Intracellular redox homeostasis is of indispensable importance in pathophysiology.In order to maintain the balance of the redox state within the cell,reactive oxygen species(ROS)and reactive sulfur species(RSS)react a...Intracellular redox homeostasis is of indispensable importance in pathophysiology.In order to maintain the balance of the redox state within the cell,reactive oxygen species(ROS)and reactive sulfur species(RSS)react and transform with each other,and their levels also directly reflect the degree of oxidative stress and disease.Hypochlorous acid(HClO)and cysteine(Cys)usually co-exist in organisms,interacting with each other in many important physiological processes and synergistically maintaining the dynamic redox balance in the body.To understand the relevance and pathophysiological effects of these two signaling molecules in oxidative stress,unique fluorescence imaging tools are required.Herein,we designed and developed a dual-channel fluorescent probe HP,for the individual and continuous detection of HClO and Cys.This probe could simultaneously monitor the changes in the concentrations of HClO and Cys in cells,and was characterized by a fast response,high sensitivity and high selectivity,especially compared with glutathione(GSH)and homocysteine(Hcy),the probe had a good specificity for Cys.Importantly,probe HP successfully observed dynamic changes in HCl O-and Cys-mediated redox status in the oxygenglucose deprivation/reperfusion(OGD/R)model of HeLa cells and dynamically monitored fluctuations in endogenous HClO levels in lipopolysaccharides(LPS)-induced peritonitis mice.展开更多
目的:基于miR-451a靶向磷酸甘油酸变位酶(PGAM5)-线粒体动力相关蛋白1(Drp1)轴调控线粒体动力学,探讨温肺降浊方对氧糖剥夺/复氧(OGD/R)诱导HT22模型的效应机制。方法:建立OGD/R诱导HT22细胞模型,设立正常对照组、OGD/R组、miR-451a过...目的:基于miR-451a靶向磷酸甘油酸变位酶(PGAM5)-线粒体动力相关蛋白1(Drp1)轴调控线粒体动力学,探讨温肺降浊方对氧糖剥夺/复氧(OGD/R)诱导HT22模型的效应机制。方法:建立OGD/R诱导HT22细胞模型,设立正常对照组、OGD/R组、miR-451a过表达组、miR-451a敲低组、温肺降浊方含药血清(WFJZF)组、miR-451a过表达+WFJZF组、miR-451a敲低+WFJZF组和miR-451a空载体组,双荧光素酶确定miR-451a和PGAM5的转录关系,免疫蛋白共沉淀确定PGAM5和Drp1的调控作用,慢病毒转染miR-451a于HT22细胞中,qRT-PCR确定转染效率,CCK-8测定缺糖缺氧状态下细胞活性的最佳时间点,qRT-PCR和Westem Blot检测HT22模型细胞内DGAM5-Drp1轴蛋白及mRNA的表达水平。结果:OGD/R诱导HT22细胞持续2 h为最佳时间点,miR-451a可以靶向调控PGAM5基因,PGAM5和Drp1具有相互调控作用。与正常对照组比较,OGD/R组细胞内PGAM5、Drp1和Fis1蛋白及mRNA表达上升,p-Drp1 Ser 616磷酸化表达上升(P<0.05),OPA1蛋白及mRNA表达下降,p-Drp1 Ser 637去磷酸化表达下降(P<0.05);与OGD/R组比较,miR-451a过表达+WFJZF组细胞内PGAM5、Drp1和Fis1蛋白及mRNA表达下降,p-Drp1 Ser 616磷酸化表达下降(P<0.05),OPA1蛋白及mRNA表达上升,p-Drp1 Ser 637去磷酸化表达上升(P<0.05)。结论:miR-451a可以靶向调控PGAM5-Drp1轴,miR-451a过表达+WFJZF组可以改善线粒体失衡状态,减少神经元过度损伤,发挥脑保护效应。展开更多
基金the National Natural Science Foundation of China(Nos.22207069,22325703,22377071,22074084)Research Project Supported by Shanxi Scholarship Council of China(No.2022-002)+2 种基金the Shanxi Province Science Foundation(Nos.20210302124012,202203021221009)Key R&D and transformation plan of Qinghai Province(No.2020-GX-101)2023 Graduate Innovation Project of Shanxi University。
文摘Intracellular redox homeostasis is of indispensable importance in pathophysiology.In order to maintain the balance of the redox state within the cell,reactive oxygen species(ROS)and reactive sulfur species(RSS)react and transform with each other,and their levels also directly reflect the degree of oxidative stress and disease.Hypochlorous acid(HClO)and cysteine(Cys)usually co-exist in organisms,interacting with each other in many important physiological processes and synergistically maintaining the dynamic redox balance in the body.To understand the relevance and pathophysiological effects of these two signaling molecules in oxidative stress,unique fluorescence imaging tools are required.Herein,we designed and developed a dual-channel fluorescent probe HP,for the individual and continuous detection of HClO and Cys.This probe could simultaneously monitor the changes in the concentrations of HClO and Cys in cells,and was characterized by a fast response,high sensitivity and high selectivity,especially compared with glutathione(GSH)and homocysteine(Hcy),the probe had a good specificity for Cys.Importantly,probe HP successfully observed dynamic changes in HCl O-and Cys-mediated redox status in the oxygenglucose deprivation/reperfusion(OGD/R)model of HeLa cells and dynamically monitored fluctuations in endogenous HClO levels in lipopolysaccharides(LPS)-induced peritonitis mice.
文摘目的:基于miR-451a靶向磷酸甘油酸变位酶(PGAM5)-线粒体动力相关蛋白1(Drp1)轴调控线粒体动力学,探讨温肺降浊方对氧糖剥夺/复氧(OGD/R)诱导HT22模型的效应机制。方法:建立OGD/R诱导HT22细胞模型,设立正常对照组、OGD/R组、miR-451a过表达组、miR-451a敲低组、温肺降浊方含药血清(WFJZF)组、miR-451a过表达+WFJZF组、miR-451a敲低+WFJZF组和miR-451a空载体组,双荧光素酶确定miR-451a和PGAM5的转录关系,免疫蛋白共沉淀确定PGAM5和Drp1的调控作用,慢病毒转染miR-451a于HT22细胞中,qRT-PCR确定转染效率,CCK-8测定缺糖缺氧状态下细胞活性的最佳时间点,qRT-PCR和Westem Blot检测HT22模型细胞内DGAM5-Drp1轴蛋白及mRNA的表达水平。结果:OGD/R诱导HT22细胞持续2 h为最佳时间点,miR-451a可以靶向调控PGAM5基因,PGAM5和Drp1具有相互调控作用。与正常对照组比较,OGD/R组细胞内PGAM5、Drp1和Fis1蛋白及mRNA表达上升,p-Drp1 Ser 616磷酸化表达上升(P<0.05),OPA1蛋白及mRNA表达下降,p-Drp1 Ser 637去磷酸化表达下降(P<0.05);与OGD/R组比较,miR-451a过表达+WFJZF组细胞内PGAM5、Drp1和Fis1蛋白及mRNA表达下降,p-Drp1 Ser 616磷酸化表达下降(P<0.05),OPA1蛋白及mRNA表达上升,p-Drp1 Ser 637去磷酸化表达上升(P<0.05)。结论:miR-451a可以靶向调控PGAM5-Drp1轴,miR-451a过表达+WFJZF组可以改善线粒体失衡状态,减少神经元过度损伤,发挥脑保护效应。
