OBJECTIVE:To explore the relationship between autophagy and apoptosis regulated by puerarin during osteoblastogenesis.METHODS:In this study,the effects of puerarin on the autophagic activity and apoptosis level of ost...OBJECTIVE:To explore the relationship between autophagy and apoptosis regulated by puerarin during osteoblastogenesis.METHODS:In this study,the effects of puerarin on the autophagic activity and apoptosis level of osteoblast precursors(MC3T3-E1 cells)was observed.Subsequently,the roles of puerarin on B-cell lymphoma-2(Bcl-2)phosphorylation at different sites in osteoblast precursors were observed.The effect of puerarin on the interaction between Bcl-2 and autophagy regulatory molecule or pro-apoptotic molecule was also investigated using Co-immunoprecipitation assays.In addition,the effect of puerarin on mitochondrial membrane potential of osteoblast precursors was also identified by mitochondrial membrane potential fluorescence probe assays.RESULTS:Our results showed that puerarin can promote the autophagic activity and apoptosis level of MC3T3-E1cells.In addition,puerarin promoted Bcl-2phosphorylation at Ser70 site,and the dissociation of Bcl-2-Beclin1 complex.Moreover,puerarin could enhance the binding of Bcl-2-Bcl-2-Associated X(Bax)complex in MC3T3-E1 cells.Furthermore,puerarin increased the mitochondrial membrane potential of MC3T3-E1 cells.CONCLUSIONS:Therefore,puerarin promotes Beclin1into autophagy flux through Bcl-2 phosphorylation at Ser70,thereby enhancing autophagy of osteoblast precursors,which mediates its anti-apoptotic role during osteoblastogenesis.Furthermore,the dissociation of Bcl-2-Beclin1 complex is conducive to the binding of Bcl-2-Bax complex,which resists the apoptosis of osteoblast precursors via the increased mitochondrial membrane potential.展开更多
【目的】本研究拟探究MG53蛋白对小鼠阿霉素心肌毒性的心脏功能的影响及机制。【方法】体内实验选择C57BL/6小鼠腹腔注射阿霉素20 mg/kg 1周诱导急性阿霉素心肌毒性模型;体外实验使用1μmol/L阿霉素处理大鼠原代心肌细胞构建DIC模型。...【目的】本研究拟探究MG53蛋白对小鼠阿霉素心肌毒性的心脏功能的影响及机制。【方法】体内实验选择C57BL/6小鼠腹腔注射阿霉素20 mg/kg 1周诱导急性阿霉素心肌毒性模型;体外实验使用1μmol/L阿霉素处理大鼠原代心肌细胞构建DIC模型。采用小动物心脏超声分析小鼠心脏功能,观察左室射血分数、缩短分数的变化;通过qPCR技术分析心脏重构相关基因ANP、BNP、α-MHC,自噬相关基因Beclin1、LC3及凋亡基因CASPASE3的表达改变;采用免疫印迹技术检测自噬相关蛋白Beclin1、LC3及凋亡相关蛋白caspase3的表达水平;采用透射电镜观察心肌组织中的自噬小体;采用TUNEL试剂盒检测原代心肌细胞的凋亡水平。【结果】心脏超声结果显示:与假手术组(Sham)相比,阿霉素组(DOX)及心肌原位注射对照腺相关病毒组(DOX+AAV9-NC)小鼠心脏功能显著下降(EF:Sham:86.06±2.08 vs. DOX:58.97±1.62,P <0.0001;Sham:86.06±2.08 vs. DOX+AAV9-NC:59.00±1.86,P <0.000 1。FS:Sham:45.47±1.95 vs. DOX:30.68±1.21,P <0.000 1;Sham:45.47±1.95 vs. DOX+AAV9-NC:30.79±1.13,P <0.000 1),而心肌原位注射腺相关病毒过表达MG53组(DOX+AAV9-MG53)小鼠的心脏功能障碍得到显著改善(EF:DOX+AAV9-MG53:66.93±1.78 vs. DOX+AAV9-NC:59.00±1.86,P <0.000 1。FS:DOX+AAV9-MG53:36.35±1.33 vs. DOX+AAV9-NC:30.79±1.13,P <0.000 1);电镜结果显示,过表达MG53后心肌细胞的自噬小体增加;qPCR结果表明过表达MG53显著下调心脏重构相关基因的表达;此外,western blot结果进一步明确过表达MG53可显著下调caspase3蛋白表达,并上调Beclin1、LC3蛋白表达(caspase:DOX+AAV9-MG53:1.49±0.13 vs.DOX+AAV9-NC:2.49±0.46,P=0.000 2;Beclin-1:DOX+AAV9-MG53:0.82±0.02 vs. DOX+AAV9-NC:0.62±0.05,P <0.000 1;LC3:DOX+AAV9-MG53:0.83±0.04 vs. DOX+AAV9-NC:0.40±0.05,P <0.000 1),而敲低MG53可显著上调caspase3蛋白表达,下调Beclin1、LC3蛋白表达(caspase:DOX+si-MG53:4.52±0.28 vs. DOX+si-NC:3.37±0.08,P<0.0001;Beclin-1:DOX+si-MG53:0.34±0.06vs.DOX+si-NC:0.54±0.07,P=0.0262;LC3:DOX+siMG53:0.41±0.12 vs. DOX+si-NC:0.70±0.07,P=0.001 5);TUNEL检测结果提示过表达MG53可显著抑制心肌细胞凋亡(DOX+Ad-MG53:9.41±0.53 vs. DOX+Ad-NC:29.34±7.29,P <0.000 1),敲低MG53可显著促进心肌细胞凋亡。(DOX+si-MG53:71.34±5.90 vs. DOX+si-NC:32.19±9.91,P <0.000 1)【结论】MG53可抑制心肌细胞凋亡,并促进自噬,改善小鼠DIC心脏重构进程,减轻心脏功能障碍。展开更多
Calpains are a group of calcium-dependent proteases that are over activated by increased intracellular calcium levels under pathological conditions. A wide range of substrates that regulate necrotic, apoptotic and aut...Calpains are a group of calcium-dependent proteases that are over activated by increased intracellular calcium levels under pathological conditions. A wide range of substrates that regulate necrotic, apoptotic and autophagic pathways are affected by calpain. Calpain plays a very important role in neuronal death and various neurological disorders. This review introduces recent research progress related to the regulatory mechanisms of calpain in neuronal death. Various neuronal programmed death pathways including apoptosis, autophagy and regulated necrosis can be divided into receptor interacting protein-dependent necroptosis, mitochondrial permeability transition-dependent necrosis, pyroptosis and poly （ADP-ribose）polymerase 1-mediated parthanatos. Calpains cleave series of key substrates that may lead to cell death or participate in cell death. Regarding the investigation of calpain-mediated programed cell death, it is necessary to identify specific inhibitors that inhibit calpain mediated neuronal death and nervous system diseases.展开更多
基金Fujian Provincial Natural Science:Exploration of Crisis Warning Values for Intraoperative Electrophysiological Monitoring in High-risk Cervical Spondylosis Patients on the Brink of Paralysis(No.2019J01548)Fujian Provincial Clinical Medical Research Center for First Aid and Rehabilitation in Orthopaedic Trauma:Study on the Treatment of Osteoporosis(No.2020Y2014)。
文摘OBJECTIVE:To explore the relationship between autophagy and apoptosis regulated by puerarin during osteoblastogenesis.METHODS:In this study,the effects of puerarin on the autophagic activity and apoptosis level of osteoblast precursors(MC3T3-E1 cells)was observed.Subsequently,the roles of puerarin on B-cell lymphoma-2(Bcl-2)phosphorylation at different sites in osteoblast precursors were observed.The effect of puerarin on the interaction between Bcl-2 and autophagy regulatory molecule or pro-apoptotic molecule was also investigated using Co-immunoprecipitation assays.In addition,the effect of puerarin on mitochondrial membrane potential of osteoblast precursors was also identified by mitochondrial membrane potential fluorescence probe assays.RESULTS:Our results showed that puerarin can promote the autophagic activity and apoptosis level of MC3T3-E1cells.In addition,puerarin promoted Bcl-2phosphorylation at Ser70 site,and the dissociation of Bcl-2-Beclin1 complex.Moreover,puerarin could enhance the binding of Bcl-2-Bcl-2-Associated X(Bax)complex in MC3T3-E1 cells.Furthermore,puerarin increased the mitochondrial membrane potential of MC3T3-E1 cells.CONCLUSIONS:Therefore,puerarin promotes Beclin1into autophagy flux through Bcl-2 phosphorylation at Ser70,thereby enhancing autophagy of osteoblast precursors,which mediates its anti-apoptotic role during osteoblastogenesis.Furthermore,the dissociation of Bcl-2-Beclin1 complex is conducive to the binding of Bcl-2-Bax complex,which resists the apoptosis of osteoblast precursors via the increased mitochondrial membrane potential.
文摘【目的】本研究拟探究MG53蛋白对小鼠阿霉素心肌毒性的心脏功能的影响及机制。【方法】体内实验选择C57BL/6小鼠腹腔注射阿霉素20 mg/kg 1周诱导急性阿霉素心肌毒性模型;体外实验使用1μmol/L阿霉素处理大鼠原代心肌细胞构建DIC模型。采用小动物心脏超声分析小鼠心脏功能,观察左室射血分数、缩短分数的变化;通过qPCR技术分析心脏重构相关基因ANP、BNP、α-MHC,自噬相关基因Beclin1、LC3及凋亡基因CASPASE3的表达改变;采用免疫印迹技术检测自噬相关蛋白Beclin1、LC3及凋亡相关蛋白caspase3的表达水平;采用透射电镜观察心肌组织中的自噬小体;采用TUNEL试剂盒检测原代心肌细胞的凋亡水平。【结果】心脏超声结果显示:与假手术组(Sham)相比,阿霉素组(DOX)及心肌原位注射对照腺相关病毒组(DOX+AAV9-NC)小鼠心脏功能显著下降(EF:Sham:86.06±2.08 vs. DOX:58.97±1.62,P <0.0001;Sham:86.06±2.08 vs. DOX+AAV9-NC:59.00±1.86,P <0.000 1。FS:Sham:45.47±1.95 vs. DOX:30.68±1.21,P <0.000 1;Sham:45.47±1.95 vs. DOX+AAV9-NC:30.79±1.13,P <0.000 1),而心肌原位注射腺相关病毒过表达MG53组(DOX+AAV9-MG53)小鼠的心脏功能障碍得到显著改善(EF:DOX+AAV9-MG53:66.93±1.78 vs. DOX+AAV9-NC:59.00±1.86,P <0.000 1。FS:DOX+AAV9-MG53:36.35±1.33 vs. DOX+AAV9-NC:30.79±1.13,P <0.000 1);电镜结果显示,过表达MG53后心肌细胞的自噬小体增加;qPCR结果表明过表达MG53显著下调心脏重构相关基因的表达;此外,western blot结果进一步明确过表达MG53可显著下调caspase3蛋白表达,并上调Beclin1、LC3蛋白表达(caspase:DOX+AAV9-MG53:1.49±0.13 vs.DOX+AAV9-NC:2.49±0.46,P=0.000 2;Beclin-1:DOX+AAV9-MG53:0.82±0.02 vs. DOX+AAV9-NC:0.62±0.05,P <0.000 1;LC3:DOX+AAV9-MG53:0.83±0.04 vs. DOX+AAV9-NC:0.40±0.05,P <0.000 1),而敲低MG53可显著上调caspase3蛋白表达,下调Beclin1、LC3蛋白表达(caspase:DOX+si-MG53:4.52±0.28 vs. DOX+si-NC:3.37±0.08,P<0.0001;Beclin-1:DOX+si-MG53:0.34±0.06vs.DOX+si-NC:0.54±0.07,P=0.0262;LC3:DOX+siMG53:0.41±0.12 vs. DOX+si-NC:0.70±0.07,P=0.001 5);TUNEL检测结果提示过表达MG53可显著抑制心肌细胞凋亡(DOX+Ad-MG53:9.41±0.53 vs. DOX+Ad-NC:29.34±7.29,P <0.000 1),敲低MG53可显著促进心肌细胞凋亡。(DOX+si-MG53:71.34±5.90 vs. DOX+si-NC:32.19±9.91,P <0.000 1)【结论】MG53可抑制心肌细胞凋亡,并促进自噬,改善小鼠DIC心脏重构进程,减轻心脏功能障碍。
基金supported by the National Natural Science Foundation of China,No.81571939&81772134the Wu Jie-Ping Medical Foundation of the Minister of Health of China,No.320.6750.14118+1 种基金the Natural Science Foundation of Hunan Province of China,No.2015JJ2187the Teacher Research Foundation of Central South University of China,No.2014JSJJ026
文摘Calpains are a group of calcium-dependent proteases that are over activated by increased intracellular calcium levels under pathological conditions. A wide range of substrates that regulate necrotic, apoptotic and autophagic pathways are affected by calpain. Calpain plays a very important role in neuronal death and various neurological disorders. This review introduces recent research progress related to the regulatory mechanisms of calpain in neuronal death. Various neuronal programmed death pathways including apoptosis, autophagy and regulated necrosis can be divided into receptor interacting protein-dependent necroptosis, mitochondrial permeability transition-dependent necrosis, pyroptosis and poly （ADP-ribose）polymerase 1-mediated parthanatos. Calpains cleave series of key substrates that may lead to cell death or participate in cell death. Regarding the investigation of calpain-mediated programed cell death, it is necessary to identify specific inhibitors that inhibit calpain mediated neuronal death and nervous system diseases.
