OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) us...OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.展开更多
信号转导及转录活化蛋白5(signal transducer and activator of transcription 5,STAT5)在乳腺发育、免疫应答、细胞代谢、造血及肿瘤的发生发展中发挥重要作用,但对胰岛细胞功能影响研究甚少。本研究旨在探讨STAT5对小鼠胰岛肿瘤MIN6...信号转导及转录活化蛋白5(signal transducer and activator of transcription 5,STAT5)在乳腺发育、免疫应答、细胞代谢、造血及肿瘤的发生发展中发挥重要作用,但对胰岛细胞功能影响研究甚少。本研究旨在探讨STAT5对小鼠胰岛肿瘤MIN6细胞胰岛素分泌的影响及作用机制。电穿孔法转染小干扰RNA(siRNA)敲减STAT5基因表达后,实时定量PCR和蛋白质印迹法显示,与对照干扰RNA转染的细胞比较,Stat5 siRNA转染细胞的mRNA及蛋白质表达水平分别约70%和67%(P<0.01),提示成功构建了Stat5基因沉默模型。酶联免疫吸附法结合蛋白质印迹法显示,与对照组细胞比较,Stat5 siRNA转染细胞在不同浓度葡萄糖刺激下,胰岛素分泌能力提高大约1倍(P<0.05);同时,钙调蛋白依赖的蛋白激酶Ⅱ(calmodulin-dependent protein kinaseⅡ,Ca MKⅡ)的磷酸化水平亦随之加强。加入Ca MKⅡ抑制剂AIPⅡ(auto camtide-2 related inhibitory peptideⅡ)可明显抑制Stat5 siRNA转染导致的细胞胰岛素分泌水平增加(P<0.01)。上述结果表明,沉默Stat5基因后可通过增强Ca MKⅡ的磷酸化促进MIN6细胞胰岛素的分泌。展开更多
目的探讨大鼠骨髓间充质干细胞(rat bone m arrow m esenchym al stem cells,MSCs)体外诱导分化为心肌样细胞内游离钙浓度([Ca2+]i)及钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的表达变化。方法取健康SD大鼠骨髓,用5-氮杂胞苷体外诱导培养。取...目的探讨大鼠骨髓间充质干细胞(rat bone m arrow m esenchym al stem cells,MSCs)体外诱导分化为心肌样细胞内游离钙浓度([Ca2+]i)及钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的表达变化。方法取健康SD大鼠骨髓,用5-氮杂胞苷体外诱导培养。取诱导培养2、3、4周的MSCs为Ⅰ、Ⅱ、Ⅲ组,另取急性分离的心肌细胞为对照组,分别用激光共聚焦技术和W estern b lot技术检测[Ca2+]i及CaMKⅡ表达水平。结果经荧光探针结合Ca2+后,用激光共聚焦技术检测发现,随诱导培养时间的延长,[Ca2+]i逐渐增加;诱导培养4周的MSCs内[Ca2+]i与对照组比较无显著差异[(100.81±17.64),(100.32±17.10),P>0.05]。各组细胞CaMKⅡ的变化趋势与[Ca2+]i定量分析结果相似,Ⅰ、Ⅱ、Ⅲ组及对照组分别为(322.45±19.43)、(434.43±16.77)、(680.91±20.61)、(682.69±21.03),Ⅲ组与对照组比较P>0.05。结论大鼠MSCs在体外诱导培养4周后已分化为心肌样细胞,其细胞内游离钙浓度和CaMKⅡ蛋白表达水平与正常心肌细胞相似。展开更多
OBJECTIVE Dorsal raphe nucleus(DRN) is the largest single collection of neurons containing5-HT in the entire brain and particularly attractive in a wide variety of complex physiological and behavioral processes,such a...OBJECTIVE Dorsal raphe nucleus(DRN) is the largest single collection of neurons containing5-HT in the entire brain and particularly attractive in a wide variety of complex physiological and behavioral processes,such as sleep-wake regulation. Calmodulin dependent kinaseⅡ(CaMKⅡ) and protein kinase C(PKC)are important signal-transducing molecules activated by Ca^(2+). Since the Ca^(2+)modulation in DRN plays an important role in sleep-wake regulation,it should be presumed that the intracellular CaMKⅡ/PKC signaling in DRN may be involved in the regulation of sleep-wake. METHODS The polysomnogram consisting of EEG and EMG was recorded for analyzing sleep architecture. Immunohistochemisrty and Western-blotting methods were used in this study to investigate the roles of Ca^(2+),CaMKⅡ and PKC in sleep-wake regulation in rat DRN. RESULTS Ca^(2+)in the DRN exert arousal effects by reducing the NREMs,SWS and REMs via up-regulating serotonergic functions and activating CaMK Ⅱ-PKC.However,inhibition of PKC leads to significant promotion of total sleep time especial y the NREM sleep,but there were no changes in sleep parameters after the inhibition of CaMKⅡ by its inhibitor KN-93 in DRN.CONCLUSION The molecular,pharmacological,and behavioral findings of this study demonstrate a novel wake promoting and sleep-suppressing role for the Ca^(2+)/CaMK Ⅱ/PKC signaling pathway in DRN. Abnormalities in CaMK Ⅱ are found in patients with several neurological disorders that are associated with disturbed sleep,such as schizophrenia,depression,and Alzheimer′s disease. Several psychotropic drugs modulate CaMK Ⅱ activity. In addition,PKC is a cellular target of most current mood stabilizing and anti-manic agents and involved in bipolar disorder. The data of the present study raise the question whether PKC or CaMKⅡ modulations may also be effective on the sleep disorders or the mood disorders associated with sleep disorders.展开更多
文摘OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.
文摘信号转导及转录活化蛋白5(signal transducer and activator of transcription 5,STAT5)在乳腺发育、免疫应答、细胞代谢、造血及肿瘤的发生发展中发挥重要作用,但对胰岛细胞功能影响研究甚少。本研究旨在探讨STAT5对小鼠胰岛肿瘤MIN6细胞胰岛素分泌的影响及作用机制。电穿孔法转染小干扰RNA(siRNA)敲减STAT5基因表达后,实时定量PCR和蛋白质印迹法显示,与对照干扰RNA转染的细胞比较,Stat5 siRNA转染细胞的mRNA及蛋白质表达水平分别约70%和67%(P<0.01),提示成功构建了Stat5基因沉默模型。酶联免疫吸附法结合蛋白质印迹法显示,与对照组细胞比较,Stat5 siRNA转染细胞在不同浓度葡萄糖刺激下,胰岛素分泌能力提高大约1倍(P<0.05);同时,钙调蛋白依赖的蛋白激酶Ⅱ(calmodulin-dependent protein kinaseⅡ,Ca MKⅡ)的磷酸化水平亦随之加强。加入Ca MKⅡ抑制剂AIPⅡ(auto camtide-2 related inhibitory peptideⅡ)可明显抑制Stat5 siRNA转染导致的细胞胰岛素分泌水平增加(P<0.01)。上述结果表明,沉默Stat5基因后可通过增强Ca MKⅡ的磷酸化促进MIN6细胞胰岛素的分泌。
文摘目的探讨大鼠骨髓间充质干细胞(rat bone m arrow m esenchym al stem cells,MSCs)体外诱导分化为心肌样细胞内游离钙浓度([Ca2+]i)及钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的表达变化。方法取健康SD大鼠骨髓,用5-氮杂胞苷体外诱导培养。取诱导培养2、3、4周的MSCs为Ⅰ、Ⅱ、Ⅲ组,另取急性分离的心肌细胞为对照组,分别用激光共聚焦技术和W estern b lot技术检测[Ca2+]i及CaMKⅡ表达水平。结果经荧光探针结合Ca2+后,用激光共聚焦技术检测发现,随诱导培养时间的延长,[Ca2+]i逐渐增加;诱导培养4周的MSCs内[Ca2+]i与对照组比较无显著差异[(100.81±17.64),(100.32±17.10),P>0.05]。各组细胞CaMKⅡ的变化趋势与[Ca2+]i定量分析结果相似,Ⅰ、Ⅱ、Ⅲ组及对照组分别为(322.45±19.43)、(434.43±16.77)、(680.91±20.61)、(682.69±21.03),Ⅲ组与对照组比较P>0.05。结论大鼠MSCs在体外诱导培养4周后已分化为心肌样细胞,其细胞内游离钙浓度和CaMKⅡ蛋白表达水平与正常心肌细胞相似。
基金The project supported by National Natural Science Foundation of China(81573407,81302746,81202511,81173031)National Mega-project of Scicence Research of China for New Drug Development(2009ZX09103-124)Research Fund for the Doctoral Program of Higher Eductaion of China(20100001110048)
文摘OBJECTIVE Dorsal raphe nucleus(DRN) is the largest single collection of neurons containing5-HT in the entire brain and particularly attractive in a wide variety of complex physiological and behavioral processes,such as sleep-wake regulation. Calmodulin dependent kinaseⅡ(CaMKⅡ) and protein kinase C(PKC)are important signal-transducing molecules activated by Ca^(2+). Since the Ca^(2+)modulation in DRN plays an important role in sleep-wake regulation,it should be presumed that the intracellular CaMKⅡ/PKC signaling in DRN may be involved in the regulation of sleep-wake. METHODS The polysomnogram consisting of EEG and EMG was recorded for analyzing sleep architecture. Immunohistochemisrty and Western-blotting methods were used in this study to investigate the roles of Ca^(2+),CaMKⅡ and PKC in sleep-wake regulation in rat DRN. RESULTS Ca^(2+)in the DRN exert arousal effects by reducing the NREMs,SWS and REMs via up-regulating serotonergic functions and activating CaMK Ⅱ-PKC.However,inhibition of PKC leads to significant promotion of total sleep time especial y the NREM sleep,but there were no changes in sleep parameters after the inhibition of CaMKⅡ by its inhibitor KN-93 in DRN.CONCLUSION The molecular,pharmacological,and behavioral findings of this study demonstrate a novel wake promoting and sleep-suppressing role for the Ca^(2+)/CaMK Ⅱ/PKC signaling pathway in DRN. Abnormalities in CaMK Ⅱ are found in patients with several neurological disorders that are associated with disturbed sleep,such as schizophrenia,depression,and Alzheimer′s disease. Several psychotropic drugs modulate CaMK Ⅱ activity. In addition,PKC is a cellular target of most current mood stabilizing and anti-manic agents and involved in bipolar disorder. The data of the present study raise the question whether PKC or CaMKⅡ modulations may also be effective on the sleep disorders or the mood disorders associated with sleep disorders.
