Objective: To investigate whether ginsenoside-Rb1(Gs-Rb1) improves the CoCl2-induced autophagy of cardiomyocytes via upregulation of adenosine 5’-monophosphate-activated protein kinase(AMPK) pathway. Methods: Ventric...Objective: To investigate whether ginsenoside-Rb1(Gs-Rb1) improves the CoCl2-induced autophagy of cardiomyocytes via upregulation of adenosine 5’-monophosphate-activated protein kinase(AMPK) pathway. Methods: Ventricles from 1-to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group(normal level oxygen), hypoxia group(500 μmol/L CoCl2), Gs-Rb1 group(200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), Ara A group(500 μmol/L Ara A + 500 μmol/L CoCl2), Ara A+ Gs-Rb1 group(500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide(AICAR)+ 500 μmol/L CoCl2], and AICAR+Gs-Rb1 group(1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2). Cel s were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay and cardiac troponin I(cTnI) levels were detected by enzyme-linked immunosorbent assay(ELISA). AMPK activity was assessed by 2’,7’-dichlorofluorescein diacetate(DCFH-DA) ELISA assay. The protein expressions of Atg4 B, Atg5, Atg6, Atg7, microtubule-associated protein 1 A/1 B-light chain 3(LC3), P62, and active-cathepsin B were measured by Western blot. Results: Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes(P<0.01). However, the viability of hypoxia-treated cardiomyocytes was significantly inhibited by Ara A(P<0.01). Gs-Rb1 increased the AMPK activity of hypoxia-treated cardiomyocytes. The AMPK activity of hypoxia-treated cadiomyocytes was inhibited by Ara A(P<0.01) and was not affected by AICAR(P=0.983). Gs-Rb1 up-regulated Atg4B, Atg5, Beclin-1, Atg7, LC3B Ⅱ, the LC3BⅡ/Ⅰ ratio and cathepsin B activity of hypoxia cardiomyocytes(P<0.05), each of these protein levels was significantly enhanced by Ara A(all P<0.01), but was not affected by AICAR(all P>0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes(P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A(P<0.05) and were not affected by AICAR(P=0.871). Conclusion: Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.展开更多
Atorvastatin is proven to ameliorate cardiac hypertrophy induced by chronic intermittent hypoxia （CIH）. However, little is known about the mechanism by which atorvastatin modulates CIH-induced cardiac hypertrophy, a...Atorvastatin is proven to ameliorate cardiac hypertrophy induced by chronic intermittent hypoxia （CIH）. However, little is known about the mechanism by which atorvastatin modulates CIH-induced cardiac hypertrophy, and whether specific hypertrophyrelated microRNAs are involved in the modulation. MiR-31 plays key roles in the development of cardiac hypertrophy induced by ischemia/hypoxia. This study examined whether miR-31 was involved in the protective role of atorvastatin against CIH-induced myocardial hypertrophy. H9c2 cells were subjected to 8-h intermittent hypoxia per day in the presence or absence of atorvastatin for 5 days. The size of cardiomyocytes, and the expression of caspase 3 and miR-31 were determined by Western blotting and RT-PCR, respectively. MiR-31 mimic or Ro 31-8220, a specific inhibitor of protein kinase C epsilon （PKCε）, was used to determine the role of miR-31 in the anti-hypertrophic effect of atorvastatin on cardiomyocytes. PKCε in the cardiomyocytes with miR-31 upregulation or downregulation was detected using RT-PCR and Western blotting. The results showed that CIH induced obvious enlargement of cardiomyocytes, which was paralleled with increased atrial natriuretic peptide （ANP）, brain natriuretic peptide （BNP）, and slow/beta cardiac myosin heavy-chain （MYHT） mRNA levels. All these changes were reversed by the treatment with atorvastatin. Meanwhile, miR-31 was increased by CIH in vitro. Of note, the atorvastatin pretreatment significantly increased the mRNA and protein expression of PKCε and decreased that of miR-31. Moreover, overexpression of miR-31 abolished the anti-hypertrophic effect of atorvastatin on cardiomyocytes. Upregulation and downregulation of miR-31 respectively decreased and increased the mRNA and protein expression of PKCε. These results suggest that atorvastatin provides the cardioprotective effects against CIH probably via up-regulating PKCε and down-regulating miR-31.展开更多
Objective:To investigate to the expression effect of hypoxia and hypoxia/reoxygenation on ROS,MAPKs and cell apoptosis in H9c2 cardiomyocytes.Methods:H9c2 cells were treated with cobalt chloride(CoCl2)to establish the...Objective:To investigate to the expression effect of hypoxia and hypoxia/reoxygenation on ROS,MAPKs and cell apoptosis in H9c2 cardiomyocytes.Methods:H9c2 cells were treated with cobalt chloride(CoCl2)to establish the chemical hypoxia and hypoxia/reoxygenation-induced cardiomyocyte injury model.CoCl2 was used to process cells at different concentrations from 150-2400μmol/L and different time from4-24 h;H9c2 cells viability was detected by MTT,and the intracellular ROS level was measured by 2’,7’-dichlorflμoresceindiacetate(DCFH-DA)and dihydroethidiμm(DHE)staining and photoflurography.The active expression level of mitogen-activated protein kinases(MAPKs)(including JNK,ERK and p38)and caspase-3.Results:At the concentration from 300 to 1200μmol/L,CoCl2 does/time-dependently inhibited the cell viability in H9c2 cells(P<0.01).Compared with control group,the ROS levels in hypoxia group were significantly increased(P<0.05).In hypoxia group,the active expression levels of p-JNK,p-p38 and caspase-3 was higher than those in control group(P<0.05).However,the expression of p-ERK wasn’t significant differernce.Furthermore,all the expression levels of ROS,p-JNK,p-ERK,p-p38 and caspase-3 in H/R group were significantly raised compared with hypoxia group(P<0.01).Conclusions:Reoxygenation further aggravate chemical hypoxia induced cardiomyocyte oxidative stress injury by activating ROS/MAPKs signals,suggesting the role of myocardial ischemia/reperfusion injury in the pathogenesis of ischemic heart disease.展开更多
Background Chronic hypoxia can cause pulmonary hypertension and pulmonary heart disease with high mortality.The signal transduction pathway of protein kinase C (PKC) plays an important role in chronic pulmonary hype...Background Chronic hypoxia can cause pulmonary hypertension and pulmonary heart disease with high mortality.The signal transduction pathway of protein kinase C (PKC) plays an important role in chronic pulmonary hypertension. So it is necessary to investigate the effect of PKC on voltage-gated potassium (K +) channels in pulmonary artery smooth muscle cells of rats exposed to chronic hypoxia. Methods Male Wistar rats were randomly divided into a control group (group A) and a chronic hypoxia group (group B). Group B received hypoxia [oxygen concentration (10±1)%] eight hours per day for four consecutive weeks. Single pulmonary artery smooth muscle cells were obtained using an acute enzyme separation method. Conventional whole cell patch clamp technique was used to record resting membrane potential,membrane capacitance and voltage-gated K + currents. The changes in voltage-gated K + currents before and after applying paramethoxyamphetamine (PMA) (500 nmol/L),an agonist of PKC,and PMA plus carbohydrate mixture of glucose,fructose and xylitol (GFX) (30 nmol/L),an inhibitor of PKC,were compared between the two groups. Results The resting membrane potential in group B was significantly lower than that of group A: -(29.0±4.8) mV (n=18) vs -(42.5±4.6) mV (n=35) ( P <0.01). But there was no change in membrane capacitance between the two groups: (17.9±4.6) pF (n=40) vs (19.7±5.8) pF (n=31) ( P >0.05). The voltage-gated K + currents were significantly inhibited by PMA in group A,and this effect was reversed by GFX. However,the voltage-gated K + currents in group B were not affected by PMA. Conclusions The resting membrane potential and voltage-gated K + currents in pulmonary artery smooth muscle cells from rats exposed to chronic hypoxia decreased significantly. It seems that PKC has different effects on the voltage-gated K + currents of pulmonary artery smooth muscle cells under different conditions.展开更多
Ischemic stroke is a major cause of morbidity and mortality,and currently there is no effective treatment.The family of protein kinase C(PKCs)could phosphorylate serine or threonine residues of its substrate proteins ...Ischemic stroke is a major cause of morbidity and mortality,and currently there is no effective treatment.The family of protein kinase C(PKCs)could phosphorylate serine or threonine residues of its substrate proteins and play a key role in the ischemia/reperfusion injury.Autophagy is essential for maintaining cell homeostasis under physiological condition and acts as a double-edged sword in the process of ischemic neuronal death.In this article,we reviewed the PKCs isoform-specific signaling pathways and PKC-modulated autophagy in ischemic stroke.展开更多
Background:Tumor necrosis factor receptor-associated protein 1(TRAP1)plays a protective effect in hypoxic cardiomyocytes,but the precise mechanisms are not well clarified.The study is aimed to identify the mechanism o...Background:Tumor necrosis factor receptor-associated protein 1(TRAP1)plays a protective effect in hypoxic cardiomyocytes,but the precise mechanisms are not well clarified.The study is aimed to identify the mechanism of TRAP1 on hypoxic damage in cardiomyocytes.Methods:In this study,the effects of TRAP1 and cytochrome c oxidase subunit Ⅱ(COXⅡ)on apoptosis in hypoxia-induced cardiomyocytes were explored using overexpression and knockdown methods separately.Results:Hypoxia induced cardiomyocyte apoptosis,and TRAP1 overexpression notably inhibited apoptosis induced by hypoxia.Conversely,TRAP1 silencing promoted apoptosis in hypoxic cardiomyocytes.Further investigation revealed that the proapoptotic effects caused by the silencing of TRAP1 were prevented by COXⅡ overexpression,whereas COXⅡ knockdown reduced the antiapoptotic function induced by TRAP1 overexpression.Additionally,changes in the release of cytochrome c from mitochondria into the cytosol and the caspase-3 activity in the cytoplasm,as well as reactive oxygen species production,were found to be correlated with the changes in apoptosis.Conclusions:The current study uncovered that TRAP1 regulates hypoxia-induced cardiomyocyte apoptosis through a mitochondria-dependent apoptotic pathway mediated by COXⅡ,in which reactive oxygen species presents as an important component.展开更多
Objective To investigate the interaction between fragile histamine triad (FHIT) and protein kinase C alpha (PKCα) in human non-small cell lung cancer tissues. Methods FHIT and PKCα double positive samples were scree...Objective To investigate the interaction between fragile histamine triad (FHIT) and protein kinase C alpha (PKCα) in human non-small cell lung cancer tissues. Methods FHIT and PKCα double positive samples were screened by immunohistochemical staining from 13 human non-small cell lung cancer tissues. Co-immunoprecipitation was performed by using anti-FHIT and anti-PKCα. The immune precipitate was analyzed by SDS-PAGE and Western blot. Results Immune precipitate staining detection showed that 3 samples out of the 13 cases were double positive for FHIT and PKCα. FHIT protein was present in the immune precipitate of anti-PKCα while there was PKCα in the immune precipitate of anti-FHITmAb. Conclusion FHIT and PKCα exist as a complex in human non-small cell lung cancer tissues, which will provide a new route for studying the pathogenesis and immunotherapy of human non-small cell lung cancer.展开更多
基金Supported by Shenyang Innovation Foundation of Science and Technology–the Application Projects of Basic Research(No.F15-199-1-06)Liaoning Province Science and Technique Foundation of China(No.2015020282)
文摘Objective: To investigate whether ginsenoside-Rb1(Gs-Rb1) improves the CoCl2-induced autophagy of cardiomyocytes via upregulation of adenosine 5’-monophosphate-activated protein kinase(AMPK) pathway. Methods: Ventricles from 1-to 3-day-old Wistar rats were sequentially digested, separated and incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum for 3 days followed by synchronization. Neonatal rat cardiomyocytes were randomly divided into 7 groups: control group(normal level oxygen), hypoxia group(500 μmol/L CoCl2), Gs-Rb1 group(200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), Ara A group(500 μmol/L Ara A + 500 μmol/L CoCl2), Ara A+ Gs-Rb1 group(500 μmol/L Ara A + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2), AICAR group [1 mmol/L 5-aminoimidazole-4-carboxamide ribonucleotide(AICAR)+ 500 μmol/L CoCl2], and AICAR+Gs-Rb1 group(1 mmol/L AICAR + 200 μmol/L Gs-Rb1 + 500 μmol/L CoCl2). Cel s were treated for 12 h and cell viability was determined by methylthiazolyldiphenyl-tetrazolium bromide(MTT) assay and cardiac troponin I(cTnI) levels were detected by enzyme-linked immunosorbent assay(ELISA). AMPK activity was assessed by 2’,7’-dichlorofluorescein diacetate(DCFH-DA) ELISA assay. The protein expressions of Atg4 B, Atg5, Atg6, Atg7, microtubule-associated protein 1 A/1 B-light chain 3(LC3), P62, and active-cathepsin B were measured by Western blot. Results: Gs-Rb1 significantly improved the cell viability of hypoxia cardiomyocytes(P<0.01). However, the viability of hypoxia-treated cardiomyocytes was significantly inhibited by Ara A(P<0.01). Gs-Rb1 increased the AMPK activity of hypoxia-treated cardiomyocytes. The AMPK activity of hypoxia-treated cadiomyocytes was inhibited by Ara A(P<0.01) and was not affected by AICAR(P=0.983). Gs-Rb1 up-regulated Atg4B, Atg5, Beclin-1, Atg7, LC3B Ⅱ, the LC3BⅡ/Ⅰ ratio and cathepsin B activity of hypoxia cardiomyocytes(P<0.05), each of these protein levels was significantly enhanced by Ara A(all P<0.01), but was not affected by AICAR(all P>0.05). Gs-Rb1 significantly down-regulated P62 levels of hypoxic cardiomyocytes(P<0.05). The P62 levels of hypoxic cardiomyocytes were inhibited by Ara A(P<0.05) and were not affected by AICAR(P=0.871). Conclusion: Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the upregulation of AMPK pathway.
基金This project was supported by the National Natural Science Foundation of China （No. 81370185 and No. 81570080）.
文摘Atorvastatin is proven to ameliorate cardiac hypertrophy induced by chronic intermittent hypoxia （CIH）. However, little is known about the mechanism by which atorvastatin modulates CIH-induced cardiac hypertrophy, and whether specific hypertrophyrelated microRNAs are involved in the modulation. MiR-31 plays key roles in the development of cardiac hypertrophy induced by ischemia/hypoxia. This study examined whether miR-31 was involved in the protective role of atorvastatin against CIH-induced myocardial hypertrophy. H9c2 cells were subjected to 8-h intermittent hypoxia per day in the presence or absence of atorvastatin for 5 days. The size of cardiomyocytes, and the expression of caspase 3 and miR-31 were determined by Western blotting and RT-PCR, respectively. MiR-31 mimic or Ro 31-8220, a specific inhibitor of protein kinase C epsilon （PKCε）, was used to determine the role of miR-31 in the anti-hypertrophic effect of atorvastatin on cardiomyocytes. PKCε in the cardiomyocytes with miR-31 upregulation or downregulation was detected using RT-PCR and Western blotting. The results showed that CIH induced obvious enlargement of cardiomyocytes, which was paralleled with increased atrial natriuretic peptide （ANP）, brain natriuretic peptide （BNP）, and slow/beta cardiac myosin heavy-chain （MYHT） mRNA levels. All these changes were reversed by the treatment with atorvastatin. Meanwhile, miR-31 was increased by CIH in vitro. Of note, the atorvastatin pretreatment significantly increased the mRNA and protein expression of PKCε and decreased that of miR-31. Moreover, overexpression of miR-31 abolished the anti-hypertrophic effect of atorvastatin on cardiomyocytes. Upregulation and downregulation of miR-31 respectively decreased and increased the mRNA and protein expression of PKCε. These results suggest that atorvastatin provides the cardioprotective effects against CIH probably via up-regulating PKCε and down-regulating miR-31.
基金The foundation project of national natural science [No.(2018)40]。
文摘Objective:To investigate to the expression effect of hypoxia and hypoxia/reoxygenation on ROS,MAPKs and cell apoptosis in H9c2 cardiomyocytes.Methods:H9c2 cells were treated with cobalt chloride(CoCl2)to establish the chemical hypoxia and hypoxia/reoxygenation-induced cardiomyocyte injury model.CoCl2 was used to process cells at different concentrations from 150-2400μmol/L and different time from4-24 h;H9c2 cells viability was detected by MTT,and the intracellular ROS level was measured by 2’,7’-dichlorflμoresceindiacetate(DCFH-DA)and dihydroethidiμm(DHE)staining and photoflurography.The active expression level of mitogen-activated protein kinases(MAPKs)(including JNK,ERK and p38)and caspase-3.Results:At the concentration from 300 to 1200μmol/L,CoCl2 does/time-dependently inhibited the cell viability in H9c2 cells(P<0.01).Compared with control group,the ROS levels in hypoxia group were significantly increased(P<0.05).In hypoxia group,the active expression levels of p-JNK,p-p38 and caspase-3 was higher than those in control group(P<0.05).However,the expression of p-ERK wasn’t significant differernce.Furthermore,all the expression levels of ROS,p-JNK,p-ERK,p-p38 and caspase-3 in H/R group were significantly raised compared with hypoxia group(P<0.01).Conclusions:Reoxygenation further aggravate chemical hypoxia induced cardiomyocyte oxidative stress injury by activating ROS/MAPKs signals,suggesting the role of myocardial ischemia/reperfusion injury in the pathogenesis of ischemic heart disease.
基金ThisworkwassupportedbyagrantfromtheNationalNaturalScienceFoundationofChina (No 3 9970 3 3 2 )
文摘Background Chronic hypoxia can cause pulmonary hypertension and pulmonary heart disease with high mortality.The signal transduction pathway of protein kinase C (PKC) plays an important role in chronic pulmonary hypertension. So it is necessary to investigate the effect of PKC on voltage-gated potassium (K +) channels in pulmonary artery smooth muscle cells of rats exposed to chronic hypoxia. Methods Male Wistar rats were randomly divided into a control group (group A) and a chronic hypoxia group (group B). Group B received hypoxia [oxygen concentration (10±1)%] eight hours per day for four consecutive weeks. Single pulmonary artery smooth muscle cells were obtained using an acute enzyme separation method. Conventional whole cell patch clamp technique was used to record resting membrane potential,membrane capacitance and voltage-gated K + currents. The changes in voltage-gated K + currents before and after applying paramethoxyamphetamine (PMA) (500 nmol/L),an agonist of PKC,and PMA plus carbohydrate mixture of glucose,fructose and xylitol (GFX) (30 nmol/L),an inhibitor of PKC,were compared between the two groups. Results The resting membrane potential in group B was significantly lower than that of group A: -(29.0±4.8) mV (n=18) vs -(42.5±4.6) mV (n=35) ( P <0.01). But there was no change in membrane capacitance between the two groups: (17.9±4.6) pF (n=40) vs (19.7±5.8) pF (n=31) ( P >0.05). The voltage-gated K + currents were significantly inhibited by PMA in group A,and this effect was reversed by GFX. However,the voltage-gated K + currents in group B were not affected by PMA. Conclusions The resting membrane potential and voltage-gated K + currents in pulmonary artery smooth muscle cells from rats exposed to chronic hypoxia decreased significantly. It seems that PKC has different effects on the voltage-gated K + currents of pulmonary artery smooth muscle cells under different conditions.
文摘Ischemic stroke is a major cause of morbidity and mortality,and currently there is no effective treatment.The family of protein kinase C(PKCs)could phosphorylate serine or threonine residues of its substrate proteins and play a key role in the ischemia/reperfusion injury.Autophagy is essential for maintaining cell homeostasis under physiological condition and acts as a double-edged sword in the process of ischemic neuronal death.In this article,we reviewed the PKCs isoform-specific signaling pathways and PKC-modulated autophagy in ischemic stroke.
基金supported by the National Natural Science Foundation of China(NSFC)(Grant No:81101426,81571898).
文摘Background:Tumor necrosis factor receptor-associated protein 1(TRAP1)plays a protective effect in hypoxic cardiomyocytes,but the precise mechanisms are not well clarified.The study is aimed to identify the mechanism of TRAP1 on hypoxic damage in cardiomyocytes.Methods:In this study,the effects of TRAP1 and cytochrome c oxidase subunit Ⅱ(COXⅡ)on apoptosis in hypoxia-induced cardiomyocytes were explored using overexpression and knockdown methods separately.Results:Hypoxia induced cardiomyocyte apoptosis,and TRAP1 overexpression notably inhibited apoptosis induced by hypoxia.Conversely,TRAP1 silencing promoted apoptosis in hypoxic cardiomyocytes.Further investigation revealed that the proapoptotic effects caused by the silencing of TRAP1 were prevented by COXⅡ overexpression,whereas COXⅡ knockdown reduced the antiapoptotic function induced by TRAP1 overexpression.Additionally,changes in the release of cytochrome c from mitochondria into the cytosol and the caspase-3 activity in the cytoplasm,as well as reactive oxygen species production,were found to be correlated with the changes in apoptosis.Conclusions:The current study uncovered that TRAP1 regulates hypoxia-induced cardiomyocyte apoptosis through a mitochondria-dependent apoptotic pathway mediated by COXⅡ,in which reactive oxygen species presents as an important component.
基金supported by the Scientific and Technological Development Research Project of Shaanxi Province (No.2005k09-G1)
文摘Objective To investigate the interaction between fragile histamine triad (FHIT) and protein kinase C alpha (PKCα) in human non-small cell lung cancer tissues. Methods FHIT and PKCα double positive samples were screened by immunohistochemical staining from 13 human non-small cell lung cancer tissues. Co-immunoprecipitation was performed by using anti-FHIT and anti-PKCα. The immune precipitate was analyzed by SDS-PAGE and Western blot. Results Immune precipitate staining detection showed that 3 samples out of the 13 cases were double positive for FHIT and PKCα. FHIT protein was present in the immune precipitate of anti-PKCα while there was PKCα in the immune precipitate of anti-FHITmAb. Conclusion FHIT and PKCα exist as a complex in human non-small cell lung cancer tissues, which will provide a new route for studying the pathogenesis and immunotherapy of human non-small cell lung cancer.
基金This work was supported by the Excellent Young Teachers Program of Ministry of Education of Chian Scientific Developing Program of Beijing Municipal Commission of Education (200KJ080+2 种基金 KM200310025100) Beijing Natural Science Foundation (7032005) Nati
