Roles of heat shock protein 90 in the blockage of H2S against cardiomyocyte injuries induced by chemical hypoxia

AIM: To explore the roles of heat shock protein 90 (HSP90) in the blockage of hydrogen sulfide (H2S) against chemical hypoxia-mimetic agent (cobalt chloride, CoCl2)-induced oxidative stress injuries in H9c2 cardiac cell. METHODS: H9c2 cells were treated with CoCl2 to set up the chemical hypoxia-induced the model of cardiomyocyte injury. Sodium hydrosulfide (NaHS, a H2S donor) was added into medium for 30 min before CoCl2 treatment. ATP content was detected by high performance liquid chromatogram (HPLC). Mitochondrial membrane potential (MMP) was measured by rhodamine123 (Rh123) staining and photofluorography. The activity of superoxide dismutase (SOD) was observed using a SOD kit. The expression of heme oxygenase-1 (HO-1) was evaluated by Western blotting. RESULTS: CoCl2 at concentration of 600 μmol/L significantly reduced SOD activity, ATP level and MMP, and enhanced the expression of HO-1 in H9c2 cells. Pretreatment with 400 μmol/L NaHS dramatically inhibited the cytotoxicity induced by CoCl2, increased SOD activity, ATP level and MMP, decreased HO-1 expression. 17-allylamino-17 demethoxygeldanamycine(17AAG), an inhibitor of HSP90, obviously blocked the inhibitory effect of H2S on the CoCl2-induced cytotoxicity, reduced the levels of ATP and MMP, increased HO-1 expression. However, no significantly influence on SOD activity was observed. CONCLUSION: HSP90 may mediate the cardioprotection of H2S via inhibiting the oxidative stress induced by chemical hypoxia.     

Hydrogen sulfide protects H9c2 cardiomyocytes against high glucose-induced injury by inhibiting necroptosis

AIM: To study whether hydrogen sulfide(H₂S) protects H9c2 cardiomyocytes against high glucose(HG)-induced injury by inhibiting necroptosis. METHODS: The protein levels of RIP3(an indicator of necroptosis) and cleaved caspase-3 were determined by Western blot. The cell viability was measured by CCK-8 assay. The intracellular le-vels of reactive oxygen species(ROS) were detected by 2', 7'-dichlorfluorescein diacetate staining followed by photofluorography. Mitochondrial membrane potential(MMP) was examined by rhodamine 123 staining followed by photofluorography. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography. RESULTS: After the H9c2 cells were treated with HG(35 mmol/L glucose) for 0~24 h, the protein expression of RIP3 in the H9c2 cells was significantly increased at 3 h, 6 h, 9 h, 12 h and 24 h, reaching the maximum level at 24 h. Pretreatment of the cells with 400μmol/L NaHS(a donor of H₂S) or co-treatment of the cells with necrostatin-1(Nec-1; a speci-fic inhibitor of necroptosis) considerably blocked the up-regulation of RIP3 protein induced by HG. Moreover, pretreatment with NaHS or co-treatment with Nec-1 obviously inhibited HG-induced injuries, leading to an increase in the cell viability, and decreases in the generation of ROS and MMP loss. On the other hand, pretreatment with NaHS also reduced the number of apoptotic cells and the protein level of cleaved caspase-3 in the HG-treated H9c2 cardiomyocytes. CONCLUSION: H₂S protects H9c2 cardiomyocytes against HG-induced injury by inhibiting necroptosis.     

Roles of CFTR Cl- channels in hydrogen sulfide-induced cardioprotection and cell proliferation in H9c2 cells

AIM: To explore the roles of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in hydrogen sulfide (H2S)-induced cardioprotection and cell proliferation in H9c2 cells. METHODS: Cobalt chloride (CoCl2) was used to set up the chemical hypoxia-induced injury model in H9c2 cells. Myocardial cell viability was detected by the CCK-8 assay kit. Apoptotic changes in H9c2 cells were observed by using Hoechst 33342 staining and photofluorography. RESULTS: At the concentrations from 400 to 2 000 μmol/L, CoCl2 dose-dependently inhibited cell viability in H9c2 cells. CoCl2 at concentration of 600 μmol/L significantly induced H9c2 cell apoptosis. Sodium hydrosulfide (NaHS) at concentrations from 100 to 400 μmol/L dose-dependently enhanced proliferation in H9c2 cells. NaHS protected H9c2 cells against CoCl2-induced injury, including an increase in cell viability and a decrease in percentage of apoptosis. 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 μmol/L), an inhibitor of CFTR Cl- channels alone did not damaged H9c2 cells, but considerably blocked the inhibitory effect of NaHS on CoCl2 cytotoxicity. However, NPPB did not antagonize the NaHS-induced antiapoptotic effect and cell proliferation in H9c2 cells. CONCLUSION: CFTR Cl- channels may be involved in the inhibitory effect of H2S on CoCl2-induced cytotoxicity in H9c2 cells.     

Critical role of cystic fibrosis transmembrane conductance regulator in hypoxia-induced apoptosis of H9c2 cardiomyocytes

AIM:To explore the roles of cystic fibrosis transmembrane conductance regulator (CFTR) in hypoxia-induced apoptosis of H9c2 cardiomyocytes and the underlying mechanisms. METHODS:The rat H9c2 cardiomyocytes were exposed to a 1% hypoxic environment in a hypoxic chamber. After CFTR overexpression, H9c2 cardiomyocytes were cultured in a hypoxic environment. The mRNA and protein levels of CFTR were examined by RT-qPCR and Western blot, respectively. The cell viability was measured by MTT assay. The apoptotic rate was determined by Hoechst 33342 and Annexin V-FITC/PI staining, and the production of reactive oxygen species (ROS) was examined by dichloro-dihydro-fluorescein diacetate (DCF-DA) staining. RESULTS:Hypoxic exposure caused the apoptosis of H9c2 cardiomyocytes, which was accompanied by the down-regulation of CFTR at mRNA and protein levels and over-production of ROS (P<0.05). After CFTR overexpression, the apoptotic rate of the H9c2 cardiomyocytes induced by hypoxia was significantly reduced, with a prominent inhibition of ROS production (P<0.05). However, pretreatment with CFTRinh-172, a specific inhibitor of CFTR, reversed the protective effect of CFTR overexpression in H9c2 cardiomyocytes. CONCLUSION:CFTR has a critical role in protecting against hypoxia-induced apoptosis of H9c2 cells, which may be through inhibiting the generation of ROS.     

miR-323 regulates hypoxia-induced apoptosis of H9C2 cardiomyocytes through FGF9

AIM: To investigate the effect of microRNA-323 (miR-323) on the apoptosis of hypoxia-induced rat H9C2 cardiomyocytes and its mechanism. METHODS: The hypoxic injury model was established in the H9C2 cells. Anti-miR-323, pcDNA-FGF9 and si-FGF9 were transfected into the H9C2 cells and cultured under hypoxic condition for 48 h. The expression of miR-323 was detected by qPCR. The protein levels of fibroblast growth factor 9 (FGF9), cleaved caspase-3, c-Jun N-terminal kinase (JNK) and p-JNK were determined by Western blot. The cell viability was measured by MTT assay, and the apoptosis was analyzed by flow cytometry. The method of bioinformatics was applied to predict the target gene of miR-323, and dual-luciferase reporter assay was used for further validation. RESULTS: Hypoxia greatly reduced the viability of H9C2 cells at 12 h, 24 h and 48 h (P<0.05), and remarkably increased apoptotic rate and the protein level of cleaved caspase-3 in a time-dependent manner (P<0.05). The expression of miR-323 and the protein level of p-JNK were up-regulated and the expression of FGF9 was down-regulated in the H9C2 cells exposed to hypoxia (P<0.05). Down-regulation of miR-323 and over-expression of FGF9 obviously increased the viability of the H9C2 cells exposed to hypoxia, and decreased the apoptotic rate and the protein level of cleaved caspase-3 (P<0.05). FGF9 was the target gene of miR-323. Down-regulation of FGF9 reversed the attenuating effect of down-regulation of miR-323 on hypoxia-induced H9C2 cell injury. miR-323 regulated FGF9 and affected p-JNK level. CONCLUSION: miR-323 affects the viability and apoptosis of H9C2 cardiomyocytes under hypoxia by targeting FGF9 and regulating JNK signaling pathway.     

Shikonin inhibits neuronal apoptosis induced by OGD through targeting PI3K/Akt signaling pathway

AIM: To explore the effect of shikonin on rat primary cortical neurons in oxygen-glucose deprivation (OGD)-induced injury model.METHODS: The neurons were pretreated with shikonin at different concentrations (0.02, 0.2, 2 and 20 μmol/L) followed by treatment with OGD. Lactate dehydrogenase (LDH) release assay and fluorescein diacetate/propidium iodide (FDA/PI) double staining were used to detect neuronal viability and apoptosis, and then the optimal concentration of shikonin was determined. LY294002 (PI3K/Akt signaling pathway inhibitor, 1 μmol/L) was added before the addition of shikonin, and the protein level of p-Akt (Ser473) in the neurons was determined by Wes-tern blot. LDH release assay and FDA/PI double staining were also used to detect neuronal viability and apoptosis.RESULTS: A certain concentration (0.2~20 μmol/L) of shikonin increased the viability of impaired neurons (P<0.05) and the protein level of p-Akt (Ser473) in the neurons (P<0.05). The effect of shikonin on neuronal p-Akt (Ser473) levels and the cell death were blocked by LY294002 (P<0.05).CONCLUSION: A certain concentration of shikonin reduces OGD-induced apoptosis of rat primary cortical neurons by activating PI3K/Akt signaling pathway.     

Tanshinone ⅡA attenuates doxorubicin-induced injury in H9c2 cardiomyocytes via AMPK-mediated autophagy

AIM: To investigate the effect of tanshinone ⅡA on doxorubicin-induced rat H9c2 cardiomyocyte injury. METHODS: The H9c2 cardiomyocytes were treated with tanshinone ⅡA and/or doxorubicin with or without AMPK inhibitor dorsomorphin. The cell viability was measured by CCK-8 assay, the LDH release was examined for evaluating the cell injury, autophagy was analyzed by immunofluorescence staining, and AMPK activation was determined by Western blot. RESULTS: Compared with control group, the viability of H9c2 cells was decreased, the release of LDH was increased, the autophagy degree was increased, and AMPK activation was inhibited after treatment with doxorubicin (P<0.05). Compared with doxorubicin group, the treatment with tanshinone ⅡA restored the cell viability, reduced the release of LDH, further increased autophagy degree, and activated AMPK in the H9c2 cells (P<0.05). AMPK inhibitor dorsomorphin attenuated the abilities of tanshinone ⅡA to restore the cell viability, reduce the release of LDH, and increase autophagy degree in the H9c2 cells (P<0.05). CONCLUSION: Tanshinone ⅡA attenuates doxorubicin-induced injury in H9c2 cardiomyocytes, and its mechanism might be related to AMPK-mediated autophagy, which provides experimental and theoretical basis for the clinical application of tanshinone ⅡA in the prevention and treatment of doxorubicin-induced cardiomyocyte injury.     

Lycopene protects primary mouse cerebrocortical neurons against t-BHP-induced damage in vitro

AIM: To investigate the protective effect of lycopene on primary mouse cerebrocortical neurons exposed to tert-butyl hydroperoxide (t-BHP) and its mechanisms of in vitro.METHODS: Primary cerebrocortical neurons of newborn C57 mice were extracted and divided into normal group, t-BHP group, lycopene+t-BHP group and lycopene group. The neuronal damage was induced by t-BHP exposure for 24 h, and the cell viability was examined by MTT assay. ROS content was measured by flow cytometry, and the protein levels of Bax, Bcl-2, caspase-3, cleaved caspase-3 and cytochrome C were examined by Western blot.RESULTS: The primary mouse cortical neurons expressed MAP-2 protein. Lycopene at concentration of 4 μmol/L reversed the decrease in cell viability. Flow cytometry revealed that lycopene treatment attenuated ROS content under the condition of t-BHP exposure. In addition, the protein level of Bcl-2 was increased, and the expression of Bax, cleaved caspase-3 and cytochrome-C was suppressed in lycopene+t-BHP group.CONCLUSION: The protective effect of lycopene on cortical neurons with t-BHP-induced injury may be involved in the mechanism of neuronal antioxidative response by down-regulating caspase-3 and Bax/Bcl-2 through the mitochondrial apoptotic pathway.     

Role of ATP-sensitive potassium channels in inhibitory effect of hydrogen sulfide on high glucose-induced injury in H9c2 cardiac cells

AIM: To investigate the roles of ATP-sensitive potassium (K_ATP) channels in high glucose-induced cardiac injury and the inhibitory effect of hydrogen sulfide (H₂S) on the cardiomyocyte injury. METHODS: The expression level of K_ATP channel protein was tested by Western blot. The cell viability was measured by CCK-8 assay. The number of apoptotic cells was observed by Hoechst 33258 nuclear staining. Mitochondrial membrane potential (MMP) was examined by JC-1 staining. RESULTS: After the H9c2 cells were treated with 35 mmol/L glucose (high glucose, HG) for 1~24 h, the protein level of K_ATP channel was significantly reduced at 6 h, 9 h, 12 h and 24 h, reaching the minimum level at 12 h and 24 h. Pretreatment of the cells with 400 μmol/L NaHS (a donor of H₂S) prior to exposure to HG for 12 h considerably blocked the down-regulation of K_ATP channels induced by HG. Pretreatment of the cells with 100 μmol/L mitochondrial K_ATP channel opener diazoxide, 50 μmol/L non-selective K_ATP channel opener pinacidil or NaHS obviously inhibited HG-induced injuries, leading to an increase in the cell viability, and decreases in the number of apoptotic cells and the MMP loss. Pretreatment with 100 μmol/L mitochondrial K_ATP channel antagonist 5-hydroxydecanoic acid or 1 mmol/L non-selective K_ATP channel antagonist glibenclamide attenuated the above cardioprotective effects of NaHS. CONCLUSION: K_ATP channels mediate the inhibitory effect of H₂S on HG-induced cardiac injury.     

Hydrogen sulfide suppresses the increase in reactive oxygen species in neurons induced by angiotensin II via ERK1/2 signal pathway

AIM: To investigate the effect of hydrogen sulfide (H₂S) on the reactive oxygen species (ROS) level in medullary neurons induced by angiotensin II (Ang II). METHODS: Primarily cultured rat medullary neurons were divided into 5 groups as follows: control group, Ang II group, sodium hydrosulfide(NaHS) group, NaHS with Ang II group, and PD98059 (an inhibitor of p-ERK1/2) with Ang II group. ROS production was measured with dihydroethidium (DHE) staining. The expression of p-ERK1/2 and ERK1/2 was determined by Western blotting. The activity of neurons was detected by CCK-8 assay. RESULTS: Ang II at concentration of 100 nmol/L significantly increased ROS level in the neurons, but the effect was inhibited by NaHS at concentrations of 50~200 μmol/L, while NaHS alone had no influence on the ROS level in neurons. Additionally, PD98059 also depressed the ROS level in neurons induced by Ang II. Furthermore, the enhanced expression of p-ERK1/2 in the neurons induced by Ang II was significantly reduced by NaHS. CONCLUSION: H₂S remarkably inhibits the ROS level in the neurons induced by Ang II via activation of MAPK signal pathways, especially p-ERK1/2, indicating that H₂S rescues neurons from oxidative stress through declining the enhanced expression of p-ERK1/2.     

Sevoflurane preconditioning inhibits brain injury in hypoxic mice

AIM: To observe the effects of sevoflurane preconditioning on brain injury in hypoxic mice and its possible mechanism. METHODS: Male C57BL/6J mice were randomly divided into control (C) group, hypoxia (H) group, 2% sevoflurane preconditioning for 30 min + hypoxia (S1+H) group, 2% sevoflurane preconditioning for 60 min+hypoxia (S2+H) group and 4% sevoflurane preconditioning for 30 min + hypoxia (S3+H) group. The hypoxia model was established by continuous inhalation of (6.5±0.1)% O₂ for 24 h. The sevoflurane preconditioning treatments, S1, S2 and S3, were conducted by inhalation of 2% sevoflurane for 30 min, 2% sevoflurane for 60 min and 4% sevoflurane for 30 min, respectively, with the carrier of (21.0±0.5)% O₂, followed by washout for 15 min and then hypoxia treatment. The histological changes of the hippocampal CA1 area were observed under light microscope and transmission electron microscope (TEM), and serum lactate dehydrogenase (LDH) activity was measured by colorimetric method. Furthermore, the protein levels of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in brain tissue homogenate were examined by ELISA, and the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured by microplate reader. RESULTS: After hypoxia for 24 h, cell edema or pyknosis in the hippocampal CA1 area was observed in H group. Sevoflurane preconditioning reduced hypoxic injury, and the cell ultrastructure under TEM was significantly improved in S2+H group. Compared with C group, the serum LDH activity and the levels of EPO, VEGF and MDA in brain tissues were significantly increased in H group, while the activity of SOD and GPx decreased. After sevoflurane pretreatment, the serum LDH activity and the levels of EPO and VEGF in brain tissues were lower than those in H group, and the most significant difference was observed in S2+H group. Moreover, the MDA content and SOD activity decreased, and the GPx activity increased in the sevoflurane preconditioning groups. CONCLUSION: Sevoflurane preconditioning attenuates brain injury in hypoxic mice by regulating antihypoxic protein synthesis and reducing oxidative stress.     

Effects of salvianolic acid B on neurons during oxygen/glucose deprivation and reintroduction

AIM: To observe the damage induced in the primary cultured rat cortical neurons by oxygen/glucose deprivation and reintroduction, and to investigate the neuroprotective mechanism of salvianolic acid B (SalB). METHODS: Primary cultured rat cortical neurons were randomly divided into the control group, the model group and the SalB group. The cell model was established by oxygen/glucose deprivation for 3 h followed oxygen/glucose reintroduction for 24 h. The cortical neurons viability was determined by MTT assay. The leakage rate of lactate dehydrogenase (LDH) was measured by chromatometry. The mitochondrial membrane potentials (MMP) and the apoptosis rate were quantitatively analyzed by flow cytometry. The cytosolic free calcium was assessed using LSCM. The morphologic changes of neuronal nuclei were observed by Hoechst 33342 fluorescence staining. RESULTS: Compared to the model group, the cortical neurons viability, the survival rate and the fluorescence value of MMP in the SalB group were obviously increased (P<0.05,P<0.01). In addition, in the SalB group, the leakage rate of LDH, the fluorescence intensity of cytosolic free calcium and the apoptosis rate were significantly lower than those in the model group (P<0.01). CONCLUSION: The neuroprotective mechanism of SalB in the oxygen/glucose deprivation and reintroduction neurons would be due to the fact that SalB maintains the MMP and the calcium homeostasis.     

Antagonistic action of Jiunaoning injection aganist oxygen/glucose- deprived and reperfusion injury-induced cultured rat cortical neurons

AIM: To determine whether Jiunaoning injection has protective effects on oxygen/glucose-deprived and reperfusion injury-induced neurons. METHODS: Various concentrations of Jiunaoning injection (0.5-5 mL/L) were used to observe their effects on cultured rat cortical neurons induced by oxygen/glucose-deprived and reperfusion injury in various time points. The neuronal metabolic rate and viability were assessed by using 3-(4,5-dimethylthiazol)-2, 5-diphenyl-tetra zoliumbromide (MTT) and lactate dehydrogenase (LDH) assay. RESULTS: Jiunaoning injection enhanced the neuronal metabolic rate in a dose-dependent manner in the range from 0.5 to 5 mL/L, and Jiunaoning injection (1.5-2.5 mL/L) enhanced the neuronal metabolic rate, decreased the cell death rate and depressed LDH leak rate significantly. CONCLUSION: Jiunaoning injection has an affirmative protective effect on oxygen/glucose-deprived and reperfusion-induced neuronal injury.     

APPL1 reduces injury of H9c2 cardiomyocytes induced by LPS

AIM:To study the effect of APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) on H9c2 cardiomyocyte injury induced by lipopolysaccharides (LPS). METHODS:The H9c2 cells were treated with LPS. RT-qPCR and Western blot were used to detect the expression of APPL1 in the H9c2 cells. The recombinant APPL1 lentiviral vector was used to transfect into the H9c2 cells. After LPS treatment, the over-expression efficiency was detected by RT-qPCR and Western blot. The viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein level of activated caspase-3 in the H9c2 cells was determined by Western blot. The content of malonaldehyde (MDA) in the H9c2 cells and the level of lactate dehydrogenase (LDH) in the culture medium were detected. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the level of reative oxygen species (ROS) in the H9c2 cells were also examined. RESULTS:The expression of APPL1 at mRNA and protein levels in LPS-treated H9c2 cells was decreased significantly (P<0.05). Over-expression of APPL1 by transfection of recombinant lentiviral vector significantly increased the level of APPL1 at mRNA and protein levels in the H9c2 cells with LPS treatment (P<0.05). LPS treatment reduced the viability, but increased the apoptotic rate of the H9c2 cells, the protein level of activated caspase-3, the content of MDA and the level of LDH in the culture medium. The activity of SOD and GSH-Px was reduced, while the level of ROS was increased as compared with control group (P<0.05). Over-expression of APPL1 elevated the viability of H9c2 cells treated with LPS, and the apoptotic rate and the protein level of activated caspase-3 were decreased. The content of MDA and the level of LDH in the culture medium were reduced, the activity of SOD and GSH-Px was elevated, and the level of ROS was reduced as compared with the H9c2 cells treated with LPS alone (P<0.05). CONCLUSION:Over-expression of APPL1 reduces oxidative damage and apoptosis of the H9c2 cells induced by LPS.     

Identification of ATTM as a novel H2S donor and investigation of its protective effect on HaCaT skin cells

AIM: To investigate the ability of a metal complex ammonium tetrathiomolybdate (ATTM) to release H₂S and its cytoprotective effect on an oxidative injury model. METHODS: Released H₂S was absorbed in a reaction flask from ATTM dissolved in the cell medium. Staining with dichlorodihydrofluorescein diacetate or rhodamine 123 followed by photofluorography was conducted for the observation of reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ_m) levels, respectively. Cell viability and release of lactate dehydrogenase (LDH) from the cells were measured with commercial kits. RESULTS: Similar to another H₂S donor GYY4137, ATTM had an ability to release H₂S in the cell medium in a dose-dependent manner. Treatment of human skin HaCaT cells with ATTM at concentrations of 25~400 μmol/L didn't significantly alter cell viability. Exposure of the cells to ultraviolet rays or a ROS donor H₂O₂ increased the intracellular ROS levels. Treatment with 400 μmol/L H₂O₂ significantly reduced the viability of HaCaT cells (P<0.01). However, before the treatment with H₂O₂, pretreatment with ATTM at 100 and 200 μmol/L markedly prevented the H₂O₂-induced cell injury (P<0.01). In addition, the treatment with H₂O₂ triggered ΔΨ_m loss (P<0.01) and LDH release from the cells (P<0.01). Prior to suffering from H₂O₂ injury, the preconditioning with 200 μmol/L ATTM significantly improved ΔΨ_m levels (P<0.05) and attenuated LDH release from the cells (P<0.01).CONCLUSION: ATTM is capable of releasing H₂S and protecting human skin cells against oxidative injury.     

Response of mitochondrial reactive oxygen species in pulmonary arterial smooth muscle cells under acute hypoxic condition

AIM:To observe the response of mitochondrial reactive oxygen species (ROS) in rat pulmonary artery smooth muscle cells (PASMCs) under acute hypoxic condition. METHODS:The cultured PASMCs were under normoxic (35 ℃, 5% CO2, 21% O2, 74% N2) or acute hypoxic (35℃, 5% CO2, 1% O2, 94% N2) condition. The cells were incubated with molecular probes chloromethyl dichlorodihydrofluorescein diacetate (CM-H2DCF/DA) and RedoxSensor Red CC-1 to detect the ROS generation by laser scanning confocal microscopy. The mitochondria were isolated and mitochondrial inhibitors were used to detect the ROS generation functional unit sites by spectrophotometry under acute hypoxic condition. RESULTS:Under acute hypoxic condition, the intracellular ROS was significantly increased in hypoxia group with 3.35 folds higher of H2O2 than that in normoxia group. The contents of H2O2 and O-·2 in hypoxia group were 1.61 folds higher than those in normoxia group. Compare with hypoxia goup, pretreatment with the mitochondrial electron transport chain (ETC) complex I inhibitor MPP, the complex II inhibitors NPA and TTFA as well as the complex III pre-ubisemiquinone site inhibitor myxothiazol all remarkably reduced hypoxia-induced increase in ROS generation in PASMCs (reduced by 60%, 73%, 75% and 61%, respectively, P<0.01), whereas the complex III postubisemiquinone site inhibitor antimycin A and the complex IV inhibitor NaN3 had no effect on hypoxia-induced increase in ROS generation (increased by 13% and 9.1%, respectively, P>0.05). Direct detection of mitochondrial ROS showed the same results as the intracellular ROS. CONCLUSION: The intracellular ROS increases significantly in rat PASMCs under acute hypoxic condition. The mitochondrial ETC complex I, complex II and complex III pre-ubisemiquinone sites increase ROS generation, whereas the complex III postubisemiquinone site and complex IV do not produce this effect under acute hypoxic condition.     

Effects of adenosine A1 receptor antagonist DPCPX on cerebral neurons after hypoxia and reoxygenation

AIM: To investigate the effects of adenosine A₁ receptor antagonist DPCPX on the release of cerebral neuronal lactate dehydrogenase (LDH), the activity of calcineurin (CaN) and acetylcholinesterase (AChE), and the level of extracellular amino acid after hypoxia and reoxygenation (H/R).METHODS: Primary cultured rat cerebral cortical neurons were used to establish an H/R injury model. Different concentrations of DPCPX (the final concentrations were as follows: 0, 25, 50 and 100 nmol/L) were added at the same time of hypoxia treatment for 8 h, 12 h or 24 h,followed by reoxygenation for 24 h. The LDH release from the neurons was measured. The effects of DPCPX (100 nmol/L) on the activity of CaN and AChE, and the level of extracellular amino acid in neurons treated with hypoxia for 12 h followed by reoxygenation were observed. RESULTS: Compared to the cells in control groups, the neurons treated with 100 nmol/L DPCPX and exposed to hypoxia for 12 h followed by reoxygenation, showed significantly higher LDH release, higher activity of CaN and AChE, and lower level of extracellular γ-aminobutyric acid.CONCLUSION: These results suggest that DPCPX increases the LDH release and the activity of CaN and AChE, decreases the level of extracellular γ-aminobutyric acid in neurons with H/R.     

Angiotensin-(1-7) protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis

AIM: To investigate whether angiotensin-(1-7)[Ang-(1-7)] protects H9c2 cardiac cells against high glucose (HG)-induced injury and inflammation by inhibiting the interaction between Toll-like receptor 4 (TLR4) activation and necroptosis. METHODS: The expression levels of receptor-interacting protein 3 (RIP3; an indicator of necroptosis) and TLR4 were determined by Western blot. Cell viability was measured by CCK-8 assay. The activity of lactate dehydrogenase (LDH) in the culture medium was measured with a commercial kit. The releases of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by ELISA. The intracellular level of reactive oxygen species (ROS) was analyzed by 2', 7'-dichlorfluorescein-diacetate (DCFH-DA) stating followed by photofluorography. Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorography. RESULTS: After the H9c2 cardiac cells were treated with HG (35 mmol/L glucose) for 24 h, the expression of RIP3 was obviously increased. Co-treatment of the cells with 30 μmol/L TAK-242 (an inhibitor of TLR4) attenuated the up-regulation of RIP3 induced by HG. Furthermore, the expression of TLR4 was significantly increased after the cells were exposed to HG for 24 h, and co-treatment of the cells with 100 μmol/L necrostatin-1 (Nec-1; a specific inhibitor of necroptosis) and HG for 24 h attenuated the up-regulation of TLR4 expression induced by HG. Moreover, 1 μmol/L Ang-(1-7) simultaneously blocked the up-regulation of the RIP3 and TLR4 induced by HG. On the other hand, co-treatment of the cells with 1 μmol/L Ang-(1-7), 30 μmol/L TAK-242 or 100 μmol/L Nec-1 and HG for 24 h attenuated HG-induced injuries and inflammatory response, leading to the increase in the cell viability, and the decreases in the activity of LDH, ROS generation, MMP loss as well as the releases of IL-1β and TNF-α. CONCLUSION: Ang-(1-7) protects H9c2 cardiac cells against HG-induced injury and inflammation by inhibiting the interaction between TLR4 activation and necroptosis.     

Effects of Homer1a over-expression on apoptosis and AMPK protein expression in mechanically injured neurons

AIM:To investigate the effects of Homer1a over-expression on the apoptosis and AMP-activated protein kinase (AMPK) protein expression in mechanically injured neurons. METHODS:The rat cortical neurons were isolated and cultured in vitro, and then ramdomly divided into control group, model group, empty vector group, and Exp-Homer1a group. Neuron models with mechanical injury were constructed and infected with the Homer1a over-expression vector. The mRNA expression of Homer1a was detected by qPCR. The cell viability in each group was detected by MTT assay. The activity of lactate dehydrogenase (LDH) in the supernatant of each group was measured by LDH test kit. The apoptosis level was analyzed by flow cytometry. The protein levels of Hormer1a, cleaved caspase-3, Bax, Bcl-2, p-AMPKα and AMPKα were determined by Western blot. RESULTS:Compared with control group, the viability of mechanically injured neurons was significantly decreased, the LDH activity in the supernatant and neuronal apoptotic rate were significantly increased (P<0.05), and Homer1a expression at mRNA and protein levels was significantly increased (P<0.05). Compared with model group, the LDH activity in the supernatant and neuronal apoptotic rate in Exp-Homer1a group were significantly decreased, the protein levels of cleaved caspase-3 and Bax were significantly decreased (P<0.05), and the protein levels of Bcl-2 and p-AMPKα were significantly increased (P<0.05). CONCLUSION:Over-expression of Homer1a may increase the viability of mechanically injured neurons and inhibit their apoptosis by promoting the activation of AMPKα phosphorylation.