目的本研究旨在探讨芒柄花黄素(formononetin,FN)通过干预ROS的生成抑制线粒体动力相关蛋白1(dynamic-related protein 1,DRP1)-NLRP3轴减轻过敏性气道炎症的有关机制。方法为建立过敏性哮喘小鼠模型,50只8周龄的BALB/c小鼠通过卵清蛋白...目的本研究旨在探讨芒柄花黄素(formononetin,FN)通过干预ROS的生成抑制线粒体动力相关蛋白1(dynamic-related protein 1,DRP1)-NLRP3轴减轻过敏性气道炎症的有关机制。方法为建立过敏性哮喘小鼠模型,50只8周龄的BALB/c小鼠通过卵清蛋白(ovalbumin,OVA)诱导后分为对照组、模型组、FN治疗组及地塞米松组。采用HE和Masson染色检测气道炎症和胶原沉积,ELISA测定支气管肺泡灌洗液(BALF)中Th2型细胞因子和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)以及IgE水平,DCFH-DA染色评估BEAS-2B细胞中ROS,免疫组织化学和免疫荧光检测肺组织和BEAS-2B细胞中DRP1表达,免疫印迹分析DRP1-NLRP3途径。结果FN治疗可有效改善哮喘小鼠模型的症状,包括减少嗜酸性粒细胞聚集、气道胶原沉积,降低Th2细胞因子和IgE水平,减少ROS和MDA生成,提高SOD和CAT活性,并调节DRP1-NLRP3途径相关蛋白表达,从而缓解炎症。结论FN通过调节DRP1-NLRP3途径改善哮喘气道炎症。展开更多
Divisions at the periphery and midzone of mitochondria are two fission signatures that determine the fate of mitochondria and cells.Pharmacological induction of excessively asymmetric mitofissionassociated cell death(...Divisions at the periphery and midzone of mitochondria are two fission signatures that determine the fate of mitochondria and cells.Pharmacological induction of excessively asymmetric mitofissionassociated cell death(MFAD)by switching the scission position from the mitochondrial midzone to the periphery represents a promising strategy for anticancer therapy.By screening a series of paninhibitors,we identified pracinostat,a pan-histone deacetylase(HDAC)inhibitor,as a novel MFAD inducer,that exhibited a significant anticancer effect on colorectal cancer(CRC)in vivo and in vitro.Pracinostat increased the expression of cyclin-dependent kinase 5(CDK5)and induced its acetylation at residue lysine 33,accelerating the formation of complex CDK5/CDK5 regulatory subunit 1 and dynaminrelated protein 1(Drp1)-mediated mitochondrial peripheral fission.CRC cells with high level of CDK5(CDK5-high)displayed midzone mitochondrial division that was associated with oncogenic phenotype,but treatment with pracinostat led to a lethal increase in the already-elevated level of CDK5 in the CRC cells.Mechanistically,pracinostat switched the scission position from the mitochondrial midzone to the periphery by improving the binding of Drp1 from mitochondrial fission factor(MFF)to mitochondrial fission 1 protein(FIS1).Thus,our results revealed the anticancer mechanism of HDACi pracinostat in CRC via activating CDK5-Drp1 signaling to cause selective MFAD of those CDK5-high tumor cells,which implicates a new paradigm to develop potential therapeutic strategies for CRC treatment.展开更多
BACKGROUND Saffron(Crocus sativus L.)has been traditionally used as food,spice,and medicine.Crocetin(CRT),as main bioactive component of saffron,has accumulated pieces of beneficial evidence on myocardial ischemia/rep...BACKGROUND Saffron(Crocus sativus L.)has been traditionally used as food,spice,and medicine.Crocetin(CRT),as main bioactive component of saffron,has accumulated pieces of beneficial evidence on myocardial ischemia/reperfusion(I/R)injury.However,the mechanisms are poorly explored.This study aims to investigate the effects of CRT on H9c2 cells under hypoxia/reoxygenation(H/R)and elucidated the possible underlying mechanism.METHODS H/R attack was performed on H9c2 cells.Cell counting kit-8 was used to detect the cell viability.Cell samples and culture supernatants were evaluated via commercial kits to measure the superoxide dismutase(SOD)activity,malondialdehyde(MDA)content,and cellular adenosine triphosphate(ATP)content.Various fluorescent probes were used to detect cell apoptosis,intracellular and mitochondrial reactive oxygen species(ROS)content,mitochondrial morphology,mitochondrial membrane potential(MMP),and mitochondrial permeability transition pore(mPTP)opening.Proteins were evaluated via Western Blot.RESULTS H/R exposure severely reduced cell viability and increased LDH leakage.Peroxisome proliferator-activated receptorγcoactivator-1α(PGC-1α)suppression and dynamin-related protein 1(Drp1)activation were coincided with excessive mitochondrial fission,mitochondrial permeability transition pore(mPTP)opening and mitochondrial membrane potential(MMP)collapse in H9c2 cells treated with H/R.Mitochondria fragmentation under H/R injury induced ROS over-production,oxidative stress,and cell apoptosis.Notably,CRT treatment significantly prevented mitochondrial fission,mPTP opening,MMP loss,and cell apoptosis.Moreover,CRT sufficiently activated PGC-1α and inactivated Drp1.Interestingly,mitochondrial fission inhibition with mdivi-1 similarly suppressed mitochondrial dysfunction,oxidative stress and cell apoptosis.However,silencing PGC-1α with small interfering RNA(siRNA)abolished the beneficial effects of CRT on H9c2 cells under H/R injury,accompanied with increased Drp1 and p-Drp1ser616 levels.Furthermore,over-expression of PGC-1αwith adenovirus transfection replicated the beneficial effects of CRT on H9c2 cells.CONCLUSIONS Our study identified PGC-1α as a master regulator in H/R-injured H9c2 cells via Drp1-mediated mitochondrial fission.We also presented the evidence that PGC-1α might be a novel target against cardiomyocyte H/R injury.Our data revealed the role of CRT in regulating PGC-1α/Drp1/mitochondrial fission process in H9c2 cells under the burden of H/R attack,and we suggested that modulation of PGC-1α level may provide a therapeutic target for treating cardiac I/R injury.展开更多
目的:在小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)中探讨动力相关蛋白1(dynamin related protein 1,Drp1)基因缺失激活线粒体未折叠蛋白反应(mitochondrial unfolded protein reaction,mtUPR)的分子机制。方法:采用不同浓度(...目的:在小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)中探讨动力相关蛋白1(dynamin related protein 1,Drp1)基因缺失激活线粒体未折叠蛋白反应(mitochondrial unfolded protein reaction,mtUPR)的分子机制。方法:采用不同浓度(0、2.5、5.0、10.0mmol/L)3-硝基丙酸(3-nitropropionic acid,3-NP)处理Drp1敲除或敲低的MEF细胞、Drp1抑制剂Mdivi-1或选择性阻断Drp1与下游蛋白相互作用的小分子多肽P110处理的MEF细胞以及相应对照,随后进行Western blot检测CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)、ATP结合盒B亚家族成员10(ATP binding cassette subfamily B member 10,ABCB10)、Lon肽酶1(Lon peptidase 1,LONP1)以及热休克蛋白60(heat shock protein 60,Hsp60)的表达。RT-qPCR检测Drp1敲低或Mdivi-1处理后MEF细胞的ABCB10的mRNA水平。同时敲低Drp1和ABCB10,Western blot检测CHOP蛋白的表达,试剂盒检测培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)含量,流式细胞术检测线粒体活性氧和线粒体膜电位水平。结果:3-NP处理后,Drp1敲除或敲低的MEF细胞以及Mdivi-1或P110处理的MEF细胞中CHOP表达呈现倍数上调。Drp1敲除或敲低的MEF细胞以及Mdivi-1或P110处理的MEF细胞中ABCB10蛋白表达上调,mtUPR效应蛋白LONP1和Hsp60表达上调。Drp1敲低的MEF细胞和Mdivi-1处理的MEF细胞中ABCB10 mRNA水平上调。同时敲低Drp1和ABCB10后,与Drp1敲低组相比,CHOP表达下调,LDH含量降低,线粒体活性氧水平降低,线粒体膜电位水平增加。结论:在MEF细胞中,Drp1表达下调可引起ABCB10表达量增加,导致mtUPR关键蛋白CHOP、LONP1和Hsp60蛋白表达上调,进而激活mtUPR。展开更多
文摘目的本研究旨在探讨芒柄花黄素(formononetin,FN)通过干预ROS的生成抑制线粒体动力相关蛋白1(dynamic-related protein 1,DRP1)-NLRP3轴减轻过敏性气道炎症的有关机制。方法为建立过敏性哮喘小鼠模型,50只8周龄的BALB/c小鼠通过卵清蛋白(ovalbumin,OVA)诱导后分为对照组、模型组、FN治疗组及地塞米松组。采用HE和Masson染色检测气道炎症和胶原沉积,ELISA测定支气管肺泡灌洗液(BALF)中Th2型细胞因子和超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、丙二醛(MDA)以及IgE水平,DCFH-DA染色评估BEAS-2B细胞中ROS,免疫组织化学和免疫荧光检测肺组织和BEAS-2B细胞中DRP1表达,免疫印迹分析DRP1-NLRP3途径。结果FN治疗可有效改善哮喘小鼠模型的症状,包括减少嗜酸性粒细胞聚集、气道胶原沉积,降低Th2细胞因子和IgE水平,减少ROS和MDA生成,提高SOD和CAT活性,并调节DRP1-NLRP3途径相关蛋白表达,从而缓解炎症。结论FN通过调节DRP1-NLRP3途径改善哮喘气道炎症。
基金supported by the National Natural Science Foundation of China(Grant Nos.:82103208,and 82002948)the Guangdong Basic and Applied Basic Research Foundation(Grant Nos.:2022A1515220212,and 2023A1515030115)+1 种基金National Key R&D Program of China(Grant No.:2020YFE0202200)Jinan University National College Students'Innovation and Entrepreneurship Training Program(Program No.:202110559085).
文摘Divisions at the periphery and midzone of mitochondria are two fission signatures that determine the fate of mitochondria and cells.Pharmacological induction of excessively asymmetric mitofissionassociated cell death(MFAD)by switching the scission position from the mitochondrial midzone to the periphery represents a promising strategy for anticancer therapy.By screening a series of paninhibitors,we identified pracinostat,a pan-histone deacetylase(HDAC)inhibitor,as a novel MFAD inducer,that exhibited a significant anticancer effect on colorectal cancer(CRC)in vivo and in vitro.Pracinostat increased the expression of cyclin-dependent kinase 5(CDK5)and induced its acetylation at residue lysine 33,accelerating the formation of complex CDK5/CDK5 regulatory subunit 1 and dynaminrelated protein 1(Drp1)-mediated mitochondrial peripheral fission.CRC cells with high level of CDK5(CDK5-high)displayed midzone mitochondrial division that was associated with oncogenic phenotype,but treatment with pracinostat led to a lethal increase in the already-elevated level of CDK5 in the CRC cells.Mechanistically,pracinostat switched the scission position from the mitochondrial midzone to the periphery by improving the binding of Drp1 from mitochondrial fission factor(MFF)to mitochondrial fission 1 protein(FIS1).Thus,our results revealed the anticancer mechanism of HDACi pracinostat in CRC via activating CDK5-Drp1 signaling to cause selective MFAD of those CDK5-high tumor cells,which implicates a new paradigm to develop potential therapeutic strategies for CRC treatment.
基金supported by the grants from National Natural Science Foundation of China(No.81903830)the Natural Science Foundation of Zhejiang Province(No.LY21H280005)+1 种基金the Research Project of Zhejiang Chinese Medical University(No.2021JKZK TS023B)the 2021 Innovation and Entrepreneurship Training Program for College Students(No.S202110344009).
文摘BACKGROUND Saffron(Crocus sativus L.)has been traditionally used as food,spice,and medicine.Crocetin(CRT),as main bioactive component of saffron,has accumulated pieces of beneficial evidence on myocardial ischemia/reperfusion(I/R)injury.However,the mechanisms are poorly explored.This study aims to investigate the effects of CRT on H9c2 cells under hypoxia/reoxygenation(H/R)and elucidated the possible underlying mechanism.METHODS H/R attack was performed on H9c2 cells.Cell counting kit-8 was used to detect the cell viability.Cell samples and culture supernatants were evaluated via commercial kits to measure the superoxide dismutase(SOD)activity,malondialdehyde(MDA)content,and cellular adenosine triphosphate(ATP)content.Various fluorescent probes were used to detect cell apoptosis,intracellular and mitochondrial reactive oxygen species(ROS)content,mitochondrial morphology,mitochondrial membrane potential(MMP),and mitochondrial permeability transition pore(mPTP)opening.Proteins were evaluated via Western Blot.RESULTS H/R exposure severely reduced cell viability and increased LDH leakage.Peroxisome proliferator-activated receptorγcoactivator-1α(PGC-1α)suppression and dynamin-related protein 1(Drp1)activation were coincided with excessive mitochondrial fission,mitochondrial permeability transition pore(mPTP)opening and mitochondrial membrane potential(MMP)collapse in H9c2 cells treated with H/R.Mitochondria fragmentation under H/R injury induced ROS over-production,oxidative stress,and cell apoptosis.Notably,CRT treatment significantly prevented mitochondrial fission,mPTP opening,MMP loss,and cell apoptosis.Moreover,CRT sufficiently activated PGC-1α and inactivated Drp1.Interestingly,mitochondrial fission inhibition with mdivi-1 similarly suppressed mitochondrial dysfunction,oxidative stress and cell apoptosis.However,silencing PGC-1α with small interfering RNA(siRNA)abolished the beneficial effects of CRT on H9c2 cells under H/R injury,accompanied with increased Drp1 and p-Drp1ser616 levels.Furthermore,over-expression of PGC-1αwith adenovirus transfection replicated the beneficial effects of CRT on H9c2 cells.CONCLUSIONS Our study identified PGC-1α as a master regulator in H/R-injured H9c2 cells via Drp1-mediated mitochondrial fission.We also presented the evidence that PGC-1α might be a novel target against cardiomyocyte H/R injury.Our data revealed the role of CRT in regulating PGC-1α/Drp1/mitochondrial fission process in H9c2 cells under the burden of H/R attack,and we suggested that modulation of PGC-1α level may provide a therapeutic target for treating cardiac I/R injury.
文摘目的:在小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)中探讨动力相关蛋白1(dynamin related protein 1,Drp1)基因缺失激活线粒体未折叠蛋白反应(mitochondrial unfolded protein reaction,mtUPR)的分子机制。方法:采用不同浓度(0、2.5、5.0、10.0mmol/L)3-硝基丙酸(3-nitropropionic acid,3-NP)处理Drp1敲除或敲低的MEF细胞、Drp1抑制剂Mdivi-1或选择性阻断Drp1与下游蛋白相互作用的小分子多肽P110处理的MEF细胞以及相应对照,随后进行Western blot检测CCAAT/增强子结合蛋白同源蛋白(CCAAT/enhancer-binding protein homologous protein,CHOP)、ATP结合盒B亚家族成员10(ATP binding cassette subfamily B member 10,ABCB10)、Lon肽酶1(Lon peptidase 1,LONP1)以及热休克蛋白60(heat shock protein 60,Hsp60)的表达。RT-qPCR检测Drp1敲低或Mdivi-1处理后MEF细胞的ABCB10的mRNA水平。同时敲低Drp1和ABCB10,Western blot检测CHOP蛋白的表达,试剂盒检测培养液中乳酸脱氢酶(lactate dehydrogenase,LDH)含量,流式细胞术检测线粒体活性氧和线粒体膜电位水平。结果:3-NP处理后,Drp1敲除或敲低的MEF细胞以及Mdivi-1或P110处理的MEF细胞中CHOP表达呈现倍数上调。Drp1敲除或敲低的MEF细胞以及Mdivi-1或P110处理的MEF细胞中ABCB10蛋白表达上调,mtUPR效应蛋白LONP1和Hsp60表达上调。Drp1敲低的MEF细胞和Mdivi-1处理的MEF细胞中ABCB10 mRNA水平上调。同时敲低Drp1和ABCB10后,与Drp1敲低组相比,CHOP表达下调,LDH含量降低,线粒体活性氧水平降低,线粒体膜电位水平增加。结论:在MEF细胞中,Drp1表达下调可引起ABCB10表达量增加,导致mtUPR关键蛋白CHOP、LONP1和Hsp60蛋白表达上调,进而激活mtUPR。
