目的:探讨敲除溶酶体跨膜蛋白175(transmembrane protein 175,TMEM175)对HeLa细胞线粒体功能的影响。方法:传代培养HeLa细胞系,分为野生型(wild-type,WT)组和TMEM175敲除(knockout,KO)组。采用Western blot技术检测TMEM175、溶酶体关联...目的:探讨敲除溶酶体跨膜蛋白175(transmembrane protein 175,TMEM175)对HeLa细胞线粒体功能的影响。方法:传代培养HeLa细胞系,分为野生型(wild-type,WT)组和TMEM175敲除(knockout,KO)组。采用Western blot技术检测TMEM175、溶酶体关联膜蛋白1(lysosomal-associated membrane protein 1,LAMP1)、LAMP2和转录因子EB(transcription factor EB,TFEB)等蛋白表达;LysoSensor染色测定溶酶体酸度变化;分别采用JC-1和Mito-SOX探针测定线粒体膜电位及活性氧(reactive oxygen species,ROS)水平;应用O2K高分辨率线粒体呼吸测定系统和ATP试剂盒测定线粒体耗氧率和ATP含量;采用Western blot技术测定线粒体动力学及能量代谢关键分子包括线粒体融合蛋白2(mitofusin 2,Mfn2)、视神经萎缩蛋白1(optic atrophy 1,OPA1)、发动蛋白相关蛋白1(dynaminrelated protein 1,Drp1)、腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)及电子呼吸链复合体等的蛋白表达。结果:与WT组相比,KO组细胞中TMEM175蛋白表达水平显著降低(P<0.01),溶酶体酸度增加(P<0.05),LAMP1表达水平降低(P<0.05),LAMP2和磷酸化TFEB蛋白水平升高(P<0.05)。缺失TMEM175后细胞线粒体膜电位降低(P<0.01),线粒体ROS水平升高(P<0.01),线粒体基础呼吸和漏呼吸能力减弱(P<0.01),ATP生成相关呼吸及ATP含量增加(P<0.05或P<0.01)。WT组和KO组细胞中Mfn2和OPA1蛋白表达无显著差异(P>0.05),KO组细胞磷酸化Drp1增加(P<0.05),AMPK蛋白表达及活性均显著增加(P<0.05),线粒体复合体I、IV蛋白表达水平降低(P<0.05或P<0.01)。结论:敲除HeLa细胞TMEM175引起溶酶体酸化,溶酶体相关蛋白表达改变,并抑制线粒体功能,激活Drp1和AMPK信号。展开更多
Recent studies have revealed a significant relationship between erectile dysfunction(ED)and lower urinary tract symptoms(LUTS),both of which commonly affect middle-aged and older men.These conditions share underlying ...Recent studies have revealed a significant relationship between erectile dysfunction(ED)and lower urinary tract symptoms(LUTS),both of which commonly affect middle-aged and older men.These conditions share underlying causes,particularly endothelial dysfunction,atherosclerosis,and chronic pelvic ischemia(CPI).This study investigated the therapeutic potential of LDD175,a large-conductance Ca2+-activated K+channel(BKCa channel)opener,in simultaneously treating both conditions using a CPI animal model of male Sprague Dawley rats.Our study investigated the induction of CPI through surgical endothelial damage combined with a high-cholesterol diet.We assessed erectile and voiding functions by measuring intracavernosal pressure(ICP)and intraurethral pressure(IUP),respectively,after nerve stimulation.We performed histological examinations of vascular changes and western blot analyses of cavernous and prostate tissues to understand the underlying mechanisms.This study evaluated the effectiveness of LDD175 compared to standard treatments,such as sildenafil for ED and tamsulosin for LUTS.Therefore,the CPI model successfully demonstrated ED and LUTS symptoms with decreased ICP and increased IUP.Analysis revealed elevated levels of hypoxia-inducible factor-1α,transforming growth factor-β1 andβ2 in cavernous tissue,and increasedα1A-adrenoceptor expression in prostate tissue.LDD175 administration showed promising results,with dose-dependent improvements in ICP and IUP,and therapeutic effects comparable to those of established treatments.Our findings suggest a novel therapeutic approach that can simultaneously address ED and LUTS,opening new possibilities for clinical application in the treatment of these interconnected conditions.展开更多
文摘目的:探讨敲除溶酶体跨膜蛋白175(transmembrane protein 175,TMEM175)对HeLa细胞线粒体功能的影响。方法:传代培养HeLa细胞系,分为野生型(wild-type,WT)组和TMEM175敲除(knockout,KO)组。采用Western blot技术检测TMEM175、溶酶体关联膜蛋白1(lysosomal-associated membrane protein 1,LAMP1)、LAMP2和转录因子EB(transcription factor EB,TFEB)等蛋白表达;LysoSensor染色测定溶酶体酸度变化;分别采用JC-1和Mito-SOX探针测定线粒体膜电位及活性氧(reactive oxygen species,ROS)水平;应用O2K高分辨率线粒体呼吸测定系统和ATP试剂盒测定线粒体耗氧率和ATP含量;采用Western blot技术测定线粒体动力学及能量代谢关键分子包括线粒体融合蛋白2(mitofusin 2,Mfn2)、视神经萎缩蛋白1(optic atrophy 1,OPA1)、发动蛋白相关蛋白1(dynaminrelated protein 1,Drp1)、腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)及电子呼吸链复合体等的蛋白表达。结果:与WT组相比,KO组细胞中TMEM175蛋白表达水平显著降低(P<0.01),溶酶体酸度增加(P<0.05),LAMP1表达水平降低(P<0.05),LAMP2和磷酸化TFEB蛋白水平升高(P<0.05)。缺失TMEM175后细胞线粒体膜电位降低(P<0.01),线粒体ROS水平升高(P<0.01),线粒体基础呼吸和漏呼吸能力减弱(P<0.01),ATP生成相关呼吸及ATP含量增加(P<0.05或P<0.01)。WT组和KO组细胞中Mfn2和OPA1蛋白表达无显著差异(P>0.05),KO组细胞磷酸化Drp1增加(P<0.05),AMPK蛋白表达及活性均显著增加(P<0.05),线粒体复合体I、IV蛋白表达水平降低(P<0.05或P<0.01)。结论:敲除HeLa细胞TMEM175引起溶酶体酸化,溶酶体相关蛋白表达改变,并抑制线粒体功能,激活Drp1和AMPK信号。
文摘Recent studies have revealed a significant relationship between erectile dysfunction(ED)and lower urinary tract symptoms(LUTS),both of which commonly affect middle-aged and older men.These conditions share underlying causes,particularly endothelial dysfunction,atherosclerosis,and chronic pelvic ischemia(CPI).This study investigated the therapeutic potential of LDD175,a large-conductance Ca2+-activated K+channel(BKCa channel)opener,in simultaneously treating both conditions using a CPI animal model of male Sprague Dawley rats.Our study investigated the induction of CPI through surgical endothelial damage combined with a high-cholesterol diet.We assessed erectile and voiding functions by measuring intracavernosal pressure(ICP)and intraurethral pressure(IUP),respectively,after nerve stimulation.We performed histological examinations of vascular changes and western blot analyses of cavernous and prostate tissues to understand the underlying mechanisms.This study evaluated the effectiveness of LDD175 compared to standard treatments,such as sildenafil for ED and tamsulosin for LUTS.Therefore,the CPI model successfully demonstrated ED and LUTS symptoms with decreased ICP and increased IUP.Analysis revealed elevated levels of hypoxia-inducible factor-1α,transforming growth factor-β1 andβ2 in cavernous tissue,and increasedα1A-adrenoceptor expression in prostate tissue.LDD175 administration showed promising results,with dose-dependent improvements in ICP and IUP,and therapeutic effects comparable to those of established treatments.Our findings suggest a novel therapeutic approach that can simultaneously address ED and LUTS,opening new possibilities for clinical application in the treatment of these interconnected conditions.
