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1.
Article in Chinese | WPRIM | ID: wpr-1014562

ABSTRACT

AIM: To investigate the relationship between vascular smooth muscle cell (VSMC) injury, organelle stress response and autophagic cell death (autophagy) and ferroptosis induced by the chemical hypoxia inducer cobalt chloride (CoCl2) through the bioinformatics analysis and in vitro cell experimentation. METHODS: The dataset GSE119226 of VSMC treated with cobalt chloride was acquired from the gene expression database (GEO). The R language was used to investigate the relationship between CoCl2 treatment and organelle stress response (Golgi stress, endoplasmic reticulum stress) and two forms of cell death (ferroptosis and autophagic cell death). With primary cultured rat VSMC (rVSMC) and CoCl2-induced anoxia model, the changes in cell viability were detected by CCK-8 method, and reactive oxygen species (ROS) levels were measured using DCFH-DA method. The expression levels of HIF-1α (a key molecule in hypoxia), Golgi stress markers GM130 and p115, endoplasmic reticulum stress markers GRP78 and CHOP, autophagy markers LC3-II / LC3-I and Beclin1, and ferroptosis markers GPx4 and xCT were detected by Western blot. The effect of inducing or inhibiting organelle stress and cell death on the CoCl2-induced cell damage was also observed. RESULTS: Differentially expressed genes analysis of GSE119226 dataset showed that CoCl2 treatment of VSMCs had significant effects on organelle function and stress response, autophagy and ferroptosis-related genes, in which endoplasmic reticulum stress, protein processing in endoplasmic reticulum, regulation of Golgi to plasma membrane protein transport, autophagy / autophagic cell death, and ferroptosis pathways were remarkably enriched. The results of in vitro experiment showed that compared with normal rVSMC, cell viability was significantly decreased after CoCl2 treatment, as well as HIF-1α protein expression and ROS levels in rVSMCs were increased. In rVSMC treated with Co-Cl2, the expression levels of Golgi structural proteins GM130 and p115 (reflecting the occurrence of Golgi stress) were decreased, while the markers GRP78 and CHOP (reflecting the occurrence of endoplasmic reticulum stress) were increased. At the same time, CoCl2 treatment also reduced the expression of autophagy markers LC3-II/LC3-I and Beclin1 (indicating the decrease levels of autophagy), while the expression of ferroptosis markers GPx4 and xCT were decreased (indicating the occurrence of ferroptosis). Compared with CoCl2 treatment group, induced Golgi stress, endoplasmic reticulum stress, or ferroptosis could further reduce cell viability, while inhibition of these processes could improve cell viability. On the other hand, increasing the level of autophagy can improve the cell viability. CONCLUSION: Hypoxia induced by cobalt chloride can lead to VSMC injury. Golgi stress, endoplasmic reticulum stress, ferroptosis, and the reduction of autophagy level play an important role in it. Inhibition of organelle stress response and ferroptosis, or increase of autophagy level can improve VSMC injury caused by cobalt chloride.

2.
Bol. latinoam. Caribe plantas med. aromát ; 22(2): 224-236, mar. 2023. tab, graf
Article in English | LILACS | ID: biblio-1555667

ABSTRACT

The objective was to evaluate plant growth regulators and ethylene inhibitors on the development and leaf abscission of Schinopsis brasiliensis Engl. Zeatin (ZEA) was evaluated in concentrations combined with concentrations of indolacetic acid (IAA), naphthalene acetic acid (NAA) and indolbutyric acid (IBA). ZEA and 6-benzylamino purine (BAP) were evaluated in concentrations plus a control. Ethylene inhibitors, silver nitrate and cobalt chloride were evaluated in four concentrations. The addition of 0.2 µL-1 of NAA to 0.4 µL-1 of ZEA promotes a greater number of baraúna sprouts. At concentrations of 5 and 10 µM, cobalt chloride is more efficient than silver nitrate for reducing leaf abscission in baraúna. IAA is the most suitable auxin to be associated with ZEA for higher shoot length and number of buds. Silver nitrate from a concentration of 20 µM completely avoids leaf abscission whilecobalt chloride has a maximum reduction in abscission at a concentration of 40 µM.


El objetivo fue evaluar reguladores de crecimiento e inhibidores de etileno sobre el desarrollo y abscisión foliar en Schinopsis brasiliensis Engl. La zeatina (ZEA) se evaluó en concentraciones combinadas con concentraciones de ácido indolacético (IAA), ácido naftaleno acético (NAA) y ácido indolbutírico (IBA). Se evaluaron ZEA y 6-bencilamino purina (BAP) en concentraciones más un control. Se evaluaron inhibidores de etileno, nitrato de plata y cloruro de cobalto, en cuatro concentraciones. La adición de 0.2 µL-1 de NAA a 0.4 µL-1 de ZEA promueve un mayor número de brotes de baraúna. A concentraciones de 5 y 10 µM, el cloruro de cobalto es más eficaz que el nitrato de plata para reducir la abscisión de las hojas en baraúna. IAA es la auxina más adecuada para asociar con ZEA para una mayor longitud de brotes y número de brotes. El nitrato de plata a partir de una concentración de 20 µM evita completamente la abscisión de las hojas, mientras que el cloruro de cobalto tiene una reducción máxima en la abscisión a una concentración de 40 µM.


Subject(s)
Anacardiaceae/growth & development , Zeatin , In Vitro Techniques , Ethylenes
3.
Article in Chinese | WPRIM | ID: wpr-742717

ABSTRACT

Objective:To observe the effect of CoCl2on the cisplatin sensitivity of human ovarian cancer SKOV3cells, and to clarify the possible mechanism.Methods:The SKOV3cells were cultured in vitro and randomly divided into control group, CoCl2 group, cisplatin (DDP) group and CoCl2 combined with DDP (combination) group.The cells in CoCl2group were cultured in normal cell medium for 20hafter cultured in 200μmol·L-1 CoCl2for 4h, the cells in DDP group were cultured in normal cell medium containing 10mg·L-1 DDP for 24h, and the cells in combination group were cultured in 10mg·L-1 DDP for 20hafter cultured in 200μmol·L-1 CoCl2for 4h.The survival rates of SKOV3cells in various groups were detected by MTT method, and the positive expression intensities of hypoxia-inducible factor-1α (HIF-1α) and inducible nitric oxide synthase (iNOS) in the cells in various groups were detected by immunofluorescence method.Rhod 2-AM fluorescence probe was used to observe the levels of Ca2+in mitochondria in the cells in various groups.Western blotting method was used to observe the expression levels of cytochrome C (cyto C) , cysteinyl aspartase 3 (caspase 3) and cleaved cysteinyl aspartase 3 (cleaved caspase 3) .Muse○R apoptosis assay kit was used to detect the apoptotic rates of cells in various groups.Results:Compared with control group, the survival rate of the cells in CoCl2group had no significant change (P>0.05) , and the survival rates of the cells in DDP and combination groups were decreased (P<0.05) ;the survival rate in combination group was higher than that in DDP group (P<0.05) .Compared with control group, the positive expression intensities of HIF-1αin CoCl2and combination groups were increased (P<0.05) .Compared with control group, the positive expressions of iNOS in DDP and combination groups were increased (P<0.05) .The Ca2+levels in the cells in DDP group and combination groups were higher than that in control group (P<0.05) and the Ca2+level in DDP group was higher than that in combination group (P<0.05) .Compared with control group, the expression levels of cyto C, caspase 3and cleaved caspase 3proteins in the SKOV3cells in CoCl2group had no significant changes (P>0.05) , and the expression levels of cyto C, caspase 3and cleaved caspase 3in DDP group were increased significantly (P<0.05) ;compared with DDP group, they were lower than those in combination group (P<0.05) .Compared with control group, the apoptotic rate of SKOV3cells in DDP group was increased significantly (P<0.05) ;the apoptotic rate of SKOV3cells in combination group was lower than that in DDP group (P<0.05) .Conclusion:CoCl2can redece the mitochondrial apoptosis of human ovarian cancer SKOV3cells by inhibiting the DDP-induced enhancement of iNOS expression and decrease the sensitivity of SKOV3cells to cisplatin.

4.
Article in Chinese | WPRIM | ID: wpr-841733

ABSTRACT

Objective: To observe the effect of CoCl on the cisplatin sensitivity of human ovarian cancer SKOV3 cells, and to clarify the possible mechanism. Methods: The SKOV3 cells were Cultured in vitro and randomly divided into control group, CoCT group, cisplatin (DDP) group and CoCT combined with DDP (combination) group. The cells in CoCL group were Cultured in normal cell medium for 20 h after cultured in 200 pmol • L CoCL for 4 h, the cells in DDP group were cultured in normal cell medium containing 10 mg • L DDP for 24 h, and the cells in combination group were cultured in 10 mg • L DDP for 20 h after cultured in 200 //mol • L CoCl • for 4 h. The survival rates of SKOV3 cells in various groups were detected by MTT method, and the positive expression intensities of hypoxia-inducible factor-1 a ( HIF-la) and inducible nitric oxide synthase (iNOS) in the cells in various groups were detected by immunofluorescence method. Rhod 2-AM fluorescence probe was used to observe the levels of Ca in mitochondria in the cells in various groups. Western blotting method was used to observe the expression levels of cytochrome C (cyto C) cysteinyl aspartasc 3 (caspasc 3) and cleaved cysteinyl aspartasc 3 (cleaved caspase 3). Muse apoptosis assay kit was used to detect the apoptotic rates of cells in various groups. Results: Compared with control group, the survival rate of the cells in CoCI group had no significant change (P> 0.05). and the survival rates of the cells in DDP and combination groups were decreased ( P0. 05) . and the expression levels of cyto C. caspase 3 and cleaved caspase 3 in DDP group were increased significantly ( P < 0.05); comparexl with DDP group, they were lower than those in combination group ( P<0. 05). Comparexl with control group∗ the apoptotic rate of SKOV3 cells in DDP group was increased significantly (P<.0. 05); the apoptotic rate of SKOV3 cells in combination group was lowe'r than that in DDP group (P<0. 05). Conclusion: CoCI can redece the mitochondrial apoptosis of human ovarian cancer SKOV3 cells by inhibiting the DDP-inducexl enhancement of iNOS expression and dccrease the sensitivity of SKOV3 cells to cisplatin.

5.
Article in Chinese | WPRIM | ID: wpr-696543

ABSTRACT

Objective To explore the effect of endogenous sulfur dioxide (SO2)on the apoptosis induced by cobalt chloride (CoCl2)in the human pulmonary arterial endothelial cells (HPAECs).Methods CoCl2was used in the primary HPAECs to mimize hypoxia-induced cell apoptosis.The aspartate aminotransferase 1(AAT1),and the key enzyme generating endogenous SO2 were over -expressed by transfecting HPAECs with lentivirus containing AAT1 cDNA.HPAECs were divided into 4 groups:vehicle group,vehicle + CoCl2 group,AAT1 group and AAT1 + CoCl2 group.The expressions of AAT1,B-cell lymphoma-2 (bcl-2),bcl-associated X protein (bax),Caspase-3 and activated Caspase-3 (cleaved Caspase-3)in the HPAECs were measured by Western blot.The AAT activity was assessed with colorimetry method.The SO2 content in the HPAECs was in situ observed by SO2-specific fluorescent probe.The HPAECs apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)assay.Results There were significant differences in the endogenous SO2 content,the expre-ssions of AAT1 and bcl-2,and the ratio of cleaved Caspase-3/Caspase-3 among 4 groups of HPAECs were (F=147.364,23.738,6.521,64.884,all P<0.05).However,there was no difference in the expression of bax among 4 groups of HPAECs (F=1.620,P>0.05).Compared with vehicle group,AAT activity [(0.96 ± 0.24)Carmen's unit/μg vs.(2.21 ± 0. 60)Carmen's unit/μg],endogenous SO2 content (40.71 ± 7.72 vs.105.60 ± 16.20)and bcl-2 expression (0.59 ± 0.19 vs.1.02 ± 0.20)in the HPAECs of vehicle +CoCl2 group were significantly de-creased,while the cell apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (1.56 ± 0.25 vs.0.95 ± 0.13)were significantly increased (all P<0.05).However,there were no differences in the expression of AAT1 (0. 50 ± 0.12 vs.0.53 ± 0.11)in the HPAECs between vehicle group and vehicle+CoCl2 group (P>0.05). The SO2 content (351.50 ± 42.43 vs.105.60 ± 16.20)and AAT1 expression (1.22 ± 0.33 vs.0.53 ± 0.11)in the HPAECs of AAT1 group were higher than those of vehicle group (all P <0. 05 ). Compared with AAT1 group, endogenous SO2content (333.50 ± 46.22 vs.351.50 ± 42.43)and the expression of AAT1 (1.26 ± 0.36 vs.1.22 ± 0.33)and bcl-2 (1.14 ± 0.38 vs.1.03 ± 0.27)in the HPAECs of AAT1 +CoCl2group did not change (all P>0. 05).Moreover,no difference was observed in the HPAECs apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (0.51 ± 0.17 vs.0.50 ± 0.11)between the two AAT1 -overexpressed groups (all P >0. 05).Conclusion Endo-genous SO2inhibited the hypoxic HPAECs apoptosis stimulated by the treatment of CoCl2.

6.
Chinese Journal of Pathophysiology ; (12): 717-722,728, 2018.
Article in Chinese | WPRIM | ID: wpr-701185

ABSTRACT

AIM: To investigate whether neural stem cell-derived exosomes promote the viability and inhibit the apoptosis of neurons under cobalt chloride(CoCl2)-induced hypoxia in vitro.METHODS:The exosomes were isolated based on ultracentrifugation.The exosomal markers,ALG-2-interacting protein X(Alix)and tumor susceptibility gene 101 (TSG101)were identified by Western blot.The shape of exosomes was observed under transmission electron microscope (TEM).The size distributions of exosomes were analyzed by nanoparticle analysis(qNano).The neurons were exposed in CoCl2at different doses(200~600 μmol/L)for 24 h.The exosomes were co-cultured with the neurons pre-treated with CoCl2.The viability and apoptosis of the neurons were measured by CCK-8 assay and TUNEL method.RESULTS: The exosomes released from the neural stem cells expressed exosomal markers Alix and TSG 101.They also displayed a cup-shaped appearance observed under TEM and their sizes were(95.0 ±23.5)nm(n=370).The neuronal viability was sig-nificantly inhibited by CoCl 2in a dose-dependent manner(P<0.05).After treatment with exosomes,the viability of the neuron pre-treated with CoCl2was increased and the apoptotic rate was decreased(P<0.05).CONCLUSION: Neural stem cell-derived exosomes promote the viability and inhibit the apoptosis of rat neurons uneder hypoxia.

7.
Article in Chinese | WPRIM | ID: wpr-505125

ABSTRACT

Objective To study the differentially expressed genes and analyze its functional pathways of retinopathy of prematurity(ROP),in order to discover the pathogenesis and provide the theoretical basis for the prevention and treatment of ROP.Methods Fetal eyeballs of induced labor were obtained and retinal microvascular endothelial cells were isolated and cultured.The endothelial cells were divided into 7 groups according to the medium of cobalt chloride (CoC12) concentration (0 μ mol/L,100 μ mol/L,150 pμmol/L,200 μmol/L,250 pμmol/L,300 μmol/L,350 μmol/L),and 150 μmol/L CoCl2 was finally used to induce ROP model in vitro.Retinal microvascular endothelial cells were verified by adopting Ⅷ factor and CD31 antibody fluorescence staining.RNA purification,gene chip hybridization and signi-ficant analysis of microarrays were performed to screen differentially expressed genes.Genes functional pathways were studied by using gene ontology analysis software.Results (1) The proliferation activity of vascular endothelial cells decreased when CoCl2 ≥ 150 μmol/L(F =21,P < 0.05).(2) In 150 μmol/L CoC]2 group,blue nucleus and green cytoplasm were visible in the second and the third generation vascular endothelial cells stained by factor Ⅷ antibody,and red fluorescence could also be observed in the cytoplasm by means of CD31 monoclonal antibody staining.However,only blue nucleus was detected in the group without CoCl2.(3)There were 326 genes differently expressed in retinal micro vascular endothelial cells induced by CoCl2 in vitro,among whom,198 genes were up-regulated and 128 genes were down-regulated.Up-regulated expression genes were 1.5 times more than those of the down-regulated genes.(4)Ten biological pathways including cell hypoxia,angiogenesis suppression and iron ion transport etc.may play important roles in ROP pathogenesis.Conclusions Hypoxic retinal microvascular endothelial cells induced by CoCl2 can successfully be used to induce cell model of ROP in vitro.Differentially expressed genes may play an important role in ROP development.Functional pathway such as hypoxic cells,inhibition of angiogenesis,iron ion transport may be associated with ROP pathogenesis.

8.
Chinese Pharmacological Bulletin ; (12): 480-486, 2017.
Article in Chinese | WPRIM | ID: wpr-511288

ABSTRACT

Aim To investigate whether necroptosis mediates chemical hypoxia-induced HT22 mouse hippocampal cell injury and inflammation.Methods HT22 hippocampal cells were exposed to cobalt chloride (CoCl2) to establish a model of the chemical hypoxia-induced injury and inflammation.The expression level of RIP3 (an index of necroptosis) was determined by Western blot.Cell counter kit-8 (CCK-8) assay was used to test the cell viability.Lactate dehydrogenase (LDH) activity in the culture medium was measured with commercial kits.Mitochondrial membrane potential (MMP) was examined by rhodamine123 staining followed by photofluorography.The intracellular level of reactive oxygen species (ROS) was detected by 2', 7'-dichlorfluorescein-diacetate (DCFH-DA) staining followed by photofluorography.The secretion levels of interleukin-1β (IL-1β) and tumor necrosis factor-a (TNF-α) were measured by ELISA.Results Treatment of HT22 hippocampal cells with 600 μmol·L-1 CoCl2 for 36 h markedly induced cytotoxicity, leading to a decrease in cell viability to (52.0±2.65) % , indicating that chemical hypoxia-induced cellular injury model was successfully set up.Besides, CoCl2 induced considerable injuries and inflammation, evidenced by increases in LDH activity, ROS production, MMP loss, as well as the secretion levels of IL-1β and TNF-α.Co-treatment of the cells with 40~100 μmol·L-1 Nec-1 (a specific inhibitor of necroptosis) and CoCl2 markedly attenuated the decrease in viability induced by CoCl2, reaching the best anti-cytotoxicity inhibitory effect at 80 μmol·L-1.Meanwhile, the co-treatment with 80 μmol·L-1 Nec-1 blocked the above injuries and inflammatory response induced by CoCl2.In addition, treatment of HT22 hippocampal cells for 6~48 h up-regulated the expression of RIP3, and Nec-1 alleviated the up-regulation of RIP3 expression level induced by CoCl2.Conclusion Necroptosis mediates chemical hypoxia-induced HT22 hippocampal cell injury and inflammation.

9.
Chongqing Medicine ; (36): 2889-2891,2894, 2017.
Article in Chinese | WPRIM | ID: wpr-617363

ABSTRACT

Objective To compare the effects of cobalt chloride (CoCl2) induced chemical hypoxia and hypoxia environment on the proliferation,migration and phenotype transformation of rat primary pulmonary artery fibroblasts (PAFs).Methods Primary PAFs were isolated and cultured.Cells were stimulated by CoCl2,or hypoxia cell culture (1% O2) was used to stimulate and induce PAFs.Then the effects of CoCI2 and hypoxia environment on PAFs were compared by CCK-8 assay,scratch assay,transwell assay,phenotype marker protein expression and PI3K/Akt signaling pathway protein expression.Results Compared with the control group,100 μmol/mL CoCl2 stimulation had no significant effect on the cell proliferation activity,cell migration ability and phenotype transformation ability of PAFs (P>0.05);while 1% O2 could significantly improve the cell proliferation and migration activities of PAFs as well as the upregulation of α-SMA expression (P<0.05).Conclusion There exist differences of effects between CoCl2 induced chemical hypoxia and hypoxia environment on promoting cell proliferation, cell migration and phenotype transformation in PAFs.

10.
Tianjin Medical Journal ; (12): 294-297, 2016.
Article in Chinese | WPRIM | ID: wpr-487601

ABSTRACT

Objective To explore the effects of cobalt chloride (CoCl2)-induced hypoxia on migration of melanoma cells, and to detect the transcription, expression and secretion of Follistatin-like 1(FSTL1) in this process. Methods B16F10 melanoma cell line was treated with CoCl2 in order to mimic hypoxia. Experimental cells were divided into three groups: 0μmol/L, 50μmol/L and 100μmol/L CoCl2 treatment groups. MTT assay was used to assure cell viability, and to determine the treatment concentration of CoCl2. Transwell assay was used to determine the migration ability of B16F10 melanoma cell line. Real-time PCR was used to measure the mRNA expression of Fstl1. Western blot assay was used to detect the intracel?lular and extracellular protein expression of FSTL1. Results The cell viability of B16F10 melanoma cell line was signifi?cantly reduced by CoCl2 treatment, with a time and concentration-dependent manner. The migration ability of B16F10 cell line was significantly increased in CoCl2 treated group compared with that of control group (P<0.05). The mRNA level of Fstl1 was obviously higher in CoCl2 treated group than that of control group (P<0.05). The intracellular expression of FSTL1 protein was consistent with the expression trend of Fstl1 mRNA. Simultaneously, the extracellular protein level of FSTL1 was significantly decreased compared with that of control group. There was no expression of FSTL1 in 100μmol/L CoCl2 treat?ment group. Conclusion The migration ability of melanoma cell line is enhanced by CoCl2 treatment, which may be associ?ated with expression and secretion of FSTL1, however, the relevant mechanism still needs further investigation.

11.
Article in English | WPRIM | ID: wpr-644856

ABSTRACT

Hypoxic condition influences biological responses in various cell types. However, a hypoxic regulating osteogenic differentiation remains controversy. Here, an influence of short-term culture in hypoxic condition on osteogenic marker gene expression by retinoic acid-treated murine gingival fibroblast-derived induced pluripotent stem cells (RA-miPS) was investigated. Results demonstrated that hypoxic condition significantly upregulated Vegf, Runx2, Osx, and Ocn mRNA expression by RA-miPS in normal culture medium at day 3. Further, desferrioxamine significantly downregulated pluripotent marker (Nanog and Oct4) and enhanced osteogenic marker (Runx2, Osx, Dlx5, and Ocn) gene expression as well as promoted in vitro mineral deposition. However, the effect of cobalt chloride on osteogenic differentiation of RA-miPS was not robust. In summary, the results imply that hypoxic condition may be useful in the enhancement of osteogenic differentiation in RA-miPS.


Subject(s)
Hypoxia , Cobalt , Deferoxamine , Gene Expression , In Vitro Techniques , Induced Pluripotent Stem Cells , Miners , Pluripotent Stem Cells , RNA, Messenger , Vascular Endothelial Growth Factor A
12.
Biol. Res ; 49: 1-10, 2016. ilus, graf
Article in English | LILACS | ID: biblio-950834

ABSTRACT

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


Subject(s)
Animals , Rats , Plant Extracts/pharmacology , Apoptosis/drug effects , Cysteine/analogs & derivatives , Basic Helix-Loop-Helix Transcription Factors/drug effects , Garlic/chemistry , Antioxidants/pharmacology , Tetrazolium Salts , Cell Hypoxia/drug effects , Cell Survival/drug effects , Cells, Cultured , Analysis of Variance , PC12 Cells , Reactive Oxygen Species/analysis , Cobalt , Cysteine/pharmacology , Flow Cytometry , Formazans
13.
Article in Chinese | WPRIM | ID: wpr-462857

ABSTRACT

AIM: To investigate the effect of cobalt chloride (CoCl2) on the apoptosis of neural stem cells (NSCs) and the expression of microRNA-26a (miR-26a) in vitro, and to explore the mechanisms of NSC apoptosis in-duced by CoCl 2 .METHODS:NSCs were exposed to CoCl 2 at different doses (200~600μmol/L) for 24 h.The cell via-bility and apoptosis were measured by CCK-8 assay and TUNEL method.The expression of miR-26a-3p, miR-26a-5p, GSK-3β, caspase-3, Bcl-2 and Bax was examined by real-time PCR.The protein levels of Bcl-2 and Bax were detected by Western blotting .RESULTS: The cell viability was inhibited and the apoptosis of NSCs was increased significantly by CoCl2 in a dose-dependent manner (P<0.05).CoCl2 at concentration of 400μmol/L for 24 h was used to induce apopto-sis and the expression of miR-26a was down-regulated compared with control (P<0.05).Exposure to CoCl2 at concentra-tion of 400μmol/L up-regulated the expression of GSK-3β, caspase-3 and Bax , down-regulated the expression of Bcl-2 and Bcl-2/Bax (P<0.05).CONCLUSION:CoCl2 at concentration of 400μmol/L induces the apoptosis of NSCs obviously . CoCl2 may induce the NSC apoptosis by mitochondrial apoptotic pathway .Declining miR-26a may be related to NSC apopto-sis.

14.
Chinese Journal of Pathophysiology ; (12): 1036-1041, 2015.
Article in Chinese | WPRIM | ID: wpr-468089

ABSTRACT

[ ABSTRACT] AIM:To investigate the effects of induced pluripotent stem cells-derived mesenchymal stem cells ( iPSC-MSCs) on cobalt chloride ( CoCl2 )-induced injuries of PC12 cells and its possible mechanism.METHODS:PC12 cells were exposed to CoCl2 to set up a chemical-induced cellular injury model and were cocultured with iPSC-MSCs.The cell viability was tested by CCK-8 assay.The apoptosis was measured by flow cytometry using Annexin V/PI staining.The mitochondrial membrane potential (MMP) was analyzed by flow cytometry using JC-1 staining.Immunofluorescence was employed to observe mitochondrial transfer from iPSC-MSCs to PC12 cells.RESULTS: Apoptosis of PC12 cells was in-creased and MMP of PC12 cells was decreased after exposed to CoCl2 at concentration of 400μmol/L for 24 h.Coculture of PC12 cells with iPSC-MSCs reduced the apoptosis and recovered the MMP of the PC12 cells.Tunneling nanotubes were formed between iPSC-MSCs and PC12 cells, through which the iPSC-MSCs transferred the mitochondria to the PC12 cells. CONCLUSION:iPSC-MSCs protect PC12 cells from CoCl2-induced injuries, which may be associated with the mitochon-drial transfer from iPSC-MSCs to PC12 cells.

15.
Article in Chinese | WPRIM | ID: wpr-474083

ABSTRACT

[ ABSTRACT] AIM:To study the effect of thrombopoietin ( TPO) on chemical hypoxia-induced apoptosis of the Rattus norvegicus adrenal pheochromocytoma (PC12) cells.METHODS:The cultured PC12 cells were randomly divided into normal control group, cobalt chloride ( CoCl2 ) group, CoCl2 +TPO group and TPO group.The cell viability was mea-sured by MTT assay.The effect of TPO on CoCl2-induced cell apoptosis was analyzed by flow cytometry with Annexin V/PI double staining.The intracellular reactive oxygen species ( ROS) were detected by fluorescence microscopy, and the chan-ges of the mitochondrial membrane potential ( MMT) were determined by flow cytometry and fluorescence microscopy.RE-SULTS:Chemical oxygen agent CoCl2 significantly inhibited the growth of PC12 cells (P<0.01).The apoptotic rate in CoCl2 group was obviously higher than that in control group ( P<0.05) , while the apoptotic rate in CoCl2 +TPO group was obviously lower than that in CoCl2 group (P<0.05).TPO decreased the production of ROS, and inhibited the decrease in MMP induced by CoCl2(P<0.01).CONCLUSION: TPO has a protective effect against CoCl2-induced apoptosis of PC12 cells by decreasing the production of ROS and inhibiting the decrease in MMP.

16.
Article in English | WPRIM | ID: wpr-51698

ABSTRACT

We evaluated the effect of cobalt chloride (CoCl2) on TNF-alpha and IFN-gamma-induced-inflammation and reactive oxygen species (ROS) in renal tubular epithelial cells (HK-2 cells). We treated HK-2 cells with CoCl2 before the administration of TNF-alpha/IFN-gamma. To regulate hemeoxygenase-1 (HO-1) expression, the cells were treated CoCl2 or HO-1 siRNA. CoCl2 reduced the generation of ROS induced by TNF-alpha/IFN-gamma. TNF-alpha/IFN-gamma-treated-cells showed an increase in the nuclear translocation of phosphorylated NF-kappaBp65 protein, the DNA-binding activity of NF-kappaBp50 and NF-kappaB transcriptional activity and a decrease in IkappaBalpha protein expression. These changes were restored by CoCl2. We noted an intense increase in monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES) production in TNF-alpha/IFN-gamma-treated cells. We demonstrated that this effect was mediated through NF-kappaB signaling because an NF-kappaB inhibitor significantly reduced MCP-1 and RANTES production. CoCl2 effectively reduced MCP-1 and RANTES production. The expression of HO-1 was increased by CoCl2 and decreased by HO-1 siRNA. However, knockdown of HO-1 by RNA interference did not affect MCP-1 or RANTES production. We suggest that CoCl2 has a protective effect on TNF-alpha/IFN-gamma-induced inflammation through the inhibition of NF-kappaB and ROS in HK-2 cells. However, CoCl2 appears to act in an HO-1-independent manner.


Subject(s)
Humans , Cell Line , Chemokine CCL2/metabolism , Chemokine CCL5/metabolism , Cobalt/pharmacology , Epithelial Cells/cytology , Heme Oxygenase-1/antagonists & inhibitors , Inflammation , Interferon-gamma/pharmacology , Kidney Tubules, Proximal/cytology , NF-kappa B/antagonists & inhibitors , NF-kappa B p50 Subunit/genetics , Oxidative Stress/drug effects , Phosphorylation , Protein Binding , RNA Interference , RNA, Small Interfering/metabolism , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/pharmacology
17.
Immune Network ; : 141-147, 2013.
Article in English | WPRIM | ID: wpr-77567

ABSTRACT

Hypoxia has been shown to promote inflammation, including the release of proinflammatory cytokines, but it is poorly investigated how hypoxia directly affects inflammasome signaling pathways. To explore whether hypoxic stress modulates inflammasome activity, we examined the effect of cobalt chloride (CoCl2)-induced hypoxia on caspase-1 activation in primary mixed glial cultures of the neonatal mouse brain. Unexpectedly, hypoxia induced by oxygen-glucose deprivation or CoCl2 treatment failed to activate caspase-1 in microglial BV-2 cells and primary mixed glial cultures. Of particular interest, CoCl2-induced hypoxic condition considerably inhibited NLRP3-dependent caspase-1 activation in mixed glial cells, but not in bone marrow-derived macrophages. CoCl2-mediated inhibition of NLRP3 inflammasome activity was also observed in the isolated brain microglial cells, but CoCl2 did not affect poly dA:dT-triggered AIM2 inflammasome activity in mixed glial cells. Our results collectively demonstrate that CoCl2-induced hypoxia may negatively regulate NLRP3 inflammasome signaling in brain glial cells, but its physiological significance remains to be determined.


Subject(s)
Animals , Mice , Hypoxia , Brain , Cobalt , Cytokines , Inflammation , Macrophages , Neuroglia
18.
Article in English | IMSEAR | ID: sea-148840

ABSTRACT

Background: Cobalt chloride can be used as an agent to stabilize hypoxia inducible factor-1α (HIF-1α) and to imitate hypoxia without low levels of oxygen inside the body. We intended to investigate if there was any regulation of renin expression by HIF-1α. Therefore, we conducted several studies to clarify this possibility starting with the induction of hypoxic mimicry in rats by intra-peritoneal (IP) injection of cobalt chloride (CoCl2) to obtain the levels and pattern of HIF-1α and renin mRNA and protein expression. Methods: Twenty-four rats were randomly divided into four groups, control group and incubation groups 2, 8, and 24 hours after intra-peritoneal injection of 30 mg CoCl2 per kg BW. After the rats were sacrificed, kidneys were excised, weighed and kidney weight compared to BW. Tissue parameters were measured such as RNA concentration, HIF-1α protein by ELISA, and renin mRNA by RT-PCR. Results: Differences between the groups in the ratios of kidney weight to BW and in the concentrations of HIF-1α protein were statistically not significant (p > 0.05). Relative expression of renin mRNA increased markedly starting 8 hours after CoCl2 IP injection (30 times over controls) and further rising until 24 hours (2465 times over controls). Correlation between HIF-1α and renin mRNA by Pearson analysis was strongly positive, but not significant (R = 0.91; p = 0.09). Conclusion: Renin gene regulation in renal hypoxic mimicry strongly correlates with HIF-1α.


Subject(s)
Mice , Kidney , Renin , Chlorides
19.
Article in Chinese | WPRIM | ID: wpr-418256

ABSTRACT

ObjectiveTo investigate the influence of cobalt chloride ( CoCl2 )-mimetic hypoxia on theproliferation,apoptosis and migration of human pancreatic cancer cell fine PANC1.MethodsPANC1 cells were treated with 0(control),100,200,400,800 μmol/L CoCl2 respectively for 24 h.Real-time RT-PCR and Western blot were used to determine hypoxia induced factor ( HIF)-1o mRNA and protein expression respectively,and cell counting kit-8(CCK-8) assays,flow cytometry and cell scratch test were used to examine the proliferation,apoptosis and migration of PANC1 cells,respectively.ResultsIn the control group and 100,200,400 and 800 μmol/L CoCl-2 groups,the expressions of HIF-1t mRNA were 1,1.08 ±0.12,1.12 ± 0.09,1.04±0.11,0.66 ±0.07,and the expressions of VEGF mRNA were 1,2.69±0.35,4.81 ±0.54,2.19 ± 0.21,0.79 ± 0.08,while the expressions of HIF-1 α protein were 0.23 ± 0.03,0.36 ± 0.04,1.15 ± 0.11,1.08 ± 0.09,0.44 ± 0.04; and the expressions of VEGF protein were 0.14 ± 0.02,0.12 ± 0.01,0.95 ±0.09,0.87 ±0.09,0.55 ±0.06; and cell viability rates were 100%,(98.43 ±2.88)%,(76.15 ± 0.70)%,(53.87 ±0.77)%,(35.23 ±0.67)% ; while cell apoptotic rates were (5.2 ±1.12)%,(5.74 ± 1.07)%,(6.82 ± 1.85)%,(12.09 ±3.53)%,(31.88 ±6.95)% ; the cell migration distance of PANC1 cells were (43.24 ±3.67)%,(59.46 ±5.39)%,(80.56 ±8.05)%,(63.89 ±5.96)%,(9.09 ± 1.59 ) %.Compared with those of control group,the expressions of VEGF mRNA,VEGF and HIF-1 α protein,cell migration distance showed a two-way variation ( ascending first and descending later) (P <0.05 ),and the expression of HIF-1α mRNA and cell proliferation rate was decreased in a dose-dependent manner,while the cell apoptosis was increased in a dose-dependent manner.Conclusions CoCl2 significantly inhibits the proliferation and promotes apoptosis of PANC1 cells at certain level.CoCl2 has a two-way effect on the migration of PANC1 cells,and it may be related to the direct injury of high concentration of CoCl2 on cells.

20.
Chinese Journal of Diabetes ; (12): 57-62, 2010.
Article in Chinese | WPRIM | ID: wpr-403888

ABSTRACT

Objective To investigate the effect and mechanism of different high concentrations glucose(Glu) on the expression of hypoxia-inducible factor 1α(HIF-1α) in cultured neonatal rat eardiomyocytes under hypoxia and non-hypoxia conditions. Methods Neonatal rat cardiomyocytes were cultured for 6 hours under different conditions and were divided into 6 groups:①Negative control group (5.5 mmol/L Glu) ; ②hypoxia mimicking cobalt chloride(Cocl_2) group(5.5 mmol/L Glu + 400 μmol/L cocl_2 ) ;③Different high concentrations Glu groups: (11.1 mmol/L, 22.2 mmol/L, 33.3 mmol/L Glu);④cocl_2 +different high concentrations Glu groups(11.1 mmol/L Glu +400 μmol/L cocl_2,22.2 mmol/L Glu +400 μmol/L cocl_2, 33.3 mmol/L Glu+400 μmol/L cocl_2);⑤High concentrations Glu + antioxidant α-tocopherol group(33.3 mmol/L Glu + 100 μmol/L α-tocopherol) ; ⑥ High concentrations Glu+antioxidant α-tocopherol+cocl_2 group(33.3 mmol/L Glu+400 μmol/L cocl_2+100 μmol/L α-tocopherol). The effect of high concentrations Glu, cocl_2 and high concentrations Glu plus cocl_2 on the expression of HIF- 1α mRNA and protein in cultured neonatal rat cardiomyocytes, as well as the effect of high concentrations Glu plus antioxidant α-tocopherol, high Glu concentrations plus cocl_2 and antioxidant α-tocopherol on the expression of HIF- 1α mRNA and protein were observed. Results 1. Compared with negative control group(5.5 mmol/L Glu), the 'expression of HIF- 1α was increased under cocl_2 mimicked hypoxia(5.5 mmol/L Glu+400 μmol/L cocl_2, 11.1 mmol/L Glu+400 μmol/L cocl_2 ,22.2 mmol/L Glu+400 μmol/L cocl_2,33.3 mmol/L Glu+400 μmol/L cocl_2). 2. The expression of HIF- 1α was increased gradually after the increasing of Glu concentrations(5.5 mmol/L, 11.1 mmol/L, 22.2 mmol/L and 33.3 mmol/L Glu).3.The expression of HIF-1α was decreased gradually after the increasing of Glu concentrations under certain cocl_2 plus different high concentrations Glu (5.5 mmol/L Glu+400 μmol/L cocl_2, 11.1 mmol/L Glu+400 μmol/L cocl_2, 22.2 mmol/L Glu+400 μmol/L cocl_2,33.3 mmol/L Glu+400 μmol/L cocl_2). 4. Under high concentration Glu plus antioxidant α-tocopherol (33.3 mmol/L Glu+100 μmol/L α-tocopherol) ,the expression of HIF- 1α was less increased than the same high Glu concentration(33.3 mmol/L Glu). 5. Under high Glu concentration plus cocl_2 (33.3 mmol/L Glu+400 μmol/L cocl_2), the expression of HIF- 1α increased less than that of the same high Glu concentration plus antioxidant α-tocopherol and cocl_2 (33.3 mmol/L Glu+400 μmol/L Cocl_2+ 100 μmol/L α-tocopherol). Conclusions High glucose increases the expression of HIF-1α under non-hypoxia, but blunts the expression of HIF-1α under hypoxia in cultured neonatal rat cardiomyocytes. Some mechanisms such as ROS (reactive oxygen species) signal transduction system and oxidative stress may be involved in it.

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