Inhibitory effect of quercetin on apoptosis of PC12 cells induced by rotenone

AIM: To observe the effects and mechanisms of quercetin on the apoptosis of PC12 cells induced by rotenone. METHODS: PC12 cells were used in the study. Quercetin at the concentration of 300 μmol/L was added into the PC12 cells cultured in DMEM-F12 medium with 10% fetal calf serum. The morphological changes of the cells were observed under fluorescence microscope. The apoptotic rate was determined by flow cytometry assay. The protein levels of Bax and Bcl-2 were determined by Western blotting, and the mitochondrial membrane potential was measured by ratiometric probe JC-1.RESULTS: In the cells treated with rotenone+quercetin, the morphology of the cells was significantly improved, and the apoptotic rate was decreased to 6.7%, significantly lower than that in the cells treated with rotenone alone (P<0.01). The expression of Bcl-2 was up-regulated and Bax was down-regulated in rotenone+quercetin group (P<0.01), while the mitochondrial membrane potential was also increased (P<0.01) as compared to those in rotenone group.CONCLUSION: Pretreatment of quercetin inhibits the development of apoptosis in PC12 cells induced by rotenone. One of the mechanisms may be correlated with up-regulating the expression of Bcl-2 and down-regulating the expression of Bax, thus maintaining mitochondrial membrane potential.     

Bone marrow-derived mesenchymal stem cells protect against hypoxia-induced injury and apoptosis of PC12 cells via up-regulation of erythropoietin expression

AIM： To investigate the effects of bone marrow-derived mesenchymal stem cells (BM-MSCs) on cobalt chloride (CoCl2)-induced injury and apoptosis of PC12 cells. METHODS：PC12 cells were divided into control group, CoCl2 group, BM-MSCs-siCTL+CoCl2 group and BM-MSCs-siEPO+CoCl2 group. The viability of the PC12 cells was measured by MTT assay. Flow cytometry and Hoechst 33258 staining were used to determine the apoptotic rate and the changes of chromatin distribution in PC12 cells. The expression of erythropoietin (EPO) in BM-MSCs was measured by RT-PCR and Western blotting. The mRNA expression of Bcl-2 and Bax in PC12 cells was detected by RT-PCR. Caspase-9 and caspase-3 assay kits were used to detect the activity of caspase-9 and caspase-3. RESULTS：The viability of PC12 cells treated with CoCl2 for 24 h and 48 h decreased to （43.0±6.4）% and （33.8±5.7）%, respectively, while 1∶15 ratio of BM-MSCs co-culture increased the cell viability to （77.9±3.8）% and （75.2±9.7）%,respectively. The expression of EPO in BM-MSCs was up-regulated after treated with 0.6 mmol/L CoCl2 for 24 h and 48 h, while EPO siRNA significantly abrogated the EPO expression in BM-MSCs. BM-MSCs-siCTL co-culture significantly inhibited the apoptosis of PC12 cells induced by CoCl2. However, EPO siRNA the protective effect of BM-MSCs. Compared with CoCl2 treatment group, BM-MSCs co-culture induced remarkable increase in the expression of Bcl-2 and decrease in the expression of Bax in PC12 cells, which was reversed by EPO siRNA. BM-MSCs-siCTL co-culture remarkably abrogated the CoCl2 induced up-regulation of caspase-9 and -3, while BM-MSCs-siEPO co-culture significantly reversed the down-regulation of caspase-9 and -3 induced by BM-MSCs-siCTL co-culture. CONCLUSION：BM-MSCs protect PC12 cells from apoptosis induced by CoCl2. The protective effect of BM-MSCs might be executed by up-regulating the expression of EPO.     

Minocycline suppresses sodium nitroprusside-induced apoptosis in PC12 cells by regulating PI3K/Akt signaling

AIM：To explore the role of PI3K/Akt signaling in the anti-apoptotic effect of minocyline (MC) on the apoptosis of PC12 cells induced by sodium nitroprusside (SNP). METHODS：PC12 cells were divided into 4 groups: blank control group, SNP (500 μmol/L) group, MC (10 μmol/L)+SNP group and LY294002+MC+SNP group. The cell viability was observed by MTT assay. The expression of Akt and p-Akt was determined by Western blotting. RESULTS： The viability of the PC12 cells decreased after exposed to 500 μmol/L SNP for 24 h. Meanwhile, MC at concentration of 10 μmol/L significantly blocked the effect of SNP, such as decreasing the cell viability. Pretreatment with LY294002 for 60 min prior to exposure of the PC12 cells to MC and SNP down-regulated the expression of p-Akt induced by SNP. CONCLUSION：Minocycline regulates PI3K/Akt signaling pathway to restrain the apoptosis of PC12 cells induced by SNP.     

Relationship between morphological changes of autophagy and apoptosis in PC12 cells induced by oxygen-glucose deprivation and reoxygenation

AIM: To investigate the relationship between morphological changes of autophagy and apoptosis in the PC12 cells induced by oxygen-glucose deprivation and reoxygenation. METHODS: The PC12 cells were randomly divided into normal control group, oxygen-glucose deprivation and reoxygenation group, autophagy inhibitor group and autophagy activator group. The cells in oxygen-glucose deprivation and reoxygenation group, autophagy inhibitor group and autophagy activator group were exposed to reoxygenation (12 h) after 3 h of oxygen-glucose deprivation, and autophagy inhibitor 3-methyladenine and autophagy activator rapamycin were added into the cells at the same time. Using transmission electron microscope and monodansylcadaverine fluorescence staining, the morphological changes of autophagosome were observed. The apoptosis of the PC12 cells were analyzed by flow cytometry with Annexin V-FITC/PI staining and TUNEL method. RESULTS: Compared with normal control group, the numbers of autophagosomes and the apoptotic rates increased in oxygen-glucose deprivation and reoxygenation group (P<0.05). Compared with oxygen-glucose deprivation and reoxygenation group, the numbers of autophagosomes decreased obviously (P<0.05) and the apoptotic rates increased markedly in autophagy inhibitor group (P<0.05). The numbers of autophagosomes increased obviously (P<0.05), the apoptotic rates decreased markedly (P<0.05), the autophagosomes became bigger in size, and autolysosomes was also found in autophagy activator group. CONCLUSION: Oxygen-glucose deprivation and reoxygenation induce autophagy in PC12 cells, and autophagy inhibits cell apoptosis to play a protective role.     

miR-422a regulates SOX6 to inhibit injury of PC12 cells induced by hydrogen peroxide

AIM: To investigate the effects of microRNA-422a (miR-422a) on the damage of rat adrenal gland pheochromocytoma PC12 cells induced by hydrogen peroxide (H₂O₂). METHODS: The expression of miR-422a in the PC12 cells treated with H₂O₂ was detected by real-time PCR. After miR-422a mimics were transfected into PC12 cells, the cell viability was measured by MTT assay, the lactate dehydrogenase (LDH) leakage rate was detected, and the apoptosis was analyzed by flow cytometry. Target gene prediction software was used to predict that sex-determining region Y box 6 (SOX6) may be the target gene of miR-422a. Luciferase reporter assay was used to identify the targeting relationship. miR-422a mimics and SOX6 over-expression vector were co-transfected into the PC12 cells. The effects of SOX6 over-expression on the viability, LDH leakage rate and apoptosis of PC12 cells treated with H₂O₂ and transfected with miR-422a mimics were evaluated. RESULTS: The expression of miR-422a in the PC12 cells was decreased after treatment with H₂O₂ (P<0.05). The viability of PC12 cells treated with H₂O₂ was decreased, and the LDH leakage rate and apoptotic rate were increased. Transfection with miR-422a mimics enhanced the viability of PC12 cells treated with H₂O₂, and the leakage rate of LDH and apoptotic rate of the PC12 cells were reduced. The expression of SOX6 was negatively regulated by miR-422a. SOX6 over-expression reversed the effects of miR-422a on PC12 cell viability, LDH leakage and apoptosis. CONCLUSION: miR-422a reduces the damage of PC12 cells induced by H₂O₂ via targeting SOX6.     

Effects of iPSC-MSCs on mitochondria of PC12 cells injured by CoCl2

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 increased and MMP of PC12 cells was decreased after exposed to CoCl₂ at concentration of 400 μmol/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 mitochondrial transfer from iPSC-MSCs to PC12 cells.     

Protective effect of progesterone on PC12 cells injured by oxygen-glucose deprivation

AIM: To investigate the effects of progesterone on the cell viability and expression of glucose transporter type 3(GLUT3) in PC12 cells injured by oxygen-glucose deprivation (OGD) in attempt to prove the neuroprotection of progesterone (PROG) against the hypoxic-ischemic injury in cultured cells in vitro. METHODS: Well-differentiated PC12 cells induced by nerve growth factor were randomly divided into 3 groups. In normal group, the cells were cultured without OGD treatment. In OGD group, the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 95% N₂ and 5% CO₂ for 30 min. After that, the cells were fed with glucose-supplemented medium and cultured under normoxic condition for 24 h. In PROG+OGD group, the cells were given the same treatments as those in OGD group except that the medium contained progesterone at concentration of 10 nmol/L. Cellular morphological changes were observed after OGD for 30 min. The cell viability was assessed by WST-8 assay. The degree of the cell damage was evaluated by determining lactate dehydrogenase (LDH) leakage. The expression of GLUT3 at mRNA and protein levels was examined by RT-PCR and Western blotting, respectively. RESULTS: Progesterone attenuated the cellular swelling, decreased the leakage of LDH and improved the viability of PC12 cells injured by OGD (P<0.01). The expression of GLUT3 at mRNA and protein levels in PC12 cells in PROG+OGD group was significantly higher than that in OGD group (P<0.05). CONCLUSION: Progesterone has protective effect on in vitro cultured PC12 cells injured by OGD. The mechanism may be related to the up-regulation of GLUT3 protein.     

Acetyl-L-carnitine attenuates H2O2-induced oxidative damage of PC12 cells

AIM: To investigate the effect of acetyl-L-carnitine (ALC) on H₂O₂-induced oxidative damage in PC12 cells and its possible mechanism. METHODS: A moderate oxidative damage PC12 cell model was induced by exposure of the PC12 cells to H₂O₂. ALC at different concentrations (100, 200 and 400 μmol/L) was applied to the PC12 cells cultured in vitro, and CCK8 assay was used to detect the cell viability. The cells were divided into control group, H₂O₂ group, and low-ALC, medium-ALC and high-ALC groups. The apoptosis of the cells was analyzed by flow cytometry. The protein levels of Nrf2 and cleaved caspase-3 were determined by Western blot. The nuclear translocation of Nrf2 was observed by immunofluorescence staining. RESULTS: ALC at different concentrations (100, 200 and 400 μmol/L) significantly inhibited H₂O₂-induced PC12 cell apoptosis, and the medium concentration group had the best effect. Compared with H₂O₂ group, low, medium and high concentrations of ALC significantly increased the viability of the PC12 cells induced by H₂O₂, inhibit cell apoptosis (P<0.05), significantly down-regulated the protein level of cleaved caspase-3 (P<0.05), up-regulated the protein level of Nrf2 (P<0.05), and promoted the translocation of Nrf2 from the cytoplasm to the nucleus. CONCLUSION: Acetyl-L-carnitine attenuates H₂O₂-induced oxidative damage of PC12 cells, inhibits the apoptosis and increases the viability, which is related to the activation of Nrf2 signaling pathway.     

Docosahexaenoic acid protects human retinal pigment epithelial cells against oxidative stress-induced apoptosis

AIM: To observe the effect of docosahexaenoic acid(DHA) on H₂O₂-induced apoptosis in human retinal pigment epithelium cells and its molecular mechanism. METHODS: Human retinal pigment epithelium cell line ARPE-19 was cultured in vitro, and 12.5 mmol/L H₂O₂ was used to mimic the oxidative stress condition. The cells were treated with 30~100μmol/L DHA for 4~24 h. The expression of heme oxygenase-1(HO-1) at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The enzymic activity of HO-1 was measured by colorimetry. Production of reactive oxygen species(ROS) was determined by fluorescent probe. Activation of NF-E2-related factor 2(Nrf2) was examined by immunofluorescence method. Apoptosis of ARPE-19 cells was analyzed by flow cytometry. RESULTS: The mRNA and protein expression and the enzymic activity of HO-1 were significantly increased in the ARPE-19 cells after DHA treatment. Meanwhile, nuclear translocation of Nrf2 was also observed. Apoptosis appeared and ROS was produced upon H₂O₂ incubation. In contrast, DHA at 100μmol/L significantly abrogated H₂O₂-induced apoptosis and ROS production. Furthermore, silencing of HO-1 by specific siRNA, or treatment with ZnPP, an inhibitor of HO-1, partly counteracted the protective effect against H₂O₂-induced apoptosis and ROS production. CONCLUSION: DHA protects retinal pigment epithelial cells against oxidative stress via induction of heme oxygenase-1 expression after Nrf2 activation.     

Protective effect of hydrogen sulfide on PC12 cells injured by ATP

AIM: To prove the purinergic signaling mechanism of the neuroprotective action of hydrogen sulfide by observing the effects of sodium hydrosulfide (NaHS), a donor of hydrogen sulfide, on the cell viability, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the change of membrane permeability in the PC12 cells injured by adenosine triphosphate (ATP). METHODS: PC12 cells in logarithmic growth phase were randomly divided into 4 groups. In control group, the cells were cultured without ATP treatment. In ATP group, the cells were treated with ATP after cultured for 24 h. In NaHS+ATP group, the cells were incubated with NaHS for 30 min before treated with ATP, and NaHS always existed in the reaction system. In KN-62+ATP group, the cells were pretreated with KN-62 for 30 min, and the other treatments were as the same as those in NaHS+ATP group. The cell viability was assessed by MTT assay. The [Ca²⁺]_i was detected by Fura-2/AM staining. The membrane permeability was observed by staining with fluorescent dye YO-PRO-1.RESULTS: ATP at concentration of 0.3 mmol/L showed no injury effect on the cells. However, the cell viability was dropped gradually in a dose-dependent manner as the ATP at doses of 1, 3, 5 and 10 mmol/L. The decline of cell viability by ATP was obviously reversed by 200 μmol/L of NaHS in the PC12 cells (P<0.05), but exasperated by 800 μmol/L of NaHS (P<0.05). At the same time, ATP evoked the increase in [Ca²⁺]_i in a dose-dependent manner, which was inhibited by NaHS (P<0.05). Furthermore, the YO-PRO-1 uptake induced by ATP in a dose-dependent and time-dependent manner was also reduced by NaHS (P<0.05). CONCLUSION: Hydrogen sulfide has protective effect on the PC12 cells injured by ATP. The mechanism may be related to the reverse of the increased [Ca²⁺]_i and YO-PRO-1 uptake.     

L-dopa induction of pheochromocytoma 12 cells apoptosis and the protective function of glutathione

AIM:To study the induction of apoptosis of pheochromocytoma (PC12) cell by L - dopa and the clinical significance. METHODS: Using PC12 cells as the medel of dopaminergic neurons,in addition to electron microscopy, agarose gel electrophoresis and flow cytometry, the apoptosis ratio by L - dopa and the effect of antioxi- dant glutathione on PC12 cells were observed. RESULTS: Treabent of PC12 cells with L - dopa in concentrations of 50 μmol/L, 100μmol/L and 150 μmol/L respectively for 24 hours, results revealed that the apoptosis ratio was 12.8%, 24.4%, 37.2%, respectively, which is in cobant with fragment pragment propartions of agarose gel electrophoresis, mere higher than that of control(2.3% ) (P < 0.01 ). The apoptosis ratio of glutathione + L - dopa group was similar to that of control (P > 0.05). CONCLUTION: Induction of apoptosis by L - dopa could be inhibited by glutathione, and oxidative damage may be involved in the pathophysiology of dopaminergic neurons death after long - term treat- ment of L - dopa.     

Phosphorylation PKCδ participates in apoptosis of PC12 cells induced by 6-hydroxydopamine

AIM: To observe the effect of phosphorylation protein kinase C delta (PKCδ) on the procedure of PC12 cells apoptosis induced by 6-hydroxydopamine(6-OHDA) and to investigate the potential molecular pathogenesis of Parkinson disease.METHODS: TUNEL staining and transmission electron microscope were applied to measure apoptosis when dopaminergic PC12 cells exposed to the excitomotors and inhibitors of PKC before 6-OHDA for 18 hours. The expression of phosphorylation of PKCδ was detected by Western blotting. RESULTS: PMA, an activating agent of PKCδ, significantly increased PC12 cell apoptosis induced by 6-OHDA. Rottlerin, an inhibitor of PKCδ, protected PC12 cells apparently. As contrast, bisindolylmaleimide I, an inhibitor of general PKC and G6976, the inhibitor of calcium-dependent PKC, did not show any protective role. CONCLUSION: The phosphorylation PKCδ is one of the important links in the process of PC12 cell apoptosis induced by 6-OHDA. PKCδ may directly participate in neurodegeneration process in parkinsonian.     

Effect of ginkgolide B on cholesterol content in PC12 cells under high cholesterol culture

AIM: To explore the effect of ginkgolide B (GB) on high cholesterol-induced dysfunction in PC12 cells. METHODS: The rat brain microvascular endothelial cells and astrocytes were used to establish the blood-brain barrier (BBB) model by in vitro co-culture in the Transwell device with PC12 cells. Cholesterol at the concentration of 40 μmol/L was added into the side of brain microvascular endothelial cells of the Transwell. Various concentrations of GB were applied to deal with the culture system. The morphological changes of the cells were observed under microscope. The safe doses of GB were screened by MTT assay. The level of cholesterol in the PC12 cells was determined by the high-performance liquid chromatography. RESULTS: The morphology of PC12 cells in control group was normal,with round,short spindle or triangle shape,cytoplasm extension,two-pole short processes,good refraction and obvious nucleus.The doses of GB determined by MTT assay were 1 μmol/L for low dose,5 μmol/L for middle dose and 25 μmol/L for high dose. Compared with control group, the cholesterol obviously increased in model group with statistical difference (P<0.05). GB at dose of 25 μmol/L for 16 h exposure had the optimum results to decrease the cholesterol level in PC12 cells. However, no significant difference between GB high dose group and simvastatin group was observed (P>0.05). CONCLUSION: The active monomer of ginkgo leaf, GB, may effectively reduce the cholesterol content in PC12 cells with the condition of BBB in vitro.     

Arachidonic acid cytochrome P450 epoxygenases protect vessel endothelial cells against apoptosis induced by tumor necrosis factor-α

AIM: To investigate whether endogenous endothelium-derived hyperpolarizing factors (EHDFs) produced by CYP epoxygenases BM₃·F87V,2C11OR or CYP2J2 transfection was able to protect endothelial cells against apoptosis induced by tumor necrosis factor alpha.METHODS: Three or four passages of cultured bovine aortic endothelial cells (BAECs) were transfected with epoxygenases or the empty vector (pCB₆).Cell viability was detected by MTT assay.Apoptosis of transfected endothelial cells was evaluated by DNA ladder assay,flow cytometry and morphological observations under fluorescence microscopy.RESULTS: Overexpression of CYP epoxygenases BM₃·F87V,2C11OR,CYP2J2 increased cell viability respectively observed by MTT assay.The percentage of cells undergoing apoptosis was decreased in 2C11OR-, BM₃F87V- or 2J2-transfected cells compared to the vector as evaluated by DNA fragment assay,flow cytometry analysis and morphological observations under fluorescence microscopy.CONCLUSION: Overexpression of CYP epoxygenases BM₃·F87V,2C11OR or 2J2 increases cell viability and protects endothelial cells against TNF-α-induced apoptosis.These findings suggest new targets to investigate the endothelium-associated disorders and provide novel therapeutic strategies to treat them by modulating cytochrome P450 epoxygenases.     

Effects of sinapic acid on proliferation and apoptosis of rat vascular smooth muscle cells induced by high glucose

AIM: To investigate the effects of sinapic acid(SA) on the proliferation and apoptosis of rat vascular smooth muscle cells(VSMCs) induced by high glucose(HG). METHODS: Cultured A7r5 cells were randomly divided and treated as indicated. The cell viability was determined by MTT assay. DNA synthesis was measured by BrdU assay. Cell cycle progression and cell apoptotic rate were determined by flow cytometry analysis. The levels of reactive oxygen species(ROS) were detected by ELISA. The protein levels of cyclin D1, P21, P27, phosphorylated protein kinase C(p-PKC), p-P38 and β-actin were evaluated by Western blot. RESULTS: Compared with control group, the viability of A7r5 cells was significantly enhanced, the DNA synthesis was increased, the cell cycle progression was promoted, the levels of ROS were elevated, the cell apoptotic rate was reduced, the protein expression of P21 and P27 was decreased, and the protein levels of cyclin D1, p-PKC and p-P38 were increased in HG group(all P<0.05). These effects were reversed by SA(0.1, 1 and 10 μmol/L) treatment in a dose-dependent manner(all P<0.05). Both P38 inhibitor SB203580 and PKC inhibitor chelerythrine significantly inhibit HG-induced PKC/P38 activation and cell viability(P<0.05).CONCLUSION: SA inhibits HG-induced VSMCs proliferation and promotes cell apoptosis via reducing PKC/P38 activation.     

Exogenous ATP induces formation of membrane pore in PC12 cells

AIM：To investigate the formation of membrane pore in PC12 cells induced by exogenous adenosine triphosphate (ATP) and to identify the key molecular targets. METHODS：PC12 cells were treated with different concentrations of ATP to establish the injury model. The morphological change was observed under an inverted phase-contrast microscope. The viability of the PC12 cells was measured by CCK-8 assay. Fluorescent dye YO-PRO-1 was used to detect the membrane permeability. The expression of P2X7 receptor and pannexin 1 (Panx1) at mRNA and protein levels was assessed by real-time PCR and Western blotting. RESULTS：After exposed to ATP (1 mmol/L, 3 mmol/L and 5 mmol/L) for 3 h, the PC12 cells became edematous, and the number of adherent cells decreased gradually in a dose-dependent manner. The cell viabilities in 3 mmol/L ATP group and 5 mmol/L ATP group were significantly decreased compared with control group (P<0.05). YO-PRO-1 uptake in the PC12 cells exposed to ATP (0, 1, 3 and 5 mmol/L) for 15 min, 30 min and 60 min increased in a dose-dependent and time-dependent manner. The cell viability increased and the intracellular fluorescence intensity induced by ATP were significantly antagonized in brilliant blue G (a P2X7 receptor inhibitor) pretreatment group (P<0.05), whereas it did not change in carbenoxolone (a Panx1 inhibitor) pretreatment group (P>0.05). The expression of P2X7 receptor at mRNA and protein levels was significantly increased (P<0.05), but the expression of Panx1 was not changed (P>0.05) when PC12 cells were exposed to ATP for 3 h. CONCLUSION：Extracellular ATP at high concentration may induce membrane pore formation with the expression and activation of P2X7 receptor in PC12 cells.     

Oxidative stress induces apoptosis in HepG2 cells

AIM: Direct exposure of cells to reactive oxygen species can induce apoptosis. In this study we investigate how oxidative stress induces cell death in HepG2 cells and characterize the molecular events involved.METHODS: Oxidative stress was created by exposing HepG2 cells to 2 mmol/L H2O2. Apoptosis was determined by analysis of DNA fragmentation by agarose gel electorphoresis. The mitochondrial membrane potential was analyzed using DePsipher fluorescent staining and the expression of cytochrome c in the cytosolic fraction was measured by Western blotting analysis. The caspase activity was detected using fluorometric assay kit by a fluorescence microplate reader.RESULTS: When HepG2 cells were treated with 2 mmol/L H2O2, the cells displayed DNA fragmentation, a typical feature of apoptosis, after 12 h. The mitochondrial membrane potential appeared different in two group of cells. H2O2-treated cells appeared green fluorescence as early as 4 h, which represents de-energized mitochondria, the untreated cells appeared red fluorescence, a feature of mitochondria with intact membrane potential. In treated cells, the expression of cytochrome c increased and accumulated in cytosolic fraction with treatment time, caspase-3 activity increased by 6.7-fold (P<0.01) at 8 h and caspase-9 activity increased by 3.6-fold (P<0.01) at 12 h, respectively, however, the activity of caspase-8 remained unchanged.CONCLUSION: These findings suggest that oxidative stress can induce apoptotic cell death in HepG2 cells, and the mechanism is related to mitochondrial pathway, which activates caspase-9 and-3, but not caspase-8.     

Effects of brain tissue extract of hypoxia-preconditioned mice at different concentrations on tolerance of PC12 cells to hypoxia

AIM: To investigate the effect of brain tissue extract of hypoxia-preconditioned mice (HP extract) on tolerance of PC12 cells to hypoxia. METHODS: The mice model of acute repetitive hypoxia was reproduced and brain tissue extracts were prepared. HP extract was added into the cultures of PC12 cells and the final concentrations of HP extracts were 0.2, 0.8, 3.2, 6.4 or 12.8 g/L (HP group), respectively. Brain tissue extract of normal mice (N extract) at the same five concentrations were used as controls (N group). The PC12 cells were cultured in hypoxia (2% O2). After hypoxia for 24 h, 48 h or 72 h, colorimetric method (A570) of tetrazolium salt MTT (3-［4,5-dimethylthiazol-2-yl］-2,5-diphenyl tetrazolium bromid) staining was adopted to determine the cell viability and lactate dehydrogenase (LDH) release percentage assay was also conducted after 24 h, 48 h or 72 h hypoxia. Besides, apoptotic percentages at early stage (24 h hypoxia) and late stage (72 h hypoxia) were detected respectively by means of annexin V-FITC/PI double-stained flow cytometry and Hoechst 33258 stained fluorescence microscopy. RESULTS: HP extract at the concentrations lower than 6.4 g/L (including 6.4 g/L) showed protective effect on PC12 cells in early stage of hypoxia (24 h). A570 values in HP group were significantly higher than those in N group, but LDH release percentages were significantly lower than those in N group after 24 h hypoxia. With hypoxia prolonging, HP extract at high concentrations gradually lost the protective effect. At the time point of 72 h hypoxia, HP extract at concentrations higher than 6.4 g/L (including 6.4 g/L) had pro-apoptotic effect. At this time point, A570 values of HP groups at these concentrations were significantly lower than those in the corresponding N group, both LDH release percentages and apoptotic percentages were significantly higher than those in the N group. CONCLUSION: The effects of HP extract on tolerance of PC12 cells to hypoxia depend on its concentrations and on the time of treatment.     

Effect of miRNA-21 on protection of PC12 cells from oxygen-glucose deprivation damage

AIM: To investigate the effect of microRNA (miRNA)-21 on the PC12 cells with hypoxic-ischemic damage.METHODS: The PC12 cells were cultured in vitro, and the cell model of oxygen-glucose deprivation (OGD) was established. In accordance with the following requirements, the cells were randomly divided into control group, OGD group, negative control sequence+OGD group, miRNA-21 inhibitor+OGD group and miRNA-21 mimic+OGD group. The effects and mechanism of miRNA-21 on the protection of PC12 cells from OGD damage were determined by CCK-8 assay, real-time PCR and Western blot.RESULTS: Decrease in the expression of miRNA-21 by transfection with miRNA-21 inhibitor inhibited the viavility of the PC12 cells subjected to OGD damage. Increase in the expression of miRNA-21 by transfection with miRNA-21 mimic promoted the viability of the PC12 cells subjected to OGD damage. It was further confirmed that miRNA-21 promoted the AKT phosphorylation in OGD-damaged PC12 cells.CONCLUSION: miRNA-21 significantly increases the viability of PC12 cells subjected to OGD damage, which may be related to the activation of PI3K/AKT signaling pathway.