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Objective To explore the effect of serum deprivation on proliferation and apoptosis of cardiomyocytes and its mechanisms. Methods Human cardiomyocytes AC16 were randomly divided into serum-deprivation group (the cells were cultured in DMEM medium without fetal bovine serum for 48 h) and control group (the cells were cultured in DMEM medium containing 10% fetal bovine serum for 48 h). CCK-8 assay was performed to test the proliferation ability, and EdU imaging was conducted to detect the DNA synthesis level. Flow cytometry was performed to analyze the apoptosis level and mitochondrial membrane potential. Western blotting was conducted to detect the protein expression levels of caspase-9, Bcl-2, P53, proliferating cell nuclear antigen (PCNA), cyclin D1, β-catenin, Wnt5a, Axis inhibition protein 1 (Axin1), dishevelled 2 (Dvl2), dishevelled 3 (Dvl3) and low-density lipoprotein receptor-related protein 6 (LRP6). Results CCK-8 assay showed that the optical density value of the control group was 1.93±0.01 and that of the serum-deprivation group was 1.08±0.08, which was 55.8% of the control group, and there was significant difference between the two groups (P0.05). Western blotting analysis showed that serum deprivation could upregulate the protein expression of caspase-9 and downregulate the protein expression of Bcl-2, P53, PCNA and cyclin D1. Serum deprivation could inhibit the protein expression of β-catenin, Wnt5a, Axin1, Dvl2, Dvl3 and LRP6 in cardiomyocytes. Conclusion Serum deprivation can inhibit proliferation and induce apoptosis of cardiomyocytes, which may be mediated by inhibition of Wnt/β-catenin signaling pathway.
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The poor survival of mesenchymal stem cells (MSCs) compromises the efficacy of stem cell therapy.Growth factor deprivation is one of the important factors that have challenged the survival of donor MSCs in cell therapy.In this study,the aim was to evaluate the effect of serum deprivation on the cell death of MSCs and to investigate the underlying mechanisms.Apoptosis of MSCs was evaluated with Hoechst 33342/PI staining.Signaling pathways involved in serum-deprivation induced apoptosis were analyzed using Western blotting.The results revealed that serum deprivation induced apoptosis in MSCs within 72 h of treatment.Serum deprivation was shown to lead to protein expression alterations in Bax,Bcl-2,casepase-3,casepase-8,GRP78,and CHOP during experiments.The data suggested that the mitochondria death pathway,the extrinsic apoptotic pathway and the endoplastic reticulum(ER) stress pathway were all involved in MSCs apoptosis.The increase in expression of CHOP and the simultaneous decrease in Bcl-2 expression suggest a synergistic effect in apoptosis induction in both the mitochondrion and the ER.
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The poor survival of mesenchymal stem cells (MSCs) compromises the efficacy of stem cell therapy.Growth factor deprivation is one of the important factors that have challenged the survival of donor MSCs in cell therapy.In this study,the aim was to evaluate the effect of serum deprivation on the cell death of MSCs and to investigate the underlying mechanisms.Apoptosis of MSCs was evaluated with Hoechst 33342/PI staining.Signaling pathways involved in serum-deprivation induced apoptosis were analyzed using Western blotting.The results revealed that serum deprivation induced apoptosis in MSCs within 72 h of treatment.Serum deprivation was shown to lead to protein expression alterations in Bax,Bcl-2,casepase-3,casepase-8,GRP78,and CHOP during experiments.The data suggested that the mitochondria death pathway,the extrinsic apoptotic pathway and the endoplastic reticulum(ER) stress pathway were all involved in MSCs apoptosis.The increase in expression of CHOP and the simultaneous decrease in Bcl-2 expression suggest a synergistic effect in apoptosis induction in both the mitochondrion and the ER.
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Objective The renin-angiotensin system ( RAS) is involved in myocardial anoxic injury .This study aimed to in-vestigate the expressions of AT 1-R, AT2-R, and angiotensin-converting enzyme ( ACE ) in bone marrow mesenchymal stem cells (MSCs) under hypoxia. Methods Rat MSCs were isolated, cultured, and identified with CD29 and CD11b/c antibodies.The is-chemic injury model was established by exposing the MSCs to hypoxia and serum deprivation ( Hypoxia/SD) for 24 hours, while the control cells were cultured in L-DMEM with 10%FBS.The vitality and apoptosis of the cells were detected by trypan blue staining , CCK8 assay, and Annexin V-FITC staining.The mRNA and protein expressions of AT 1-R, AT2-R, and ACE were determined by real-time quantitative PCR and Western blot , respectively. Results The positive rate of CD29 was >97%and that of CD11b/c was <1% in the MSCs.Compared with the control group, Hypoxia/SD significantly increased the rate of cell apoptosis ([6.73 ±0.78]%vs [19.93 ±4.92]%, P<0.01), decreased the rate of cell viability ([78.49 ±4.94]%vs [37.33 ±2.91]%, P<0.01), and up-regulated the mRNA and protein expressions of AT 1-R, AT2-R, and ACE. Conclusion Hypoxia/SD activates the RAS in MSCs and improves the protective function of the cells against myocardial anoxic injury .
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Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.
Subject(s)
Animals , Cattle , Apoptosis/physiology , /metabolism , Chondrocytes/metabolism , Lentivirus/genetics , RNA Interference/physiology , Starvation/metabolism , Blotting, Western , Cartilage/metabolism , Caspase 9/metabolism , /metabolism , Flow Cytometry , Genetic Vectors/metabolism , Microscopy, Fluorescence , Primary Cell Culture , Propidium , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Serum/physiology , TransfectionABSTRACT
Objective To explore the effect of HeLa cells infected with Coxsackie virus B3 (CVB3) on the changes of mTOR signal pathway under different nutritional conditions.Methods The HeLa cells were cultured under conventional culture and serum starvation culture.(1) For the conventional method,the medium with 10 g/L fetal bovine serum was added for 24 h after the Hela cells were fused into 40％ to 50％,and the medium was changed on the next day,then the virus group was infected with CVB3 of 50％ tissue culture infective dose (TCID50).However,the control group was cultured by 2 g/L fetal bovine serum.(2) For the serum starvation method,HeLa cells were cultured with the medium without fetal bovine serum for 24 h.Then the virus group was infected with CVB3 of TCID50.The cells in control group were cultured by 2 g/L fetal bovine serum.Cell morphology changes were observed by inverted microscope,and the expressions of the mTOR,p70S6K mRNA were detected with Real-time PCR at 3 h,6 h,9 h,12 h,24 h respectively in both conventional culture and serum starvation groups.Results The expressions of mTOR and p70S6K mRNA were lower in the virus group than those in control group at 12 h and the 24 h (all P ＜0.05) in the conventional culture group.And the expressions of mTOR and p70S6K mRNA in the virus group were lower than those in the control group at every time points (all P ＜ 0.05) in serum starvation group.The expressions of mTOR and p70S6K mRNA in group with serum starvation virus and the control groups were higher than those in conventional culture group in all time points,but only the expressions of mTOR mRNA were significantly different between the 2 groups (all P ＜0.05),however,the expressions of p70S6K mRNA had no significant difference between the 2 groups (all P ＞ 0.05).Conclusion CVB3 may be able to down-regulate the expressions of mTOR and p70S6K mRNA.
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Objective To investigate the effect of serum-deprivation on the changes of [ Ca2+ ] i and the protein release of S100A13 and fibroblast growth factor 1 ( FGF-1 ) in thyroid cancer TT cell,and to reveal the role of Ca2+in the protein release of S100Al3 and FGF-1.Methods The protein expressions of FGF-1 and S100A13 in TT cells under serum-deprivation were detected by Western blot.The released FGF-1 protein from TT cells in the supernatant fluid was detected by ELISA.Realtime dynamic examinations on the change of 1 h [ Ca2+ ] i in TT cells under serum-deprivation were detected by confocal laser scanning microscopy.Then,the effect of EGTA( 2.5 mmol/ L),BAPTA-AM (2.5 μmol/L)on distributions of the fluorescence of S100AI 3 and FGF-1 in TT cells under serumdeprivation for6 h were detected by indirect immunofluorescence.Results The expressions of FGF-1 and S100A13 in TT cells after serum-deprivation for4 h and 6 h were reduced( P＜0.05 or P＜0.01 ),but the released FGF-1 protein from TT cells in the supernatant fluid was elevated ( P＜0.05 or P＜0.01).Confocal laser scanning of Ca2+ imaging indicated that [ Ca2+ ] i of serum-deprivation TT cells maintained the relative stabilization within 23 win,but the rapid rise of [ Ca2+ ] i achieved peak value 1.6 μmol/L after 30 min,and remained stable for about 17 win,and thereafter 40 win slowly dropped to a low level From 40 win to 60 win the [ Ca2+ ] i was about 0.3-0.6 μ mol/L.The average [ Ca2+ ] i was higher than that in normal group,EGTA group,and BAPTA-AM group within 1 h.The protein expressions of S100A13 and FGF-1 did not drop obviously in EGTA group and BAPTA-AM group.Conlusion The release of S100A13 and FGF-1 from TT cell under serum-deprivation is possibly related with the change of [ Ca2+ ]i.Both Ca2+-chelating agents EGTA and BA PTA-AM are able to inhibit the rise of [ Ca2+ ] i and release of S100A 13 and FGF-1 from TT cells under serum-deprivation.
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Objective To investigate the expression of insulin-like growth factor 2 receptor(IGF-2R)induced by hypoxia and serum deprivation(H/SD)in cultured neonatal rat cardiomyocytes in vitro.Methods Cultured neonatal rat cardiomyocytes were randomly divided into control group and H/SD group.Expression of IGF-2R mRNA was measured by RT-PCR and real time RT-PCR.Level of IGF-2R protein was measured by enzyme-linked immunosorbent assay(ELISA).Results Compared with control group,the expression of IGF-2R mRNA increased significantly (P ＜0.01)in cardiomyocytes cultured in H/SD condition for 12 h and 24 h,and IGF-2R protein was upregulated in cardiomyocytes cultured in H/SD condition for 24 h(P ＜0.05).Conclusion H/SD may induce IGF-2R expression,and IGF-2R may potentially explain the mechanism of cardiomyocyte damage induced by ischemia.
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We elucidated the effects of various components of ischemic medium on the outcome of simulated ischemia-reperfusion injury. Hypoxia for up to 12 hours induced neither apoptotic bodies nor LDH release. However, reoxygenation after 6 or 12 hours of hypoxia resulted in a marked LDH release along with morphological changes compatible with oncotic cell death. H9c2 cells were then subjected to 6 hours of simulated ischemia by exposing them to modified hypoxic glucose-free Krebs-Henseleit buffer. Lowered pH (pH 6.4) of simulated-ischemic buffer resulted in the generation of apoptotic bodies during ischemia, with no concomitant LDH release. The degree of reperfusion-induced LDH release was not affected by the pH of ischemic buffer. Removal of sodium bicarbonate from the simulated ischemic buffer markedly increased cellular damages during both the simulated ischemia and reperfusion. Addition of lactate to the simulated ischemic buffer increased apoptotic cell death during the simulated ischemia. Most importantly, concomitant acidosis and high lactate concentration in ischemic buffer augmented the reperfusion-induced oncotic cell death. These results confirmed the influences of acidosis, bicarbonate deprivation and lactate on the progression and outcome of the simulated ischemia-reperfusion, and also demonstrated that concomitant acidosis and high lactate concentration in simulated ischemic buffer contribute to the development of reperfusion injury.
Subject(s)
Acidosis , Hypoxia , Apoptosis , Cell Death , Hydrogen-Ion Concentration , Ischemia , Lactic Acid , Myocytes, Cardiac , Reperfusion , Reperfusion Injury , Sodium BicarbonateABSTRACT
Oxidative damage to mitochondria is a critical mechanism in necrotic or apoptotic cell death induced by many kinds of toxic chemicals. Thioredoxin (Trx) family proteins are known to play protective roles in organisms under oxidative stress through redox reaction by using reducing equivalents of cysteines at a conserved active site, Cys-X-X-Cys. Whereas biological and physiological properties of Trx1 are well characterized, significance of mitochondrial thioredoxin (Trx2) is not well known. Therefore, we addressed physiological role of Trx2 in PC12 cells under oxidative stress. In PC12 cells, transiently overexpressed Trx2 significantly reduced cell death induced by hydrogen peroxide, whereas mutant Trx2, having serine residues instead of two cysteine residues at the active site did not. In addition, stably expressed Trx2 protected PC12 cells from serum deprivation. These results suggest that Trx2 may play defensive roles in PC12 cells by reducing oxidative stress to mitochondria.
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Animals , Humans , Catalytic Domain , Cell Death , Cysteine , Hydrogen Peroxide , Hydrogen , Mitochondria , Oxidation-Reduction , Oxidative Stress , PC12 Cells , Serine , ThioredoxinsABSTRACT
PURPOSE: Thioredoxin is an endogenous antioxidant. It regulates the activities of transcriptional factors such as NF-kB(nuclear factor kappa B)and AP-1(activator protein-1) and it increases the synthesis of cytokines, preventing cellular proliferation and apoptosis. The aim of this study was to clarify the role of thioredoxin on apoptosis-inducing neuronal cell injury. We investigated the protective effects of thioredoxin against apoptosis-inducing neuronal cell injury through intracellular mechanism by 6-hydroxydopamine and serum deprivation. METHODS: PC 12 cells were cultured in RPMI 1640 media containing 10% fetal calf serum and subcultured in 96-well plates. Each well contained 30,000 cells. Cells were treated with hydrogen peroxide, diamide or 6-hydroxydopamine 30 minutes after thioredoxin treatment and then incubated for 24 hours. Cytotoxicity and cellular viability were assessed by measuring lactate dehydrogenase(LDH) release and MTT reduction. RESULTS: Thioredoxin increased cytotoxicity of PC cells treated with 6-hydroxydopamine by increasing LDH release and decreasing MTT reduction. In the serum deprivation condition, thioredoxin increased cytotoxicity of PC cells by increasing LDH release. CONCLUSION: Thioredoxin potentiates oxidative injury through intracellular mechanisms by 6-hydroxydopamine and serum deprivation instead of protecting. The cytotoxicity of thioredoxin may be mediated by decreasing the activity of NF-kB, which has been reported recently to protect against cellular apoptosis. Evidence suppors that the cytotoxic effect was not increased in the presence of serum in this study. Therefore, we found that the antioxidant effects of thioredoxin depended on mechanisms of injuries.
Subject(s)
Antioxidants , Apoptosis , Cell Proliferation , Cytokines , Diamide , Hydrogen Peroxide , Lactic Acid , Neurons , NF-kappa B , Oxidopamine , ThioredoxinsABSTRACT
Objective To study the protective effect of(-)clausenamide on the damage of PC12 cells induced by serum deprivation and to explore its related mechanism. Methods The cell viability was detected by MTT assay and morphological observation. The effect of(-)clausenamide on the PC12 cell apoptosis was analyzed by flow cytometry. Then western blotting and confocal microscope was used for the further study of effect of(-)clausenamide on the protein expression of GSK-3?,Bax and Bcl-2. Results(-)Clausenamide remarkably increased PC12 cell survival rate through inhibiting the PC12 cell apoptosis induced by serum deprivation at the concentration of 1 or 10 ? mol/L(P