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.     

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.     

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.     

Rapamycin reduces SH-SY5Y cell damage induced by oxygen-glucose deprivation

AIM: To observe the effect of rapamycin (Rapa) on human neuroblastoma SH-SY5Y cell injury induced by oxygen-glucose deprivation (OGD), and to explore the role of autophagy in this process. METHODS: The SH-SY5Y cells were randomly divided into 4 groups:normal control group:the cells were cultured without OGD treatment; Rapa group:the cells were pretreated with Rapa for 1 h; 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 1% O₂, 94% N₂ and 5% CO₂ for 12 h; Rapa+OGD group:the cultured cells were treated with Rapa for 1 h, and then were given the same treatments as those in OGD group. The cell viability was assessed by MTT assay. The degree of the cell damage was evaluated by determining the leakage of lactate dehydrogenase (LDH). The enzyme activity of caspase-3 was detected. TUNEL staining were used to detect the variation of cell apoptosis. The protein levels of apoptosis-related proteins Bax and Bcl-2, autophagy-related protein beclin-1 and autophagy marker protein LC3B were determined by Western blot. RESULTS: Compared with OGD group, the viability of the SH-SY5Y cells was significantly increased, and the activity of caspase-3 was significantly reduced in Rapa+OGD group (P<0.05). The SH-SY5Y cell injury was apparent after OGD with a great increase in the apoptotic rate (P<0.05). Compared with OGD group, the apoptotic rate significantly decreased in Rapa+OGD group (P<0.05). Compared with control group, the protein level of Bcl-2 was significantly decreased (P<0.05) and the protein level of Bax was significantly increased in OGD group. Compared with OGD group, the levels of Bcl-2, beclin-1 and LC3B-Ⅱ were significantly increased and the protein level of Bax was significantly increased in Rapa+OGD group (P<0.05). CONCLUSION: Rapamycin has a protective effect on in vitro cultured SH-SY5Y cells injured by OGD. The mechanism may be related to the promotion of autophagy.     

DL-3-n-butylphthalide attenuates mitochondrial damage in vascular endothelial cells under the condition of oxygen-glucose deprivation

AIM:To investigate the mechanism in which DL-3-n-butylphthalide (NBP) protects the mitochondria from the damage of oxygen-glucose deprivation (OGD). METHODS:Human umbilical vein endothelial cells (HUVECs) were exposed to OGD to induce endothelial damage. Mitochondrial morphology and mitochondrial reactive oxygen species (ROS) were examined using MitoTracker Green and MitoSOX Red, respectively. The activity of superoxide dismutase (SOD) was evaluated by SOD assay kit. RESULTS:NBP significantly attenuated OGD-induced mitochondrial fragmentation, reduced the content of mitochondrial ROS and increased the activity of SOD. CONCLUSION:NBP alleviates OGD-induced damage in the mitochondria. Reduction of mitochondrial ROS and enhancement of SOD activity may be the mechanism in which NBP protects mitochondria.     

Effect of rosuvastatin on cerebral microvascular endothelial cell injury induced by oxygen-glucose deprivation/reoxygenation

AIM: To explore the effect of rosuvastatin on the oxygen-glucose deprivation (OGD)/reoxygenation induced injury of cerebral microvascular endothelial cells (BMECs). METHODS: BMECs derived from BALB/c mice were isolated and cultured. BMECs were pretreated with rosuvastatin, followed by OGD for 3 h or 6 h and reoxygenation for 24 h. The morphological changes of BMECs were observed under light microscope. MTT assay was used to measured the cell viability, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining was used to assess the proliferation of BMECs. The protein levels of cleaved caspase-3 was observed by immunofluorescence staining. The protein levels of Bcl-2, Bax, matrix metalloproteinase (MMP) 2, MMP9, phosphorylated nuclear factor kappa B (p-NF-κB), phosphorylated P38 mitogen-activated protein kinase (p-P38) and phosphorylated c-Jun N-terminal kinase (p-JNK) were determined by Western blot. RESULTS: Rosuvastatin at 10 μmol/L improved the viability of the BMECs with OGD/reoxygenation-induced damage, and maintained the structure of BMECs. Moreover, rosuvastatin significantly prohibited the protein levels of cleaved caspase-3, MMP2, MMP9, p-NF-κB, p-P38 and p-JNK, and up-regulated the ratio of Bcl-2/Bax (P<0.05). CONCLUSION: Rosuvastatin reduces OGD/reoxygenation-induced injury of BMECs by inhibiting the expression of apoptosis-related proteins and MMPs, suggesting that rosuvastatin has potential value for the maintenance of blood-brain barrier.     

Hypoxic preconditioning protects rat myocardial microvascular endothelial cells against endoplasmic reticulum stress-induced injury

AIM：To investigate the effect of hypoxic preconditioning (HPC) on endoplasmic reticulum stress (ERS)-induced injury in cultured microvascular endothelial cells (MVECs) from rat hearts. METHODS：MVECs injury was induced by an ERS inductor thapsigargin (TG). Lactate dehydrogenase (LDH) leakage and apoptotic rate were detected to evaluate the injury of MVECs. Cytoskeleton and endoplasmic reticulum (ER) in MVECs were observed by phalloidin-FITC fluorescence staining and ER staining, respectively. Two-dimensional electrophoresis and mass spectrometry (MS) were used to identify proteomic profile in MVECs treated with TG. Western blotting was used to detect the expression of ERS markers, calreticulin (CRT) and glucose-regulated protein 78 (GRP78). RESULTS：TG induced the increase in LDH activity in medium and the apoptosis of MVECs in a dose-dependent manner. TG treatment up-regulated the expression of CRT and GRP78, while HPC attenuated the ERS-induced injury and the up-regulation of ERS markers in MVECs. CONCLUSION：HPC protects MVECs from ERS-induced injury.     

Mild hypothermia protects against injury of rat hippocampal neurons induced by oxygen-glucose deprivation

AIM：To study the protective effect of mild hypothermia (31~32 °C) on rat hippocampal neurons against oxygen-glucose deprivation (OGD)-induced injury and its possible mechanisms. METHODS：An OGD experimental model of rat hippocampal neurons in vitro was established to simulate cerebral ischemic-hypoxic injury. The rat hippocampal neurons were randomly divided into 4 groups：control group, mild hypothermia group, OGD group and mild hypothermia+OGD group. The cell morphology was observed under light and electron microscopes. The neuronal apoptosis was detected by flow cytometry. The activity of caspase-3 in the cytoplasm was measured by colorimetry. RESULTS：The neuronal injury was apparent after OGD, with a great increase in apoptotic rate (P<0.01). Compared with OGD group, the morphology of neuronal injury in mild hypothermia+OGD group was attenuated, and the neuronal apoptotic rate and the activity of caspase-3 in the cytoplasm decreased. The activity of caspase-3 in the cytoplasm increased after OGD, and was positively correlated with the neuronal apoptotic rate (r=0.823, P<0.05). The activity of caspase-3 in the cytoplasm also increased after mild hypothermia and OGD, and was also positively correlated with the neuronal apoptotic rate (r=0.841, P<0.05). CONCLUSION：OGD can increase caspase-3 activity in the neuronal cytoplasm and induce neuronal apoptosis. Restraint on caspase-3 activity in the neuronal cytoplasm may be the mechanism by which mild hypothermia protects against neuronal injury induced by OGD.     

EGCG antagonizes hypoxia-induced autophagy and apoptosis in PC12 cells by mTOR regulation

AIM:Hypoxia (evoked by CoCl2)-induced apoptosis and autophagy are emerging as crucial events in the etiopathology of many neurodegenerative diseases. Epigallocatechin gallate (EGCG) is the active ingredient in tea polyphenols with abilities of anti-apoptosis and anti-autophagy, but the mechanism has not been fully elucidated. In recent years, studies have reported that the mammalian target of rapamycin (mTOR) involved in the regulation of a variety of neurological like differentiation and maturation of nerve cells, anti-oxidative stress, etc. Therefore, we investigate that whether EGCG protects PC12 from hypoxia-induced apoptosis and autophagy by enhancing mTOR expression. METHODS:The expression of mTOR and beclin-1 were detected by Western blotting. The expression of caspase-3 was measured by ELISA. The cell viability was detected by CCK-8 assay. The LC-3 expression in nucelus was observed by immunofluorescence. RESULTS:Hypoxia induced apoptosis and autophagy in PC12 cells. EGCG antagonized hypoxia-induced apoptosis and autophagy by enhancing mTOR expression. Blocking the pathway of mTOR reversed the protective effect of EGCG on PC12 cells. CONCLUSION: EGCG antagonizes hypoxia-induced autophagy and apoptosis in PC12 cells by controlling mTOR regulation.     

Panax notoginseng saponins suppress oxygen-glucose deprivation/reoxygenation-induced pyroptosis of SH-SY5Y cells

AIM To investigate the effect of Panax notoginseng saponins (PNS) on pyroptosis of SH-SY5Y cells induced by oxygen-glucose deprivation/reoxygenation (OGD/R). METHODS The OGD/R was conducted to induce ischemia/reperfusion injury in SH-SY5Y cells. The effects of PNS on the viability (detected by CCK-8 assay) and membrane permeability [indicated by lactate dehydrogenase (LDH) leakage and propidium iodide (PI) staining positive cell proportion] of OGD/R-induced SH-SY5Y cells were observed. The protein levels of gasdermin D (GSDMD), GSDMD N-terminal fragment (GSDMD-N), caspase-1 and caspase-4, and the release of interleukin-1β (IL-1β) and IL-18 in the cells were also determined. RESULTS After exposure to OGD/R, the viability of SH-SY5Y cells dramatically decreased (P<0.01), while the LDH leakage, the PI staining positive cell proportion, the protein levels of GSDMD, GSDMD-N, caspase-1 and caspase-4, and the release of IL-1β and IL-18 were significantly increased (P<0.01). However, PNS treatment enhanced the viability of SH-SY5Y cells inhibited by OGD/R (P<0.01), but reduced the leakage of LDH and the percentage of PI staining positive cells (P<0.05 or P<0.01). Moreover, PNS reversed the increases in the protein levels of GSDMD, GSDMD-N, caspase-1 and caspase-4 and the release of IL-1β and IL-18 in OGD/R-induced SH-SY5Y cells (P<0.05 or P<0.01). CONCLUSION Treatment with PNS alleviates OGD/R-induced injury in SH-SY5Y cells. Its mechanism may be related to inhibition of SH-SY5Y cell pyroptosis induced by OGD/R.     

Protective effect of aliskiren on SH-SY5Y cells injured by oxygen-glucose deprivation and its possible mechanisms

AIM: To investigate the effects of aliskiren on the injury of SH-SY5Y cells induced by oxygen-glucose deprivation (OGD) and its possible mechanisms. METHODS: The SH-SY5Y cells were randomly divided into control group, OGD group and aliskiren (5.0, 10.0 and 20.0 μmol/L) groups. The cell viability was measured by CCK-8 assay. The levels of excitatory amino acid transporter 2 (EAAT2/GLT-1), EAAT3/EAAC1, EAAT4, endothelin-1 (ET-1) and S100 calcium-binding protein β subunit (S-100β) in the SH-SY5Y cells were detected by ELISA. The morphological changes of the cells were observed by Hoechst 33258 staining. Meanwhile, the content of lactic acid (LD) and activity of Na⁺-K⁺-ATPase were also analyzed. RESULTS: The viability of SH-SY5Y cells was not more than 60% after OGD injury for 4 h, so the appropriate time for OGD injury was 4 h. Compared with control group, the protein levels of GLT-1, EAAC1 and EAAT4 in the SH-SY5Y cells of OGD group were significantly decreased (P<0.05), but the protein levels of ET-1 and S-100β were significantly increased (P<0.05). Compared with OGD group, treatment with aliskiren dose-dependently increased the protein levels of GLT-1, EAAC1 and EAAT4 in the SH-SY5Y cells, but decreases in the levels of ET-1 and S-100β were observed (P<0.05). The results of Hochest 33258 staining showed that aliskiren significantly reduced the apoptosis of SH-SY5Y cells. Compared with control group, a significant increase in the content of LD (P<0.05) and a significant decrease in Na⁺-K⁺-ATPase activity (P<0.05) were found in the SH-SY5Y cells of OGD group. Compared with OGD group, aliskiren dose-dependently decreased the content of LD, but increased the Na⁺-K⁺-ATPase activity in the SH-SY5Y cells (P<0.05). CONCLUSION: Aliskiren has good neuroprotective effects on SH-SY5Y cells after OGD injury. The underlying mechanisms may be associated with the increases in the protein levels of GLT-1, EAAC1 and EAAT4, the enhancement of Na⁺-K⁺-ATPase activity, and the decreases in the levels of ET-1 and S-100β and the content of LD.     

Inhibition of Toll-like receptor 9 activation in microglia after oxygen-glucose deprivation and reoxygenation protects neurons from damage

AIM: To observe the Toll-like receptor 9 (TLR9) activation in microglia BV-2 cells after oxygen-glucose deprivation and reoxygenation (OGDR), and its effects on neuronal apoptosis. METHODS: The BV-2 cell supernatants were collected after the corresponding treatment and added to mouse primary cortical neurons after OGDR for 4 h, followed by normal culture for 24 h. The cells were divided into normal BV-2 group, NC-siRNA group, TLR9-siRNA group, OGDR group, OGDR+NC-siRNA group, OGDR+TLR9-siRNA group and control group (without adding BV-2 cell supernatant). The changes of the neuronal morphology were observed under an inverted phase- contrast microscope, and the neuronal apoptosis was detected by TUNEL. The protein expression of cleaved caspase-3 was detected by Western blotting. RESULTS: After OGDR, the axon turned thin, twisted and broken, and neuronal swelling, decrease in refraction and vacuolar degeneration were observed. The green-stained apoptotic bodies in the neurons in all groups were positive. Compared with control group, the caspase-3 protein levels in other groups were increased. Compared with the normal BV-2 group, the caspase-3 protein in OGDR group and TLR9-siRNA group was increased. Compared with OGDR+TLR9-siRNA group, the caspase-3 protein in TLR9-siRNA group and OGDR group was decreased. CONCLUSION: After OGDR, TLR9 activation in BV-2 cells induces neuronal apoptosis with the increase in caspase-3 protein level. Inhibition of TLR9 expression reduces neuronal damage.     

Influence of Bcl-2 inhibitor on Astragalus injection-reduced expression of caspase-3 in rat hippocampal neurons with oxygen-glucose deprivation and reoxygenation

AIM: To observe the influence of Bcl-2 inhibitor on the expression of caspase-3 reduced by Astra-galus injection in rat hippocampal neurons with oxygen-glucose deprivation and reoxygenation (OGD/R). METHODS: The primary rat hippocampal neurons cultured in vitro for 8 d were chosen and randomly divided into 6 groups: normal control group, model group (OGD/R group), Astragalus injection group, Astragalus injection solvent (sterile deionized water)group, Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group. The cells in all groups were tested 24 h after they were treated with reoxygenation after deprived of oxygen and glucose for 30 min except normal control group. The cell type and rate of positive cells were observed by immunohistochemistry. The protein levels of Bcl-2 and cleaved caspase-3 in the hippocampal neurons were measured by Western blotting. The mRNA expression of caspase-3 was detected by RT-PCR. RESULTS: Compared with normal control group, the caspase-3 positive rate of the cells, the protein levels of Bcl-2 and cleaved caspase-3, and the mRNA expression of caspase-3 in model group enhanced significantly (P < 0.05). Compared with model group, the expression of Bcl-2 in Astragalus injection group obviously enhanced, while the caspase-3 positive rate of the cells, the protein level of cleaved caspase-3 and the mRNA expression of caspase-3 in the Astragalus injection group decreased significantly (P < 0.05). No significant difference in injection solvent group, Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group was observed (P > 0.05). The expression of Bcl-2 was decreased sharply in Bcl-2 inhibitor group and Bcl-2 inhibitor with Astragalus injection group. CONCLUSION: Bcl-2 inhibitor antagonizes the inhibitory effect of Astragalus injection on caspase-3 expression in rat hippocamal neurons with OGD/R, which may be one of the possible target for the inhibitory action of Astragalus injection on the apoptosis of rat hippocampal neurons induced by OGD/R.     

ZFP580, a novel transcription factor,is involved in cardioprotection of hypoxic preconditioning against hypoxia-reoxygenation injury in myocardial cells

AIM：To elucidate whether ZFP580 is involved in the cardioprotective effects of hypoxic preconditioning (HPC) against hypoxia-reoxygenation (H/R) injury in H9c2 myocardial cells. METHODS：Rat heart-derived H9c2 cells were cultured in DMEM. H/R was induced by incubation under ischemic hypoxia for 3 h and reoxygenation for 2 h. HPC was induced by exposing the H9c2 cells to 10 min of hypoxia and 20 min of reoxygenation for 3 cycles before H/R treatment. MTT staining and LDH leakage detection were used to evaluate the effects of HPC. Western blotting was used to detect the protein levels of ZFP580, phosphorylated ERK1/2 and cleaved caspased-3. The effects of ZFP580 overexpre-ssion or knockdown on H/R induced apoptosis were determined. RESULTS：The results of MTT staining and LDH leakage detection showed evidence of HPC cytoprotection against H/R-induced cell death in H9c2 cells. ZFP580 protein level and ERK1/2 phosphorylation were significantly increased in the HPC group compared with control group and H/R group. PD98059, an inhibitor of ERK1/2 phosphorylation, significantly suppressed the HPC-induced up-regulation of ZFP580 protein expression. ZFP580 overexpression significantly inhibited apoptosis and caspase-3 activation in H9c2 cells. CONCLUSION：HPC exhibits cytoprotection against H/R and leads to high level of ZFP580 protein in H9c2 cells. ZFP580 is regulated by ERK1/2 activation and mediates the anti-apoptotic effect of the ERK1/2 signaling pathway in HPC cytoprotection.     

Effects of NBP on expression of VEGF and HIF-1α in HUVECs under the condition of oxygen-glucose deprivation

AIM: To investigate the mechanism that dl-3-n-butylphthalide (NBP) protects cells from the induced by oxygen-glucose deprivation (OGD).METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to OGD to induce endothelial damage. Endothelial injury was assessed by measuring the changes of chromatin morphology and MTT method. The protein levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were determined by immunofluorescence and quantitatively analyzed with the software IPP. Western blotting was applied to verify the results.RESULTS: NBP at the concentrations of 0.01 to 100 μmol/L dose-dependently protected against OGD-induced cell damage. Compared with OGD group, NBP enhanced OGD-induced the expression of VEGF and HIF-1α, and the difference was statistically significant. The expression of VEGF and HIF-1α reached to the peak at the time points of 6 h and 8 h after OGD, respectively.CONCLUSION: Under the condition of OGD, NBP enhances the expression of HIF-1α in HUVECs, subsequently promotes the expression of downstream VEGF, and eventually elevates the survival of the cells.     

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.     

Effects of AZD8055 on autophagy and apoptosis in cholangiocarcinoma cells

AIM: To explore the effects of mammalian target of rapamycin (mTOR) double inhibitor AZD8055 on autophagy and apoptosis of human cholangiocarcinoma cell line HuCCT1. METHODS: The effect of AZD8055 on the viability of HuCCT1 cells was detected by MTT assay. Autophagosome was detected by acridine orange (AO) staining. After treated with AZD8055, the expression levels of apoptosis-related proteins Bcl-2, Bax and cleaved caspase-3 and auto-phagy marker proteins beclin 1, LC3 and p62 were determined by Western blot. Apoptotic rate was analyzed by flow cyto-metry with Annexin V-FITC/PI double staining. RESULTS: AZD8055 significantly inhibited the viability of HuCCT1 cells (P<0.05). AO staining showed that AZD8055 significantly increased orange granules in the cytoplasm. After treated with AZD8055, compared with the control group, the protein level of beclin 1 and the ratio of LC3-Ⅱ/LC3-I were enhanced, while p62 was attenuated (P<0.05). The protein expression level of pro-apoptotic regulator Bax was down-regulated and anti-apoptotic regulator Bcl-2 was increased. The protein level of cleaved caspase-3 was reduced (P<0.05). The results of flow cytometry showed that AZD8055 inhibited cell apoptosis. CONCLUSION: AZD8055 inhibits the viability of cholangiocarcinoma cells, and the mechanism is closely related with autophagy induced by AZD8055.     

Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in cardiomyocytes via inducing autophagy

AIM: This study aims to explore the effect of abietic acid (AA) on advanced glycosylation end products (AGEs)-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes. METHODS: H9c2 cells were divided into 5 groups. The cells in control group were treated with saline for 24 h. The cells in AGEs treatment group were treated with AGEs (100 mg/L) for 24 h. The cells in AGEs+AA (10, 25 and 50 μmol/L) groups were simulta-neously treated with AGEs (100 mg/L) and AA (10, 25 and 50 μmol/L) for 24 h. The cell viability was measured by MTT assay. The protein levels of myoglobin (Mb), creatine kinase MB isoenzyme (CK-MB), cardiac troponin I (cTnI), C/EBP homologous protein (CHOP), cleaved caspase-12, GADD34, BiP, LC3, P62 and beclin 1 were determined by Western blot. The levels of lactate dehydrogenase (LDH) were measured by ELASA. The apoptosis was analyzed by flow cytometry. RESULTS: The low concentration (<50 μmol/L) of abietic acid had no obvious effect on the viability of H9c2 cells. The high concentration (>50 μmol/L) of abietic acid decreased the viability of H9c2 cells. The levels of Mb, CK-MB, cTnI and LDH in AGEs group were higher than those in control group (P<0.05). Compared with AGEs group, the levels of Mb, CK-MB, cTnI and LDH in AGEs+AA (10, 25 and 50 μmol/L) groups were obviously reduced (P<0.05). Abietic acid at concentrations of 10, 25 and 50 μmol/L inhibited AGEs-induced apoptosis, elevated the protein levels of CHOP and cleaved caspase-12, and attenuated expression of GADD34 and BiP (P<0.05). Moreover, abietic acid at concentrations of 10, 25 and 50 μmol/L suppressed AGEs-induced decreased ratio of LC3-Ⅱ/LC3-Ⅰ and expression of beclin 1, and enhanced the expression of P62 (P<0.05). 3-Methyladenine, an inhibitor of autophagy, reversed the effect of abietic acid on the protein levels of LC3, Mb, cleaved caspase-12 and BiP (P<0.05). CONCLUSION: Abietic acid alleviates AGEs-induced apoptosis and endoplasmic reticulum stress in H9c2 cardiomyocytes via inducing autophagy.     

Protective effect of autophagy on nucleus pulposus cells under starvation

AIM: To explore the possibility that the starvation environment induces autophagy of nucleus pulposus cells. METHODS: Primary rat nucleus pulposus cells was cultured and stained with toluidine blue, Alcian blue and immunocytochemistry for typeⅡ collagen. The cultured cells were divided into 4 groups: control group, 3-methyladenine (3-MA)+DMEM group, 3-MA+EBSS group and EBSS group. The cells were detected for autophay using monodansylcadaverine (MDC) staining, electron microscopy and Western blotting. At the same time, the inhibitory rate and apoptotic rate of the cells were detected by Cell Counting Kit-8(CCK-8) assay and TUNEL staining, respectively. RESULTS: Compared with control group, the autophagosomes were observed in nucleus pulposus cells under electron microscope and fluorescence microscope in EBSS group, and the 3-MA+EBSS treatment suppressed the formation of autophagosomes. The results of Western blotting analysis showed that the ratios of LC3-II/LC3-I and Beclin-1/β-actin in EBSS treatment group were higher than those in control group and 3-MA+EBSS treatment group. However, the apoptotic rate of nucleus pulposus cells and the inhibitory rate of cell viability were increased in 3-MA+EBSS treatment group. CONCLUSION: Autophagy of nucleus pulposus cells is induced by nutrient starvation, and 3-MA suppresses the response. Autophagy may have a protective effect on nucleus pulposus cells under the condition of starvation.