目的·探讨沉默信息调节因子2相关酶5(sirtuin 5,SIRT5)在脓毒症肺血管内皮细胞损伤中的作用及机制。方法·对野生型(wild-type,WT)和Sirt5基因敲除的C57BL/6雄性小鼠行盲肠结扎穿孔术(cecal ligation and puncture,CLP),对小...目的·探讨沉默信息调节因子2相关酶5(sirtuin 5,SIRT5)在脓毒症肺血管内皮细胞损伤中的作用及机制。方法·对野生型(wild-type,WT)和Sirt5基因敲除的C57BL/6雄性小鼠行盲肠结扎穿孔术(cecal ligation and puncture,CLP),对小鼠安乐死后进行肺组织取材。苏木精-伊红(hematoxylin and eosin staining,H-E)染色评估肺组织炎症水平;伊文思蓝(Evans blue,EB)染色评估血管渗漏;肺组织免疫荧光染色评估小鼠凝血功能改变情况;采用免疫组化染色检测血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)蛋白表达,评估CLP小鼠内皮细胞炎症水平。采用基因编辑技术对人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)SIRT5基因进行敲减或者过表达,并使用脂多糖(lipopolysaccharide,LPS)刺激诱导内皮细胞炎症。采用蛋白质印迹法检测VCAM-1、组织因子(tissue factor,TF)等内皮细胞损伤标志物,通过实时荧光定量聚合酶链反应(quantitative real-time PCR,qPCR)检测白细胞介素-6(interleukin 6,IL-6)、IL-1β等炎症指标。对SIRT5过表达的HUVEC进行转录组测序,并通过qPCR验证关键基因如F2R样凝血酶或胰蛋白酶受体3(F2R like thrombin or trypsin receptor 3,F2RL3)或serpin家族成员3(serpin family A member 3,SERPINA3)以及转化生长因子-β2/3(transforming growth factorβ2/3,TGF-β2/3)的表达。结果·Sirt5基因敲除显著加重CLP小鼠肺损伤,使其生存率降低(P<0.001);H-E染色显示小鼠肺组织炎症浸润增加,EB染色观察到血管渗漏增加(P<0.001),免疫荧光染色可见纤维蛋白原沉积升高。SIRT5基因敲减的HUVEC中VCAM-1、TF蛋白及IL-6、IL-1β、VCAM-1、E选择素(E-selectin)等炎症因子mRNA表达上调(均P<0.001),而SIRT5过表达可逆转此效应。转录组测序分析表明SIRT5通过抑制F2RL3/SERPINA3/TGF-β通路调控内皮炎症与凝血反应。结论·SIRT5通过负向调控F2RL3/SERPINA3/TGF-β信号轴减轻内皮炎症及促凝血反应,提示其在脓毒症肺损伤中的潜在保护作用。展开更多
Tetrandrine(TET),a natural bisbenzyl isoquinoline alkaloid extracted from Stephania tetrandra S.Moore,has diverse pharmacological effects.However,its effects on melanoma remain unclear.Cellular prolif-eration assays,m...Tetrandrine(TET),a natural bisbenzyl isoquinoline alkaloid extracted from Stephania tetrandra S.Moore,has diverse pharmacological effects.However,its effects on melanoma remain unclear.Cellular prolif-eration assays,multi-omics analyses,and xenograft models were used to determine the effect of TET on melanoma.The direct target of TET was identified using biotin-TET pull-down liquid chromatograph-mass spectrometry(LC-MS),cellular thermal shift assays,and isothermal titration calorimetry(ITC)analysis.Our findings revealed that TET treatment induced robust cellular autophagy depending on activating transcription factor 6(ATF6)-mediated endoplasmic reticulum(ER)stress.Simultaneously,it hindered autophagic flux by inducing cytoskeletal protein depolymerization in melanoma cells.TET treatment resulted in excessive accumulation of reactive oxygen species(Ros)and simultaneously triggered mitophagy.Sirtuin 5(SIRT5)was ultimately found to be a direct target of TET.Mechanistically,TET led to the degradation of SIRT5 via the ubiquitin(Ub)-26S proteasome system.SIRT5 knockdown induced ROS accumulation,whereas SIRT5 overexpression attenuated the TET-induced ROS accumula-tion and autophagy.Importantly,TET exhibited anti-cancer effects in xenograft models depending on SIRT5 expression.This study highlights the potential of TET as an antimelanoma agent that targets SIRT5.These findings provide a promising avenue for the use of TET in melanoma treatment and underscore its potential as a therapeutic candidate.展开更多
目的探讨乳腺癌中SIRT5的表达及其与PKM2及HK2相关性及临床意义。方法采用免疫组化SP法检测130例乳腺癌组织及癌旁正常组织中SIRT5、PKM2及HK2的表达,应用RT-PCR法检测60例乳腺癌组织及癌旁正常组织SIRT5、PKM2及HK2的基因表达,分析SIRT...目的探讨乳腺癌中SIRT5的表达及其与PKM2及HK2相关性及临床意义。方法采用免疫组化SP法检测130例乳腺癌组织及癌旁正常组织中SIRT5、PKM2及HK2的表达,应用RT-PCR法检测60例乳腺癌组织及癌旁正常组织SIRT5、PKM2及HK2的基因表达,分析SIRT5表达与乳腺癌临床病理特征的关系。结果乳腺癌组织中SIRT5、PKM2及HK2的平均光密度值均显著高于癌旁正常组织(5824.4±163.1 vs 2629.9±132.3、5980.3±232.2 vs 2164.9±121.3、6005.1±197.5 vs 2196.7±155.4,P<0.01),乳腺癌组织SIRT5、PKM2及HK2 mRNA的表达均高于癌旁正常组织(P<0.01)。乳腺癌组织中SIRT5与PKM2、HK2蛋白间呈正相关(r=0.647、r=0.600,P<0.01)。SIRT5阳性率与患者年龄、淋巴结转移、TNM分期、ER及PR表达差异无统计学意义(P=0.859,P=0.248,P=0.986,P=0.489,P=0.882);与肿瘤大小、组织学分级及HER-2表达差异有统计学意义(P=0.003,P=0.001,P=0.037)。结论SIRT5的过表达对判断乳腺癌的发生、发展及预后具有一定价值,并为SIRT5可能通过有氧糖酵解影响乳腺癌的发生、发展奠定基础。展开更多
文摘目的·探讨沉默信息调节因子2相关酶5(sirtuin 5,SIRT5)在脓毒症肺血管内皮细胞损伤中的作用及机制。方法·对野生型(wild-type,WT)和Sirt5基因敲除的C57BL/6雄性小鼠行盲肠结扎穿孔术(cecal ligation and puncture,CLP),对小鼠安乐死后进行肺组织取材。苏木精-伊红(hematoxylin and eosin staining,H-E)染色评估肺组织炎症水平;伊文思蓝(Evans blue,EB)染色评估血管渗漏;肺组织免疫荧光染色评估小鼠凝血功能改变情况;采用免疫组化染色检测血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1)蛋白表达,评估CLP小鼠内皮细胞炎症水平。采用基因编辑技术对人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)SIRT5基因进行敲减或者过表达,并使用脂多糖(lipopolysaccharide,LPS)刺激诱导内皮细胞炎症。采用蛋白质印迹法检测VCAM-1、组织因子(tissue factor,TF)等内皮细胞损伤标志物,通过实时荧光定量聚合酶链反应(quantitative real-time PCR,qPCR)检测白细胞介素-6(interleukin 6,IL-6)、IL-1β等炎症指标。对SIRT5过表达的HUVEC进行转录组测序,并通过qPCR验证关键基因如F2R样凝血酶或胰蛋白酶受体3(F2R like thrombin or trypsin receptor 3,F2RL3)或serpin家族成员3(serpin family A member 3,SERPINA3)以及转化生长因子-β2/3(transforming growth factorβ2/3,TGF-β2/3)的表达。结果·Sirt5基因敲除显著加重CLP小鼠肺损伤,使其生存率降低(P<0.001);H-E染色显示小鼠肺组织炎症浸润增加,EB染色观察到血管渗漏增加(P<0.001),免疫荧光染色可见纤维蛋白原沉积升高。SIRT5基因敲减的HUVEC中VCAM-1、TF蛋白及IL-6、IL-1β、VCAM-1、E选择素(E-selectin)等炎症因子mRNA表达上调(均P<0.001),而SIRT5过表达可逆转此效应。转录组测序分析表明SIRT5通过抑制F2RL3/SERPINA3/TGF-β通路调控内皮炎症与凝血反应。结论·SIRT5通过负向调控F2RL3/SERPINA3/TGF-β信号轴减轻内皮炎症及促凝血反应,提示其在脓毒症肺损伤中的潜在保护作用。
基金This work was supported by funding from Natural Science Foundation of China(Grant Nos.:82372519 and 81902664)the PostdoctoralFellowshipProgramof CPSF(GrantNo.:GZB20240544)+3 种基金the China Postdoctoral Science Foundation(Grant No.:2024M752432)the Natural Science Foundation of Hebei Province(Grant Nos.:H2022206368 and H2022206446)Medical Science Research Program of the Hebei Provincial Health Commission(Grant No.:20241603)Pilot Program of Southwest University(Program No.:SWU-XDZD22006)。
文摘Tetrandrine(TET),a natural bisbenzyl isoquinoline alkaloid extracted from Stephania tetrandra S.Moore,has diverse pharmacological effects.However,its effects on melanoma remain unclear.Cellular prolif-eration assays,multi-omics analyses,and xenograft models were used to determine the effect of TET on melanoma.The direct target of TET was identified using biotin-TET pull-down liquid chromatograph-mass spectrometry(LC-MS),cellular thermal shift assays,and isothermal titration calorimetry(ITC)analysis.Our findings revealed that TET treatment induced robust cellular autophagy depending on activating transcription factor 6(ATF6)-mediated endoplasmic reticulum(ER)stress.Simultaneously,it hindered autophagic flux by inducing cytoskeletal protein depolymerization in melanoma cells.TET treatment resulted in excessive accumulation of reactive oxygen species(Ros)and simultaneously triggered mitophagy.Sirtuin 5(SIRT5)was ultimately found to be a direct target of TET.Mechanistically,TET led to the degradation of SIRT5 via the ubiquitin(Ub)-26S proteasome system.SIRT5 knockdown induced ROS accumulation,whereas SIRT5 overexpression attenuated the TET-induced ROS accumula-tion and autophagy.Importantly,TET exhibited anti-cancer effects in xenograft models depending on SIRT5 expression.This study highlights the potential of TET as an antimelanoma agent that targets SIRT5.These findings provide a promising avenue for the use of TET in melanoma treatment and underscore its potential as a therapeutic candidate.
文摘目的探讨乳腺癌中SIRT5的表达及其与PKM2及HK2相关性及临床意义。方法采用免疫组化SP法检测130例乳腺癌组织及癌旁正常组织中SIRT5、PKM2及HK2的表达,应用RT-PCR法检测60例乳腺癌组织及癌旁正常组织SIRT5、PKM2及HK2的基因表达,分析SIRT5表达与乳腺癌临床病理特征的关系。结果乳腺癌组织中SIRT5、PKM2及HK2的平均光密度值均显著高于癌旁正常组织(5824.4±163.1 vs 2629.9±132.3、5980.3±232.2 vs 2164.9±121.3、6005.1±197.5 vs 2196.7±155.4,P<0.01),乳腺癌组织SIRT5、PKM2及HK2 mRNA的表达均高于癌旁正常组织(P<0.01)。乳腺癌组织中SIRT5与PKM2、HK2蛋白间呈正相关(r=0.647、r=0.600,P<0.01)。SIRT5阳性率与患者年龄、淋巴结转移、TNM分期、ER及PR表达差异无统计学意义(P=0.859,P=0.248,P=0.986,P=0.489,P=0.882);与肿瘤大小、组织学分级及HER-2表达差异有统计学意义(P=0.003,P=0.001,P=0.037)。结论SIRT5的过表达对判断乳腺癌的发生、发展及预后具有一定价值,并为SIRT5可能通过有氧糖酵解影响乳腺癌的发生、发展奠定基础。
