Energy status is linked to the production of reactive oxygen species(ROS)in macrophages,which is elevated in obesity.However,it is unclear how ROS production is upregulated in macrophages in response to energy overloa...Energy status is linked to the production of reactive oxygen species(ROS)in macrophages,which is elevated in obesity.However,it is unclear how ROS production is upregulated in macrophages in response to energy overload for mediating the development of obesity.Here,we show that the Rab-GTPase activating protein(Rab GAP)TBC1D1,a substrate of the energy sensor AMP-activated protein kinase(AMPK),is a critical regulator of macrophage ROS production and consequent adipose inflammation for obesity development.TBC1D1 deletion decreases,whereas an energy overload-mimetic non-phosphorylatable TBC1D1^(S231A)Amutation increases,ROS production and M1-like polarization in macrophages.Mechanistically,TBC1D1 and its downstream target Rab8a form an energy-responsive complex with NOX2 for ROS generation.Transplantation of TBC1D1^(S231A)bone marrow aggravates diet-induced obesity whereas treatment with an ultra-stable Tt SOD for removal of ROS selectively in macrophages alleviates both TBC1D1~(S231A)mutation-and diet-induced obesity.Our findings therefore have implications for drug discovery to combat obesity.展开更多
Much effort has been made to uncover the cellular heterogeneities of human hearts by single-nucleus RNA sequencing.However,the cardiac transcriptional regulation networks have not been systematically described because...Much effort has been made to uncover the cellular heterogeneities of human hearts by single-nucleus RNA sequencing.However,the cardiac transcriptional regulation networks have not been systematically described because of the limitations in detecting transcription factors.In this study,we optimized a pipeline for isolating nuclei and conducting single-nucleus RNA sequencing targeted to detect a higher number of cell signal genes and an optimal number of transcription factors.With this unbiased protocol,we characterized the cellular composition of healthy human hearts and investigated the transcriptional regulation networks involved in determining the cellular identities and functions of the main cardiac cell subtypes.Particularly in fibroblasts,a novel regulator,PKNOX2,was identified as being associated with physiological fibroblast activation in healthy hearts.To validate the roles of these transcription factors in maintaining homeostasis,we used single-nucleus RNA-sequencing analysis of transplanted failing hearts focusing on fibroblast remodelling.The trajectory analysis suggested that PKNOX2 was abnormally decreased from fibroblast activation to pathological myofibroblast formation.Both gain-and loss-of-function in vitro experiments demonstrated the inhibitory role of PKNOX2 in pathological fibrosis remodelling.Moreover,fibroblast-specific overexpression and knockout of PKNOX2 in a heart failure mouse model induced by transverse aortic constriction surgery significantly improved and aggravated myocardial fibrosis,respectively.In summary,this study established a high-quality pipeline for single-nucleus RNA-sequencing analysis of heart muscle.With this optimized protocol,we described the transcriptional regulation networks of the main cardiac cell subtypes and identified PKNOX2 as a novel regulator in suppressing fibrosis and a potential therapeutic target for future translational studies.展开更多
目的:观察Nox2对AngⅡ活化的人肾上腺皮质腺癌细胞(H295R细胞)醛固酮合成的影响。方法:将H295R细胞分为正常对照组、AngⅡ、AngⅡ+gp91ds-tat(Nox2抑制剂)组、AngⅡ+PEG-Cat(H_(2)O_(2)清除剂)组、AngⅡ+Nox2 si RNA组及不同时间的AngⅡ...目的:观察Nox2对AngⅡ活化的人肾上腺皮质腺癌细胞(H295R细胞)醛固酮合成的影响。方法:将H295R细胞分为正常对照组、AngⅡ、AngⅡ+gp91ds-tat(Nox2抑制剂)组、AngⅡ+PEG-Cat(H_(2)O_(2)清除剂)组、AngⅡ+Nox2 si RNA组及不同时间的AngⅡ组,采用Q-PCR和western blot检测醛固酮合成酶(CYP11B2)和Nox2基因及蛋白水平;放免法检测细胞上清液醛固酮浓度;应用流式细胞术和酶标仪检测细胞内Nox2来源的ROS和H_(2)O_(2)的含量。结果:10 nmol/L AngⅡ以时间依赖性增加H295R细胞内ROS和H_(2)O_(2)含量、Nox2和CYP11B2表达、醛固酮合成(P<0.05)。与正常对照组相比,gp91ds-tat和PEG-Cat明显降低AngⅡ诱导的细胞内ROS和H_(2)O_(2)含量(P<0.05),而gp91ds-tat组和PEG-Cat组AngⅡ诱导的细胞内ROS和H_(2)O_(2)抑制作用无差别(P>0.05)。10nmol/L AngⅡ处理24 h诱导H295R细胞CYP11B2表达(P<0.05),而gp91ds-tat组、PEG-Cat组和Nox2 si RNA组明显抑制AngⅡ诱导H295R细胞CYP11B2表达(P<0.05)。结论:Nox2来源的ROS在AngⅡ诱导的醛固酮合成过程中起主要调控作用。展开更多
基金the Ministry of Science and Technology of China(Grant Nos.2018YFA0801100 and 2021YFF0702100)the National Natural Science Foundation of China(Grant Nos.32025019 and 31970719 to S.C.,31971067)the Fundamental Research Funds for the Central Universities(021414380533,021414380505)for financial support。
文摘Energy status is linked to the production of reactive oxygen species(ROS)in macrophages,which is elevated in obesity.However,it is unclear how ROS production is upregulated in macrophages in response to energy overload for mediating the development of obesity.Here,we show that the Rab-GTPase activating protein(Rab GAP)TBC1D1,a substrate of the energy sensor AMP-activated protein kinase(AMPK),is a critical regulator of macrophage ROS production and consequent adipose inflammation for obesity development.TBC1D1 deletion decreases,whereas an energy overload-mimetic non-phosphorylatable TBC1D1^(S231A)Amutation increases,ROS production and M1-like polarization in macrophages.Mechanistically,TBC1D1 and its downstream target Rab8a form an energy-responsive complex with NOX2 for ROS generation.Transplantation of TBC1D1^(S231A)bone marrow aggravates diet-induced obesity whereas treatment with an ultra-stable Tt SOD for removal of ROS selectively in macrophages alleviates both TBC1D1~(S231A)mutation-and diet-induced obesity.Our findings therefore have implications for drug discovery to combat obesity.
基金supported by the National Natural Science Foundation of China(82100377)National High Level Hospital Clinical Research Funding(2023-GSP-RC-01,2023-GSP-ZD-2)the Beijing Nova Program(Z211100002121046,20220484205).Figure 10 was created with BioRender.com.
文摘Much effort has been made to uncover the cellular heterogeneities of human hearts by single-nucleus RNA sequencing.However,the cardiac transcriptional regulation networks have not been systematically described because of the limitations in detecting transcription factors.In this study,we optimized a pipeline for isolating nuclei and conducting single-nucleus RNA sequencing targeted to detect a higher number of cell signal genes and an optimal number of transcription factors.With this unbiased protocol,we characterized the cellular composition of healthy human hearts and investigated the transcriptional regulation networks involved in determining the cellular identities and functions of the main cardiac cell subtypes.Particularly in fibroblasts,a novel regulator,PKNOX2,was identified as being associated with physiological fibroblast activation in healthy hearts.To validate the roles of these transcription factors in maintaining homeostasis,we used single-nucleus RNA-sequencing analysis of transplanted failing hearts focusing on fibroblast remodelling.The trajectory analysis suggested that PKNOX2 was abnormally decreased from fibroblast activation to pathological myofibroblast formation.Both gain-and loss-of-function in vitro experiments demonstrated the inhibitory role of PKNOX2 in pathological fibrosis remodelling.Moreover,fibroblast-specific overexpression and knockout of PKNOX2 in a heart failure mouse model induced by transverse aortic constriction surgery significantly improved and aggravated myocardial fibrosis,respectively.In summary,this study established a high-quality pipeline for single-nucleus RNA-sequencing analysis of heart muscle.With this optimized protocol,we described the transcriptional regulation networks of the main cardiac cell subtypes and identified PKNOX2 as a novel regulator in suppressing fibrosis and a potential therapeutic target for future translational studies.
文摘目的:观察Nox2对AngⅡ活化的人肾上腺皮质腺癌细胞(H295R细胞)醛固酮合成的影响。方法:将H295R细胞分为正常对照组、AngⅡ、AngⅡ+gp91ds-tat(Nox2抑制剂)组、AngⅡ+PEG-Cat(H_(2)O_(2)清除剂)组、AngⅡ+Nox2 si RNA组及不同时间的AngⅡ组,采用Q-PCR和western blot检测醛固酮合成酶(CYP11B2)和Nox2基因及蛋白水平;放免法检测细胞上清液醛固酮浓度;应用流式细胞术和酶标仪检测细胞内Nox2来源的ROS和H_(2)O_(2)的含量。结果:10 nmol/L AngⅡ以时间依赖性增加H295R细胞内ROS和H_(2)O_(2)含量、Nox2和CYP11B2表达、醛固酮合成(P<0.05)。与正常对照组相比,gp91ds-tat和PEG-Cat明显降低AngⅡ诱导的细胞内ROS和H_(2)O_(2)含量(P<0.05),而gp91ds-tat组和PEG-Cat组AngⅡ诱导的细胞内ROS和H_(2)O_(2)抑制作用无差别(P>0.05)。10nmol/L AngⅡ处理24 h诱导H295R细胞CYP11B2表达(P<0.05),而gp91ds-tat组、PEG-Cat组和Nox2 si RNA组明显抑制AngⅡ诱导H295R细胞CYP11B2表达(P<0.05)。结论:Nox2来源的ROS在AngⅡ诱导的醛固酮合成过程中起主要调控作用。
