Protective effect of HSP70 on injuried myocardial cells induced by H2O2

AIM: To investigate the role of heat-shock protein 70(HSP₇₀) in the protection of myocardial cells against ischemic injury.METHODS: Myocardial cells were cultured in vitro. HSP₇₀ was induced by hyperthermia. H₂O₂-injured myocardial cells were divided into different groups. Flow cytometry, DNA Ladder and biochemistry methods were employed to observe the myocardial cells of different groups.RESULTS: Immunohistochemistry showed hyperthermia induced the up-regulation of HSP₇₀ in myocardial cells. Apoptotic rate, activity analysis of cytochrome C and succinic dehydrogenase in H₂O₂-injuried and HSP₇₀-protected groups were obviously different. Electron micrograph shomed hyperthermia alliviated myocardial cell injury induced by H₂O₂. CONCLUSION: HSP₇₀ delays apoptosis and protects against H₂O₂-induced myocardial cell injury.     

Astragalosides inhibit autophagy and apoptosis of rat cardiomyocytes induced by H2O2 through mTOR signaling pathway

AIM: To investigate the effects of astragalosides on autophagy and apoptosis of rat cardiomyocytes induced by hydrogenperoxide (H₂O₂).METHODS: The injury model of H9c2 cells induced by H₂O₂ was established, and the cells in astragalosides group and rapamycin group were treated with 20 mg/L astragalosides and 0.1 mg/L rapamycin, respectively. The apoptotic rate was detected by flow cytometry. The autophagy was observed by acridine orange staining. Western blot was used to detect the protein levels of p-mTOR, P70S6K, LC3 and caspase-3. RESULTS: Compared with H₂O₂ group and rapamycin group, the viability of H9c2 cells in astragalosides group was significantly increased (P<0.05). The shape of the H9c2 cells in astragalosides group was complete, the nuclei were stained with yellow-green fluorescence, and the chromatin was distributed evenly. The protein levels of p-mTOR and P70S6K in the H9c2 cells of astragalosides group were significantly increased (P<0.05), whereas the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H9c2 cells of astragalosides group were decreased significantly (P<0.05). CONCLUSION: Astragalosides enhance the viability, inhibit the apoptosis, increase the protein levels of p-mTOR and P70S6K, and decrease the protein levels of LC3, cleaved caspase-3 and caspase-3 in the H₂O₂-induced rat myocardial H9c2 cells. The mechanism is related to the mTOR signaling pathway.     

Effect of CLCN2 antisense oligonucleotide on U251 cells injury by cisplatin

AIM: To investigate the effect of chloride channel CLCN2 antisense oligonucleotide on the cell injury of malignant U251 glioma cells induced by cisplatin (DDP). METHODS: The experiment was divided into 4 groups: control group (nonsense oligonucleotide), CLCN2 antisense oligonucleotide group, DDP group (DDP＋nonsense oligonucleotide), DDP＋CLCN2 antisense oligonucleotide group. The viability of U251 cells was measured by MTT assay, CLCN2 mRNA level was determined by RT-PCR, cell apoptosis was measured by TUNEL assay. RESULTS: Compared to the control group, the cell viability, CLCN2 and cyclinD1 mRNA decreased in CLCN2 antisense oligonucleotide group, DDP treated group and CLCN2 antisense oligonucleotide with DDP treated group, cells apoptosis increased. Compared to DDP group, the cell viability (P<0.05) and CLCN2 mRNA decreased in CLCN2 antisense oligonucleotide with DDP treated group, and cells apoptosis increased (P<0.01). Compared to CLCN2 antisense oligonucleotide group, CLCN2 mRNA significantly decreased (P<0.01) in CLCN2 antisense oligonucleotide with DDP treated group. CONCLUSION: CLCN2 antisense oligonucleotide inhibits the expression of CLCN2 mRNA in U251 cells. Inhibition of CLCN2 mRNA facilitates the cell injury of U251 cells induced by DDP. The decrease in CLCN2 mRNA is involved in the mechanism of cell injury by DDP.     

Effects of luteolin on endothelial cell injury induced by H2O2

AIM: To study the effect of luteolin on endothelial cell injuries induced by H₂O₂. METHODS: Endothelial cells were cultured in vitro and divided into seven groups: control; solvent; H₂O₂; quercetin+H₂O₂; luteolin-L+H₂O₂; luteolin-M+H₂O₂; luteolin-H+H₂O₂ group, respectively. Endothelial cells were incubated with 750 μmol/L H₂O₂ for 18 h or 14 h in the absence or presence of various concentrations of luteolin and quercetin. The expression of caspase-3, the pro-apoptotic factors, was detected by immunohistochemical technique, and the apoptotic index was detected by flow cytometry. Meanwhile, the amount of malondialdehyde (MDA), nitric oxide (NO), lacate dehydrogenase (LDH) in serum, and cell viability were measured. RESULTS: Incubation of endothelial cells with H₂O₂ for 18 h elicited the increase in the extent of immunostaining for caspase-3, the rate of apoptosis, the release of LDH, and the number of dead cells accompanied by the decrease in the content of NO in the medium, which were inhibited by pretreatment of luteolin at concentrations of 0.5-50 μmol/L in a concentration-dependent manner (P<0.01). CONCLUSION: Luteolin possesses a protective effect against endothelial cell injuries induced by H₂O₂, which may be related with anti-oxidation, increasing the concentration of NO and inhibiting the activation of caspase-3.     

Sodium dichloroacetate improves radiosensitivity of U251 cells

AIM: To study the change of radiosensitivity of U251 cells after treated with sodium dichloroacetate (DCA) and further to explore the possible mechanism.METHODS: The U251 cells were divided into 4 groups: control group, DCA group, X-ray irradiation without DCA pretreatment (IR) group and X-ray irradiation with DCA pretreatment (DIR) group. MTT assay was applied to determine the cell viability. The intracellular reactive oxygen species (ROS) were detected by DHE fluorescence. The expression level of Bcl-2 was assessed by Western blot. The percentage of apoptosis of cells was determined by flow cytometry. RESULTS: No difference between control group and DCA group in cell viability (P>0.05) was observed. However, the cell viability of both IR group and DIR group was markedly reduced compared with control group (P<0.05). Furthermore, the viability of DIR group was significantly decreased compared with IR group (P<0.05). The percentage of ROS positive cells was obviously increased in DIR group compared with IR group (P<0.05). The expression level of Bcl-2 was sharply decreased in DIR group (P<0.05) and the percentage of apoptosis of cells was significantly elevated (P<0.05) in DIR group compared with IR group.CONCLUSION: The better antitumor effect was obtained by improving the radiosensitivity through pretreating the cells with DCA, and the possible mechanism was down-regulation of the Bcl-2 expression by developing the intracellular ROS.     

Effect of sinapine on H2O2-induced adipogenic differentiation of bone marrow mesenchymal stem cells

AIM:To investigate the effects of sinapine, an effective monomer of Chinese medicine, on hydrogen peroxide (H₂O₂)-induced adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).METHODS:The undifferentiated rat BMSCs were identified and screened by flow cytometry. The adipogenic differentiation of BMSCs was induced by H₂O₂, and the toxicity of sinapine on BMSCs was tested by CCK-8 assay. After the modeling method and the concentration range of sinapine were determined, the lipid droplets in the cells were detected by Oil Red O semi-quantitative assay, and the optimal drug concentration was selected. Finally, Oil Red O assay was observed 24 h after drug intervention, and the expression of adipogenic differentiation-related proteins, adipocyte protein 2 (aP2), peroxisome proliferator-activated receptor γ (PPARγ) and glucose transporter 4 (Glut4), at mRNA and protein levels in the BMSCs was determined by qPCR and Western blot.RESULTS:Treatment with H₂O₂ at 200 μmol/L for 1 h induced BMSCs to differentiate into adipocytes. Below the concentration of 40 μmol/L, sinapine had no toxicity to BMSCs. The best inhibitory concentration of sinapine on adipogenic differentiation was at 15 μmol/L. The number of lipid droplets in sinapine (15 μmol/L) group was significantly lower than that in model group. In sinapine group, the expression of aP2, PPARγ and Glut4 at mRNA and protein levels was lower than that in model group (P<0.01).CONCLUSION:Sinapine inhibits H₂O₂-induced adipogenic differentiation of rat BMSCs. The mechanism may be related to the PPARγ/AMPK signaling pathway.     

Induction of apoptosis with Salvia miltiorrhiza monomer IH764-3 via downregulating focal adhesion kinase in H2O2-stimulated hepatic stellate cells

AIM: To investigate the effect of IH764-3 on the apoptosis of H₂O₂-stimulated hepatic stellate cells(HSCs) and the alteration of focal adhesion kinase (FAK). METHODS: The proliferation of HSCs was examined by direct cell count and the apoptosis was determined by Annexin-V/PI labeling, while the morphological change was observed by light microscopy and transmission electron microscopy. In addition, FAK mRNA was detected by RT-PCR. RESULTS: H₂O₂ promoted the proliferation of HSCs. IH764-3 induced the apoptosis of HSCs in a dose-dependent manner. The HSC apoptotic rates of different groups were 6. 35%,9. 28%,15. 10%,19. 69%,respectively, after treated with different concentrations of IH764-3 for 48 h while H₂O₂ group showed 2. 30%. In 30 mg/L group, the apoptosis rates were 6. 73%、10. 34%、15. 10% for the indicated time periods(12 h, 24 h, 48 h). In the presence of IH764-3, FAK mRNA decreased. The FAK mRNA reduction began at 2 h after adding IH764-3. CONCLUSION: IH764-3 induced the apoptosis of HSCs. Down-regulating the expression of FAK mRNA may be one of its mechanisms.     

Notch1 regulates stemness and chemotherapeutic sensitivity of human glioma U251 cells

AIM: To investigate whether Notch1 changes stemness and chemotherapeutic sensitivity in human glioma U251 cells. METHODS: The lentiviral vectors, which expressed Notch1-shRNA or Notch1 intracellular domain (NICD), were transfected into U251 cells. Western blot and immunofluorescence staining were applied to monitor the validity of the cells, down-regulation of Notch1 expression or over-expression of NICD. The proportion of CD133⁺ cells was analyzed by flow cytometry. The expression of nestin and GFAP was identified by immunofluorescence staining. The formation rate of tumor cell spheres and the implanted tumor growth in SCID mice were observed. MTT assay was performed to evaluate the chemotherapeutic sensitivity to VM-26 and BCNU of the cells with different treatments. RESULTS: Stemness was significantly enhanced in the cells over-expressing NICD. For example, the proportion of CD133⁺ cells was increased, the expression of nestin was up-regulated, the expression of GFAP was down-regulated, and the formation rate of tumor cell spheres and implanted tumor growth were increased. The chemotherapeutic sensitivity to VM-26 and BCNU of the cells was decreased. In the cells with Notch1 gene down-regulation by RNAi, the stemness was inhibited and chemotherapeutic sensitivity was increased. CONCLUSION: Notch1, which leads to the change of stemness and chemotherapeutic sensitivity in human glioma U251 cells, is likely to be a potential molecular target for treatment of glioma.     

Adiponectin attenuates H2O2-induced SH-SY5Y cell injury and tau hyperphosphorylation via activating PP2A

AIM: To study the effects of adiponectin on H₂O₂-induced cell injury and tau hyperphosphorylation in human neuroblastoma SH-SY5Y cells. METHODS: Cell viability was determined by MTT assay. H₂O₂-induced cell injury and morphological changes in the SH-SY5Y cells with or without adiponectin treatment were observed. The level of tau phosphorylation as well as the activities of protein phosphatase 2A(PP2A) and of glycogen synthase kinase-3β(GSK-3β) were examined by Western blotting. RESULTS: Adiponectin significantly attenuated H₂O₂-induced cell injury(P<0.01). Adiponectin upregulated the activity of PP2A and decreased phosphorylation levels of tau under the stimulation with H₂O₂ (P<0.01). Okadaic acid, a specific inhibitor of PP2A, blocked the protective effects of adiponectin(P<0.01). Adiponectin increased the phosphorylation level of GSK-3β at Ser9 site under H₂O₂ stimulation(P<0.01). CONCLUSION: Adiponectin protects SH-SY5Y cells against H₂O₂-induced cell injury and decreases tau hyperphosphorylation by activating PP2A and inactivating GSK-3β.     

Limb ischemic preconditioning protects human umbilical vein endothelial cells from oxidative stress induced by H2O2

AIM: To investigate the effects of the sera from the rats after limb ischemic preconditioning (LIPC) on human umbilical vein endothelial cells (HUVECs) injured by hydrogen peroxide (H₂O₂). METHODS: The HUVECs were divided into 5 groups: the cells in control group were cultured without any intervention; the cells in model group (M) were damaged by 1 mmol/L H₂O₂ for 2 h; the cells in early preconditioning serum (EPS) group, delayed preconditioning serum (DPS) group or sham limb ischemic preconditioning serum (SPS) group were treated with the corresponding serum at 5% for 12 h, respectively, and then treaed with H₂O₂ for 2 h. The viability of the HUVECs was analyzed by flow cytometry. The lactate dehydrogenase (LDH) in the culture media was detected. The cell adhesion molecules in the HUVECs were detected by real-time PCR. The mRNA and protein expression of heme oxygenase-1 (HO-1) was also determined. RESULTS: The viability of HUVECs incubated with 1 mmol/L H₂O₂ for 2 h significantly decreased compared with the control cells, which was accompanied with the augmentations of LDH in the medium and the cell adhesion molecules in cells, such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Preincubation with EPS and DPS derived from the rats subjected LIPC attenuated these injuries. Furthermore, pretreatment with EPS and DPS increased the expression of HO-1 at mRNA and protein levels. CONCLUSION: LIPC protects the HUVECs from H₂O₂-induced injury.     

H2O2 degradation is suppressed in kumquat leaves infected with Xanthomonas axonopodis pv. citri

We found in a previous study that after leaves of kumquat [Fortunella margarita (Lour.) Swingle] cv ‘Nagami’ were inoculated with Xanthomonas axonopodis pv. citri (Xac), total superoxide dismutase activity (SOD) increased to promote higher H₂O₂ concentrations that coincided with a 4-fold decline in Xac populations ( Kumar et al., 2011a). The objective of the current study was to determine how activities and isoforms of important enzymes that catabolize H₂O₂, specifically catalase (CAT), ascorbate peroxidase (APOD), and the Class III peroxidases (POD) that are located in the apoplast, change in infected kumquat leaves to affect concentration and compartmentalization of H₂O₂. DAB (3,3-diaminobenzidine) staining of the Xac-infected leaves confirmed higher overall concentration of H₂O₂ as in our earlier study. One day after inoculation (dai), APOD activity declined below the controls and declines steadily up to 10 dai when the experiment was terminated. CAT activity was similar to the controls until 4 dai then declined rapidly to about 60% the activity of the controls by 6 dai, after which it remained fairly constant until 10 dai. There were 4 CAT isoforms in control leaves and 5 isoforms in infected leaves. The CAT-1 isoform band was much smaller in infected plants than the control at all sampling times. The CAT-3 isoform band disappeared at 10 dai. The CAT-5 isoform band, which was not observed in control leaves, appeared only at 4 dai in infected leaves. POD activity of infected leaves increased above the controls starting 1 dai and reached a maximum of about 3-fold higher than the controls 8 dai after which it declined. Two POD isoforms were detected in control and infected plants. This study demonstrated that the higher accumulation of H₂O₂ in kumquat leaves infected with Xac was promoted during pathogenesis first by the suppression of APOD activity and later by suppression of CAT activity. We propose that the higher SOD and lower APOD and CAT activities in the symplast contributed H₂O₂ substrate for the higher POD activity in the apoplast, which is known to be involved in plant defense against pathogens.     

MK-2206, an inhibitor of Akt,induced cell apoptosis and autophagy in U2OS cells

AIM：To observe the effect of MK-2206, an inhibitor of Akt, on the cell apoptosis and autophagy of U2OS cells. METHODS：The cell viability was detected by MTT assay. The cell apoptosis was analyzed by TdT-mediated dUTP nick end labeling assay. The expression of LC3-II was examined by Western blotting. RESULTS：MK-2206 inhibited the cell viability in a dose-dependent manner. MK-2206 induced the cell apoptosis via activation of caspase-3, caspase-9 and PARP. MK-2206 treatment substantially induced the U2OS cell autophagy by increasing in the levels of LC3-II. Blockage of autophagy using chloroquine magnified MK-2206-induced cell death in U2OS cells. CONCLUSION：The Akt inhibitor MK-2206 induces cell apoptosis and autophagy. Blocking autophagy magnifies MK-2206-induced the inhibition of the viability in U2OS cells.     

Overexpression of TAP1 up-regulates HLA-Ι in human glioma U251 cells

AIM：To investigate the relationship between the overexpression of transporter associated with antigen processing 1 (TAP1) and the human leukocyte antigen I (HLA-I).METHODS：The full length of TAP1 gene was obtained from the cDNA library. The lentiviral vector pSIN-EF2-IRES-GFP-puro was digested by BamH I and EcoR I, and the full length of TAP1 gene was inserted into the vector by T4 DNA ligase. Subsequently, the recombinant plasmid was transformed into Escherichia coli DH5α cells and the correct transformant was selected. The recombinant plasmid and the Lenti-X HTX packaging mixture were co-transfected into 293T cells, and the virus particle was acquired. Human glioma U251 cells were transfected with the lentivirus. The expression of TAP1 and HLA-I was determined by real-time fluorescence quantitative PCR, Western blotting and flow cytometric analysis.RESULTS：TAP1 gene was successfully transfected into the U251 cells and stably expressed in the cell line. The expression of TAP1 in U251 cells at mRNA and protein levels increased by (8.73±1.07) and (11.71±0.83) folds, respectively. As a result, the mRNA expression of HLA-A, HLA-B, HLA-C (heavy chain) and β2-microglobulin (light chain) was up-regulated by (3.51±0.36), (4.78±0.85), (2.94±0.28) and (3.23±0.24) folds, respectively. The protein expression of HLA-I also increased to (3.14±0.53) fold. The surface expression of HLA-I on the U251 cells transfected with TAP1 gene was largely enhanced as well.CONCLUSION：Overexpression of TAP1 up-regulates the expression of HLA-I. TAP1 plays an important role in HLA-I processing pathway.     

USP14 regulates H2O2 induced oxidative stress in H9c2 cells

AIM:To evaluate the effect of inhibiting ubiquitin-specific protease 14(USPl4) activity on oxidative stress induced by H₂O₂ of H9c2 cells.METHODS:The H9c2 cells were incubated with H₂O₂ at 25 μmol/L for 2 h to establish the oxidative stress injury model.The cells were divided into control group,H₂O₂ group,IU1 group (25 μmol/L or 50 μmol/L) and IU1+H₂O₂ group.The H9c2 cells activity was measured by MTS assay.The level of intracellular reactive oxygen species (ROS) and cell survival rate were analyzed by flow cytometry assay.The changes of the mitogen-activated protein kinase (MAPK) family related proteins were detected by Western blot.RESULTS:Compared with control group,the cell activity and the viability rate in H₂O₂ group were decreased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were increased (P<0.05).Compared with H₂O₂ group,the cell activity and the viability rate of the H9c2 cells in IU1+H₂O₂ group were increased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were decreased (P<0.05).CONCLUSION:Inhibition of USPl4 activity reduces the oxidative stress injury of the H9c2 cells.The mechanism may be related to inhibition of the MAPK signaling and down-regulation of apoptosis related proteins.     

Role of mtCLIC/CLIC4 in injured C6 glioma cells induced by hydrogen peroxide

AIM: To explore the changes and the possible function of mtCLIC/CLIC4 (mitochondrial chloride intracellular channel 4) proteins in malignant C6 glioma cells treated with hydrogen peroxide (H₂O₂). METHODS: The viability of C6 cells was measured by MTT assay, LDH release rate was detected by ultraviolet spectrophotometry, CLIC4 mRNA level was determined by RT-PCR and CLIC4 protein level was measured by Western blotting. RESULTS: Compared with the control group, the cell viability was constant, the LDH release rate increased obviously, and the CLIC4 protein level also increased significantly in 500 μmol/L H2O2 treated group (P<0.05, respectively). However, the cell viability decreased, LDH release rate increased significantly (P<0.01, respectively), and the CLIC4 protein level increased obviously in 1 000 μmol/L H₂O₂ treated group (P<0.05). CONCLUSION: The CLIC4 protein may be involved in the process of C6 injuries induced by H₂O₂.     

Down-regulation of miR-153-3p attenuates H2O2-induced H9C2 cell injury by promoting Nrf2 expression

AIM To study the effect of microRNA-153-3p (miR-153-3p) knock-down on oxidative injury of H9C2 cells induced by H₂O₂ and its specific mechanism. METHODS The oxidative stress injury of H9C2 cell model was induced by H₂O₂, and then the cell viability and the expression of miR-153-3p were detected by MTT assay and RT-qPCR, respectively. The effects of miR-153-3p knock-down on the H9C2 cell injury under oxidative stress were studied by RNA interference technology. The targets of miR-153-3p were identified by Western blot and dual-luciferase reporter assay. RESULTS MTT assay showed that the viability of H9C2 cells was decreased with the increase in H₂O₂ concentration (P<0.05). The results of RT-qPCR showed that the expression of miR-153-3p was increased with the increase in H₂O₂ concentration (P<0.05). Knock-down of miR-153-3p increased the viability of H9C2 cells under oxidative stress, decreased the cell apoptosis and the content of malondialdehyde (MDA), and increased the activity of superoxide dismutase (SOD). The expression of nuclear factor E2-related factor 2（Nrf2） and antioxidant response element（ARE） activity were increased with the increase in H₂O₂ concentration (P<0.01). TargetScan analysis and dual-luciferase reporter assay showed that Nrf2 was one of the potential target genes of miR-153-3p. The results of Western blot further showed that over-expression of miR-153-3p inhibited the expression of Nrf2 (P<0.01), while down-regulation of miR-153-3p increased the expression of Nrf2 (P<0.01). Dual interference with Nrf2 and miR-153-3p significantly reduced H9C2 cell viability, promoted the apoptosis, increased MDA content, and decreased SOD activity in the presence of H₂O₂ (P<0.01). CONCLUSION Inhibition of miR-153-3p expression attenuates the injury of H9C2 cells induced by H₂O₂ through up-regulating Nrf2/ARE signaling pathway.     

Protective effect of BNDF on vascular endothelial cells with H2O2-induced oxidative injury

AIM: To study the protective effect of brain-derived neurotrophic factor (BDNF) on vascular endothelial cells with H₂O₂-induced oxidative injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro, and the oxidation injury model of HUVECs was established by treatment with H₂O₂. The oxidatively injured HUVECs were cultured with different concentrations (1, 10 and 100 μg/L) of BDNF. At the same time, the control group (no injury), PBS treatment after H₂O₂ injury group and TrkB inhibitor group (with 100 μg/L BDNF and 1: 1 000 TrkB inhibitor) were also set up. The viability of the HUVECs was detected by MTT assay. The levels of LDH, MDA, SOD and GSH were measured. The releases of NO, ET-1 and ICAM-1 were analyzed by ELISA. The changes of ROS production and cell apoptosis were evaluated by flow cytometry. The protein levels of TrkB, p-TrkB, cleaved caspase-3, Bcl-2 and Bax were determined by Western blot. RESULTS: Compared with uninjured control group, in H₂O₂ oxidative injury plus PBS treatment group, the viability of the cells was decreased significantly, the LDH and MDA levels were increased significantly and the activities of SOD and GSH were decreased significantly. The NO secretion was decreased, and the ET-1 and ICAM-1 concentrations were increased significantly. The ROS content and apoptotic rate were increased significantly. The protein levels of cleaved caspase-3 and Bax were increased but Bcl-2 protein expression was decreased significantly. Compared with PBS treatment group, in H₂O₂-injured HUVECs treated with different concentrations of BDNF, the cell viability was gradually increased, the LDH and MDA levels were decreased and the activities of SOD and GSH were increased gradually. The secretion of NO was increased but ET-1 and ICAM-1 were decreased gradually. The ROS content and apoptotic rate were decreased significantly. The TrkB and p-TrkB levels were significantly increased significantly, the protein expression of cleaved-caspase 3 and Bax was decreased gradually and the Bcl-2 protein expression increased gradually. The role of BDNF was inhibited by TrkB inhibitor. CONCLUSION: BDNF protects HUVECs from oxidative injury by binding with TrkB to activate the BDNF-TrkB signaling pathways.     

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.     

Effect of autophagic inhibitor 3-methyladenine on apoptosis of hippo-campal neurons induced by Aβ(25-35)

AIM: To investigate the influence of autophagy on the apoptosis of hippocampal neurons in the rat model of Alzheimer disease.METHODS: Sprague-Dawley rats were divided into model group, autophagic inhibitior 3-methyladenine (3-MA) pretreatment group and control group.In model group, the rats were anesthetized and placed in a stereotaxic apparatus.Hippocampus CA1 area microinjection was performed and Aβ(25-35) was applied to establish the model of AD.3-MA in 0.9% saline was administered by the same way prior to Aβ(25-35) infusion.The learning and memory ability of the rats was observed by Morris water maze.The ultrastructure of the hippocampal neurons, the formation of autophagic vesicles, beclin-1 expression and cell apoptosis were detected after behavioral experiment.RESULTS: Compared with model group, the learning and memory ability of the rats in 3-MA group significantly impaired (P<0.05) and the apoptotic rate of the hippocampal neurons significantly increased (P<0.05).Moreover, the expression of beclin-1 was declined.In model group, hippocampal neurons showed double membrane wrapped in the autophagic vacuoles, and the neuronal damages were significantly milder than that in 3-MA group.CONCLUSION: Decrease in the levels of neuronal autophagy increases the neuronal apoptosis, indicating that increasing neuronal autophagy may have therapeutic potential for AD.