Cells, Cultured , Cell Hypoxia/genetics
6.
Journal of Southern Medical University ; (12): 354-359, 2022.
Article in Chinese | WPRIM | ID: wpr-936323

ABSTRACT

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.


Subject(s)
Animals , Rats , Cell Differentiation/drug effects , Cell Hypoxia/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Leptin/pharmacology , PTEN Phosphohydrolase/metabolism , Rats, Sprague-Dawley , Retina/metabolism , Stem Cells/metabolism , Tubulin
7.
Journal of Zhejiang University. Science. B ; (12): 204-217, 2022.
Article in English | WPRIM | ID: wpr-929052

ABSTRACT

It has been revealed that hypoxia is dynamic in hypertrophic scars; therefore, we considered that it may have different effects on hypoxia-inducible factor-1α (HIF-1α) and p53 expression. Herein, we aimed to confirm the presence of a teeterboard-like conversion between HIF-1α and p53, which is correlated with scar formation and regression. Thus, we obtained samples of normal skin and hypertrophic scars to identify the differences in HIF-1α and autophagy using immunohistochemistry and transmission electron microscopy. In addition, we used moderate hypoxia in vitro to simulate the proliferative scar, and silenced HIF-1α or p53 gene expression or triggered overexpression to investigate the changes of HIF-1α and p53 expression, autophagy, apoptosis, and cell proliferation under this condition. HIF-1α, p53, and autophagy-related proteins were assayed using western blotting and immunofluorescence, whereas apoptosis was detected using flow cytometry analysis, and cell proliferation was detected using cell counting kit-8 (CCK-8) and 5-bromo-2'-deoxyuridine (BrdU) staining. Furthermore, immunoprecipitation was performed to verify the binding of HIF-1α and p53 to transcription cofactor p300. Our results demonstrated that, in scar tissue, HIF-1α expression increased in parallel with autophagosome formation. Under hypoxia, HIF-1α expression and autophagy were upregulated, whereas p53 expression and apoptosis were downregulated in vitro. HIF-1α knockdown downregulated autophagy, proliferation, and p300-bound HIF-1α, and upregulated p53 expression, apoptosis, and p300-bound p53. Meanwhile, p53 knockdown induced the opposite effects and enhanced HIF-1α, whereas p53 overexpression resulted in the same effects and reduced HIF-1α. Our results suggest a teeterboard-like conversion between HIF-1α and p53, which is linked with scar hyperplasia and regression.


Subject(s)
Humans , Apoptosis , Autophagy , Cell Hypoxia , Fibroblasts/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Tumor Suppressor Protein p53/metabolism
8.
Journal of Experimental Hematology ; (6): 386-392, 2022.
Article in Chinese | WPRIM | ID: wpr-928725

ABSTRACT

OBJECTIVE@#To explore the effect of hypoxia on the chemosensitivity of B-acute lymphoblastic leukemia (B-ALL) cells to Vincristine (VCR) and the mechanisms.@*METHODS@#B-ALL cells SUP-B15, Nalm-6 and RS4;11 were selected as the research objects. The cells were divided into the control group and the hypoxia mimic group (CoCl2 pretreatment). The two groups were treated with VCR at different concentrations for 24 hours, CCK-8 was used to detect cell viability, flow cytometry was used to detect cell apoptosis, and Western bolt method was used to detect hypoxia inducible factor (HIF-1α), BAX, Bcl-2 and β-actin protein expression. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect BAX and β-actin mRNA levels.@*RESULTS@#CoCl2 could simulate hypoxic environment to induce the expression of HIF-1α. The cells SUP-B15 and RS4;11 of the hypoxia mimic group were lower sensitivity to VCR as compared with the control group; the apoptosis rate of the hypoxia mimic group was lower than that of the control group after 80 nmol/L VCR treatment. The expression levels of BAX protein and mRNA in the hypoxia mimic group were lower than those of the control group, and there was no significant difference in the expression levels of Bcl-2 protein between two groups.@*CONCLUSION@#Under hypoxic conditions, HIF-1α may mediate VCR resistance in B-ALL cells by downregulating the pro-apoptotic protein BAX.


Subject(s)
Humans , Actins/pharmacology , Apoptosis , Cell Hypoxia , Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit , Proto-Oncogene Proteins c-bcl-2 , RNA, Messenger , Vincristine/pharmacology , bcl-2-Associated X Protein/pharmacology
9.
Journal of Central South University(Medical Sciences) ; (12): 1629-1636, 2022.
Article in English | WPRIM | ID: wpr-971345

ABSTRACT

OBJECTIVES@#Fluorouracil chemotherapeutic drugs are the classic treatment drugs of gastric cancer. But the problem of drug resistance severely limits their clinical application. This study aims to investigate whether hypoxia microenvironment affects gastric cancer resistance to 5-fluorouracil (5-FU) and discuss the changes of gene and proteins directly related to drug resistance under hypoxia condition.@*METHODS@#Gastric cancer cells were treated with 5-FU in hypoxia/normoxic environment, and were divided into a Normoxic+5-FU group and a Hypoxia+5-FU group. The apoptosis assay was conducted by flow cytometry Annexin V/PI double staining. The real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the expression level of hypoxia inducible factor-1α (HIF-1α), multidrug resistance (MDR1) gene, P-glycoprotein (P-gp), and vascular endothelial growth factor (VEGF) which were related to 5-FU drug-resistance. We analyzed the effect of hypoxia on the treatment of gastric cancer with 5-FU.@*RESULTS@#Compared with the Normoxic+5-FU group, the apoptosis of gastric cancer cells treated with 5-FU in the Hypoxia+5-FU group was significantly reduced (P<0.05), and the expression of apoptosis promoter protein caspase 8 was also decreased. Compared with the the Normoxic+5-FU group, HIF-1α mRNA expression in the Hypoxia+5-FU group was significantly increased (P<0.05), and the mRNA and protein expression levels of MDR1, P-gp and VEGF were also significantly increased (all P<0.05). The increased expression of MDR1, P-gp and VEGF had the same trend with the expression of HIF-1α.@*CONCLUSIONS@#Hypoxia is a direct influencing factor in gastric cancer resistance to 5-FU chemotherapy. Improvement of the local hypoxia microenvironment of gastric cancer may be a new idea for overcoming the resistance to 5-FU in gastric cancer.


Subject(s)
Humans , Fluorouracil/therapeutic use , Vascular Endothelial Growth Factor A/metabolism , Stomach Neoplasms/drug therapy , Drug Resistance, Multiple , Vascular Endothelial Growth Factors/metabolism , Hypoxia , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Cell Line, Tumor , Cell Hypoxia , RNA, Messenger/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Tumor Microenvironment
10.
Chinese Journal of Applied Physiology ; (6): 68-74, 2022.
Article in Chinese | WPRIM | ID: wpr-927900

ABSTRACT

Objective: To investigate the mechanism that hypoxia promotes the migration of lung adenocarcinoma A549 cells. Methods: A549 cells were cultured and cells that knockdown of acetyl-CoA carboxylase 1 (ACC1) were obtained by transfection with lentivirus, and cells that knockdown of sterol regulatory element-binding proteins-1 (SREBP-1) were obtained by treated with si-RNA. A549 cells were treated with hypoxia combined with hypoxia inducible factor-1α (HIF-1α) inhibitor PX-478 (25 μmol); Hypoxia combined with linoleic acid (LA) (20 μmol) treated A549 cells with ACC1 knockdown, and A549 cells with SREBP-1 knockdown were treated by hypoxia. Transwell migration assay was used to detect cell migration. Western blot was conducted to detect HIF-1α, ACC1 and epithelial mesenchymal transition (EMT) related proteins, Vimentin, E-Cadherin and SREBP-1; Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was performed to detect the changes of ACC1 and SREBP-1 mRNA in A549 cells after hypoxia and HIF-1α inhibitor PX-478 (25 μmol) treatment. Each experiment was repeated three times. Results: Compared with the normoxic control group, hypoxia promoted the migration of A549 cells (P<0.01), and up-regulated the expressions of ACC1, HIF-1α (all P<0.01) and SREBP-1 (P<0.05). PX-478 (25 μmol) inhibited the migration of A549 cells induced by hypoxia and down-regulated the expression of SREBP-1 (all P<0.05). ACC1 mRNA and SREBP-1 mRNA levels were increased after hypoxia treatment of A549 cells (all P<0.05). The levels of ACC1 mRNA and SREBP-1 mRNA were decreased after A549 cells treated with hypoxia combined with PX-478 (25 μmol) for 24 h (P<0.05, P<0.01). Knockdown of SREBP-1 in A549 cells was obtained by transfection with si-RNA. Transwell migration assay showed the number of cell migration in si-SREBP-1 group was less than that in normoxia control group (P<0.01). The si-SREBP-1 group and the si-NC group were treated with hypoxia. Compared with the control group, the number of cell migration in the si-SREBP-1 group was decreased (P<0.01), however, the difference was not statistically significant compared with the normoxia si-SREBP-1 group (P>0.05). Western blot showed that the expression of ACC1 in the si-SREBP-1 group was lower than that in the control group (P<0.01). Compared with the control group, the expression of ACC1 was decreased after si-SREBP-1 group treated with hypoxia (P<0.01). Knockdown of ACC1 inhibited the migration of A549 cells (P<0.05). After knockdown of ACC1, the migration number of A549 cells under normoxia and 5% O2 conditions had no significant difference (P>0.05). Application of LA under hypoxia condition rescued ACC1-knockdown induced inhibitory effect on hypoxia-promoted A549 cell migration (P<0.05). Conclusion: Hypoxia promotes migration of lung adenocarcinoma A549 cells by regulating fatty acid metabolism through HIF-1α/SREBP-1/ACC1 pathway.


Subject(s)
Humans , A549 Cells , Acetyl-CoA Carboxylase , Adenocarcinoma of Lung , Cell Hypoxia/physiology , Cell Line, Tumor , Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit , Lung Neoplasms , RNA/metabolism , RNA, Messenger/metabolism , Sterol Regulatory Element Binding Protein 1/metabolism
11.
Acta Physiologica Sinica ; (6): 26-34, 2021.
Article in Chinese | WPRIM | ID: wpr-878232

ABSTRACT

Intermittent hypoxia (IH) could induce cognitive impairment through oxidative stress and inflammation. However, the degree of cell damage is closely related to the IH stimulus frequency. IH stimulation with different frequencies also induces opposite results on neuronal cell lines. Therefore, this study was aimed to compare the effects of IH stimulation with three different frequencies on murine hippocampal neuronal HT22 cell activity, and to explore the molecular mechanism of the IH stimulus frequency-related neuron injury. HT22 cells were cultured and divided into control group and three IH stimulation groups with different frequencies. Oxygen concentration in the chamber was circulated between 21% and 1% (IH1 group, 6 cycles/h; IH2 group, 2 cycles/h; IH3 group, 0.6 cycle/h). Cell morphology was observed at 6, 12, 24 and 48 h of IH treatment. Cell viability was determined by the CCK-8 kit, lactate dehydrogenase (LDH) content in cell supernatant was determined by LDH kit, oxidative stress level was detected by the reactive oxygen species (ROS) probe, and protein expression levels of hypoxia inducible factor-1α (Hif-1α) and phosphorylated nuclear factor κB (p-NF-κB) were detected by Western blot. The results showed that, compared with control group, cell number and activity in the three IH groups were decreased, LDH content and ROS levels were increased with the prolongation of IH stimulation time, and the changes were most obvious in the IH1 group among those of the three IH groups. Hif-1α expression and the p-NF-κB/NF-κB ratio were also up-regulated with the prolongation of IH stimulation time, and the changes of IH1 group were the most significant. These results suggest that IH stimulation induces oxidative stress injury in HT22 cells, which is related to increased Hif-1α expression and NF-κB phosphorylation. Moreover, the higher frequency of IH stimulation induces more serious cell injury.


Subject(s)
Animals , Mice , Cell Hypoxia , Cell Survival , Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , NF-kappa B/metabolism , Oxidative Stress , Reactive Oxygen Species
12.
China Journal of Chinese Materia Medica ; (24): 3257-3269, 2021.
Article in Chinese | WPRIM | ID: wpr-887975

ABSTRACT

Cardiovascular diseases seriously endanger human health and life. The accompanying myocardial injury has been a focus of attention in society. Chinese medicine,serving as a natural and precious reservoir for the research and development of new drugs,is advantageous in resisting myocardial injury due to its multi-component,multi-pathway,and multi-target characteristics. In recent years,with the extensive application of culture method for isolated cardiomyocytes,a cost-effective,controllable in vitro model of cardiomyocyte injury with uniform samples is becoming a key tool for mechanism research on cardiomyocyte injury and drug development.A good in vitro model can reduce experimental and manpower cost,and also accurately stimulate clinical changes to reveal the mechanism. Therefore,the selection and establishment of in vitro model are crucial for the in-depth research. This study summarized the modeling principles,evaluation indicators,and application of more than ten models reflecting different clinical conditions,such as injuries induced by hypoxia-reoxygenation,hypertrophy,oxidative stress,inflammation,internal environmental disturbance,and toxicity. Furthermore,we analyzed advantages and technical difficulties,aiming to provide a reference for in-depth research on myocardial injury mechanism and drug development.


Subject(s)
Humans , Apoptosis , Cell Hypoxia , Myocardium , Myocytes, Cardiac , Oxidative Stress
13.
Journal of Zhejiang University. Medical sciences ; (6): 614-620, 2021.
Article in English | WPRIM | ID: wpr-922252

ABSTRACT

To construct a hypobaric hypoxia-induced cell injury model. Rat pheochromocytoma PC12 cells were randomly divided into control group, normobaric hypoxia group and hypobaric hypoxia group. The cells in control group were cultured at normal condition, while cells in other two groups were cultured in normobaric hypoxia and hypobaric hypoxia conditions, respectively. CCK-8 method was used to detect cell viability to determine the optimal modeling conditions like the oxygen concentration, atmospheric pressure and low-pressure hypoxia time. The contents of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by microplate method. The apoptosis ratio and cell cycle were analyzed by flow cytometry. The hypobaric hypoxia-induced cell injury model can be established by culturing for 24 h at 1% oxygen concentration and 41 kPa atmospheric pressure. Compared with the control group and normobaric hypoxia group, the activity of LDH and the content of MDA in hypobaric hypoxia group were significantly increased, the activity of SOD was decreased, the percentage of apoptosis was increased (all <0.05), and the cell cycle was arrested in G0/G1 phase. A stable and reliable cell injury model induced by hypobaric hypoxia has been established with PC12 cells, which provides a suitable cell model for the experimental study on nerve injury induced by hypoxia at high altitude.


Subject(s)
Animals , Rats , Cell Hypoxia , Hypoxia , Malondialdehyde , PC12 Cells , Superoxide Dismutase/metabolism
14.
Bol. méd. Hosp. Infant. Méx ; 77(4): 186-194, Jul.-Aug. 2020. graf
Article in English | LILACS | ID: biblio-1131975

ABSTRACT

Abstract Background: Acute lymphoblastic leukemia (ALL) is an aggressive malignant disease with high prevalence in pediatric patients. It has been shown that the downregulation of Fas expression is correlated with an inadequate response in ALL, although these mechanisms are still not well understood. Several reports demonstrated that hypoxia is involved in dysfunctional apoptosis. Yin-Yang-1 (YY1) transcription factor is involved in resistance to apoptosis, tumor progression, and it is increased in different types of cancer, including leukemia. The regulatory mechanism underlying YY1 expression in leukemia is still not understood, but it is known that YY1 negatively regulates Fas expression. The study aimed to evaluate the effect of YY1 on Fas expression under hypoxic conditions in ALL. Methods: Leukemia cell line RS4; 11 was cultured under normoxic and hypoxic conditions. YY1, Fas receptor, and hypoxia-inducible factor (HIF-1α) expression were analyzed. After treatment with a Fas agonist (DX2), apoptosis was analyzed through the detection of active caspase 3. Data were analyzed using Pearson’s correlation. Results: Leukemia cells co-expressed both HIF-1α and YY1 under hypoxia, which correlated with a downregulation of Fas expression. During hypoxia, the levels of apoptosis diminished after DX2 treatment. The analysis revealed that patients with high levels of HIF-1α also express high levels of YY1 and low levels of Fas. Conclusions: These results suggest that YY1 negatively regulates the expression of the Fas receptor, which could be involved in the escape of leukemic cells from the immune response contributing to the ALL pathogenesis.


Resumen Introducción: La leucemia linfoblástica aguda (LLA) es una enfermedad con alta prevalencia en la población pediátrica. El mecanismo por el cual el receptor de Fas participa en la regulación inmunitaria en los tumores es desconocido, pero se sabe que está subexpresado en LLA. El factor de transcripción Ying-Yang-1 (YY1) está involucrado en la resistencia a la apoptosis y la progresión tumoral; se encuentra aumentado en diferentes tumores, incluida la LLA. Aunque los mecanismos que regulan la expresión de YY1 en LLA son desconocidos, se sabe que YY1 regula la expresión del receptor de Fas. El objetivo de este trabajo fue evaluar el efecto de YY1 en la expresión de Fas en condiciones de hipoxia en la LLA. Métodos: Se cultivaron células RS4;11 en condiciones de hipoxia y se analizó la expresión de YY1, receptor de Fas y HIF-1α. La apoptosis fue inducida usando un agonista de Fas (DX2) y se analizó con la detección de caspasa 3 activa. Los datos se analizaron mediante correlación de Pearson. Resultados: Las células RS4;11 coexpresaron HIF-1αy YY1 en hipoxia, lo cual correlaciona con una baja expresión de Fas. La apoptosis se encontró disminuida durante condiciones de hipoxia, después del tratamiento con DX2. El análisis bioinformático mostró que los pacientes con altos niveles de HIF-1αpresentan YY1 elevado y bajos niveles del receptor de Fas. Conclusiones: Estos resultados sugieren que YY1 regula negativamente la expresión del receptor de Fas, lo cual podría estar involucrado en el escape de las células leucémicas a la respuesta inmunitaria, contribuyendo a la patogénesis de la LLA.


Subject(s)
Child , Humans , Cell Hypoxia/physiology , Apoptosis/physiology , fas Receptor/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , YY1 Transcription Factor/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Down-Regulation , Gene Expression , fas Receptor , Cell Line, Tumor , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , YY1 Transcription Factor/genetics , Caspase 3/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Immune Evasion , Tumor Hypoxia/physiology , Immunologic Surveillance
15.
Journal of Central South University(Medical Sciences) ; (12): 1223-1227, 2020.
Article in English | WPRIM | ID: wpr-880589

ABSTRACT

Cardiomyocytes injury model has been widely used in the study for the molecular mechanism of cardiovascular diseases and drug action. It is very important to select the appropriate model due to the different formation mechanisms for various models. Clinical cardiovascular pathological change is relatively complex. Currently used models according to the characteristics of clinical cardiovascular diseases mainly include hydrogen peroxide-induced myocardial cell damage model, hypoxia reoxygenation injury model, adriamycin-induced myocardial cell damage model, high sugar high fat-induced myocardial cell damage model, and isoprenaline-induced myocardial cell damage model. Every model has its advantages as well as its disadvantages. The suitable model of myocardial cell injury can be selected according to the research purpose.


Subject(s)
Animals , Rats , Cell Hypoxia , Myocardial Reperfusion Injury/metabolism , Myocardium , Myocytes, Cardiac/metabolism , Rats, Sprague-Dawley , Research
16.
Journal of Zhejiang University. Medical sciences ; (6): 942-948, 2020.
Article in Chinese | WPRIM | ID: wpr-828513

ABSTRACT

OBJECTIVE@#To investigate the effect of periostin on hypoxia-induced oxidative stress and apoptosis in human periodontal ligament fibroblasts and the molecular mechanism involved.@*METHODS@# cultured human periodontal ligament fibroblasts were placed in an anaerobic gas-producing bag for hypoxia treatment for 48 h followed by treatment with periostin at low (25 ng/mL), moderate (50 ng/mL) or high (100 ng/mL) doses. MTT assay was used to measure the cell viability, and the cell apoptosis rate was determined using flow cytometry. The contents of IL-1β, IL-6 and TNF-α in the cells were determined with ELISA, and ROS levels were measured using a fluorescent plate reader. The intracellular SOD activity was detected using ELISA. The expressions of HIF-1α, P21, cyclin D1, Bax, cleaved caspase-3, Bcl-2, P38MAPK and p-p38 MAPK proteins in the cells were detected with Western blotting.@*RESULTS@#Hypoxia treatment significantly reduced the cell viability ( < 0.05), increased P21, Bax, and cleaved caspase-3 protein levels ( < 0.05), promoted cell apoptosis ( < 0.05), and decreased cyclin D1 and Bcl-2 protein levels ( < 0.05) in the cells. Compared with the hypoxic group, the cells treated with periostin at different concentrations showed significantly increased cell viability ( < 0.05) with significantly lowered apoptotic rates ( < 0.05) and decreased expression levels of Bax and cleaved caspase-3 ( < 0.05) but significantly increased expression levels of cyclin D1 and Bcl-2 ( < 0.05). Hypoxic exposure of the cells resulted in significantly increased expression levels of HIF-1α and p-p38 MAPK ( < 0.05) and increased levels of IL-1β, IL-6, TNF-α and ROS ( < 0.05) but decreased SOD activity ( < 0.05). Periostin treatment at different concentrations significantly lowered the expression levels of HIF-1α and p-p38 MAPK ( < 0.05) and the levels of IL-1β, IL-6, TNF-α and ROS ( < 0.05) and significantly increased SOD activity in the hypoxic cells ( < 0.05).@*CONCLUSIONS@#Periostin promotes the proliferation, inhibits apoptosis, enhances cellular antioxidant capacity, and reduces inflammatory damage in human periodontal ligament fibroblasts exposed to hypoxia possibly by inhibiting the activation of the p38 MAPK signaling pathway.


Subject(s)
Humans , Apoptosis , Cell Adhesion Molecules , Cell Hypoxia , Fibroblasts , Oxidative Stress , Periodontal Ligament , Cell Biology , Signal Transduction , p38 Mitogen-Activated Protein Kinases
17.
China Journal of Chinese Materia Medica ; (24): 2960-2965, 2020.
Article in Chinese | WPRIM | ID: wpr-828061

ABSTRACT

The aim of this paper was to investigate whether the mechanism of salvianolic acid B in protecting H9 c2 cardiomyocytes from hypoxia/reoxygenation injury is related to the regulation of mitochondrial autophagy mediated by NIX. H9 c2 cardiomyocytes were cultured in vitro and divided into normal group, model group and salvianolic acid B group(50 μmol·L~(-1)). Hypoxia/reoxygenation injury model was established by hypoxia for 4 h and reoxygenation for 2 h. In normal group, high glucose DMEM medium was used for culture. Those in model group were cultured with DMEM medium without glucose and oxygen, and no drugs for hypoxia and reoxyge-nation. In salvianolic acid B group, salvianolic acid B prepared by glucose-free DMEM medium was added during hypoxia, and the other process was as same as the model group. The cell viability was evaluated by CCK-8 assay. The leakage of lactate dehydrogenase(LDH) was detected by microplate method. The levels of intracellular reactive oxygen species(ROS) and mitochondrial membrane potential(ΔΨm) were measured by chemical fluorescence method. The level of intracellular adenosine triphosphate(ATP) was mea-sured by fluorescein enzyme method. The autophagy related proteins LC3-Ⅰ, LC3-Ⅱ, apoptosis related protein cleaved caspase-3 and mitochondrial autophagy receptor protein NIX were detected by Western blot. As compared with the normal group, the activity of H9 c2 cardiomyocytes and ATP level were decreased(P<0.05); LDH leakage and ROS production were increased(P<0.01); ΔΨm was decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio, cleaved caspase-3 and NIX protein expression levels were increased(all P<0.05) in the model group. As compared with the model group, the activity of cells and ΔΨm were significantly increased(P<0.01); ATP level was increased(P<0.05); LDH leakage and ROS generation were decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio was decreased(P<0.01); cleaved caspase-3 and NIX expression levels were decreased(P<0.05) in the salvianolic acid B group. The protective effect of salvianolic acid B on hypoxia/reoxygenation injury of H9 c2 cardiomyocytes may be associated with inhibiting mitochondrial auto-phagy. The specific mechanism may be related to inhibiting the activation of mitochondrial autophagy mediated by NIX, increasing ΔΨm, reducing ROS production, reducing the expression of cleaved caspase-3, LC3-Ⅱ, and increasing cell viability.


Subject(s)
Humans , Apoptosis , Autophagy , Benzofurans , Cell Hypoxia , Cell Survival , Hypoxia , Myocytes, Cardiac
18.
Chinese Journal of Contemporary Pediatrics ; (12): 491-496, 2019.
Article in Chinese | WPRIM | ID: wpr-774046

ABSTRACT

OBJECTIVE@#To study the effect of 280 nm-LED ultraviolet irradiation on the proliferation of acute promyelocytic leukemia (APL) HL-60 cells under hypoxic conditions and related mechanism.@*METHODS@#HL-60 cells in the logarithmic growth phase were selected and divided into control, hypoxia, ultraviolet and hypoxia+ultraviolet groups. The cells in the hypoxia group were treated with cobalt chloride (with a final concentration of 150 μmol/L), those in the ultraviolet group were irradiated by 280 nm-LED ultraviolet with an energy intensity of 30 J/m, and those in the hypoxia+ultraviolet group were treated with cobalt chloride and then irradiated by 280 nm-LED ultraviolet. After 48 hours of treatment, the cells were placed under an invert microscope to observe cell morphology. CCK-8 assay was used to measure the inhibition rate of cell proliferation. Annexin V-FITC/PI double staining flow cytometry was used to evaluate cell apoptosis. Quantitative real-time PCR was used to measure the mRNA expression of Bcl-2. Each experiment above was repeated three times independently.@*RESULTS@#Compared with the control group, the experimental groups showed shrinkage, decreased brightness, and disordered arrangement of cells, and the number of cells decreased over the time of culture. There were significant differences in the inhibition rate of cell proliferation and cell apoptosis rate among the groups (P<0.01), and the hypoxia+ultraviolet group showed the strongest inhibition of cell proliferation and induction of cell apoptosis, followed by the ultraviolet group and the hypoxia group. Compared with the control group, the other three groups had a gradual reduction in the mRNA expression of Bcl-2, and the hypoxia+ultraviolet group had a significantly greater reduction than the hypoxia and ultraviolet groups (P<0.01).@*CONCLUSIONS@#Both hypoxia and ultraviolet irradiation can inhibit the proliferation of HL-60 cells and induce cell apoptosis, and ultraviolet irradiation has a better effect on proliferation inhibition and cell apoptosis under hypoxic conditions than under normoxic conditions, possibly by downregulating the mRNA expression of Bcl-2.


Subject(s)
Humans , Apoptosis , Cell Hypoxia , Cell Proliferation , Leukemia, Promyelocytic, Acute
19.
Chinese journal of integrative medicine ; (12): 23-30, 2019.
Article in English | WPRIM | ID: wpr-773974

ABSTRACT

OBJECTIVE@#To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role.@*METHODS@#An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting.@*RESULTS@#SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002.@*CONCLUSION@#SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.


Subject(s)
Animals , Rats , Adenosine Triphosphate , Animals, Newborn , Caffeic Acids , Pharmacology , Cell Hypoxia , Cells, Cultured , Glycogen Synthase Kinase 3 beta , Physiology , Lactates , Pharmacology , Mitochondria, Heart , Physiology , Mitochondrial Membrane Transport Proteins , Myocytes, Cardiac , Proto-Oncogene Proteins c-akt , Physiology , Rats, Sprague-Dawley , Reactive Oxygen Species , Metabolism , Signal Transduction , Physiology
20.
Journal of Southern Medical University ; (12): 898-903, 2019.
Article in Chinese | WPRIM | ID: wpr-773515

ABSTRACT

OBJECTIVE@#To investigate the effect of miR-186 inhibition on the expression of hypoxia-inducible factor-1α (HIF-α) and mitochondrial function in hypoxic vascular endothelial cells.@*METHODS@#Human umbilical vein endothelial cells (HUVECs) cultured in routine or hypoxic conditions for 6 h were examined for the expression of miR-186. A miR-186 inhibitor was transfected in the HUVECs, and the cells were subsequently cultured in hypoxic condition for 6 h to observe the changes in the mitochondrial structure under an electron microscope. The changes in the mRNA and protein expressions of HIF-1α in response to miR-186 interference were tested using real-time fluorescent quantitative PCR and Western blotting.@*RESULTS@#The expression of miR-18 was mildly increased in HUVECs after hypoxic exposure for 6 h (=0.0188). Interference of miR-186 expression obviously promoted the mRNA and protein expressions of HIF-1α in HUVECs. In hypoxic conditions, miR-186 interference significantly reduced mitochondrial damage in HUVECs as observed under electron microscope (=0.0297).@*CONCLUSIONS@#Inhibition of miR-186 protects vascular endothelial cells against hypoxic injuries by promoting HIF-α expression to lessen mitochondrial damage, suggesting the possibility of targeted miR-186 interference for treatment of hemorrhagic shock.


Subject(s)
Humans , Cell Hypoxia , Human Umbilical Vein Endothelial Cells , Hypoxia , Hypoxia-Inducible Factor 1, alpha Subunit , MicroRNAs , Mitochondria , Umbilical Veins
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