目的通过体外细胞培养和建立大鼠胆管结扎(b ile duct ligation,BDL)所致阻塞性胆汁淤积动物模型,在蛋白水平观察多耐药相关蛋白MRP3(mu ltidrug resistance-assoc iated prote in 3,MRP3)和核受体RXRα(retinoid-X receptor al-pha,RXR...目的通过体外细胞培养和建立大鼠胆管结扎(b ile duct ligation,BDL)所致阻塞性胆汁淤积动物模型,在蛋白水平观察多耐药相关蛋白MRP3(mu ltidrug resistance-assoc iated prote in 3,MRP3)和核受体RXRα(retinoid-X receptor al-pha,RXRα)表达的关系。方法用鹅去氧胆酸(chenodeoxycholic ac id,CDCA)或苯巴比妥(phenobarb ital,PB)分别刺激培养的肝癌细胞HepG2并建立DBL阻塞性胆汁淤积大鼠模型后,分别抽提HepG2细胞总蛋白、核蛋白和大鼠肝脏细胞膜蛋白和核蛋白,W estern b lot方法检测MRP3和RXRα蛋白表达水平的变化。结果在细胞水平,CDCA和PB可诱导HepG2细胞膜MRP3蛋白表达增高,并抑制细胞核RXRα蛋白表达;在动物体内,BDL大鼠肝脏MRP3明显增加,同时RXRα表达明显下降。结论肝细胞膜蛋白MRP3表达的上调可能与核受体RXRα表达抑制有关。展开更多
Fructus Psoraleae,which is commonly consumed for the treatment of osteoporosis,bone fracture,and leucoderma,induces liver injury.This study investigated the pathogenesis of the ethanol extract of Fructus Psoraleae(EEF...Fructus Psoraleae,which is commonly consumed for the treatment of osteoporosis,bone fracture,and leucoderma,induces liver injury.This study investigated the pathogenesis of the ethanol extract of Fructus Psoraleae(EEFP)-induced liver injury in rats.EEFP(1.35,1.80,and 2.25 g·kg^–1)was administrated to Sprague Dawley(SD)rats for 30 d.We measured liver chemistries,histopathology,and quantitative isobaric tags for relative and absolute quantitation(iTRAQ)-based protein profiling.EEFP demonstrated parameters suggestive of liver injury with changes in bile secretion,bile flow rate,and liver histopathology.iTRAQ analysis showed that a total of 4042 proteins were expressed in liver tissues of EEFP-treated and untreated rats.Among these proteins,81 were upregulated and 32 were downregulated in the treatment group.KEGG pathway analysis showed that the drug metabolic pathways of cytochrome P450,glutathione metabolism,glycerolipid metabolism,and bile secretion were enriched with differentially expressed proteins.The expression of key proteins related to the farnesoid X receptor(FXR),i.e.,the peroxisome proliferators-activated receptor alpha(PPAR-α),were downregulated,and multidrug resistance-associated protein 3(MRP3)was upregulated in the EEFP-treated rats.Our results provide evidence that EEFP may induce hepatotoxicity through various pathways.Furthermore,our study demonstrates changes in protein regulation using iTRAQ quantitative proteomics analysis.展开更多
目的体外HepG2细胞培养观察芒果苷(mangiferin)对多耐药相关蛋白3(multidrug resistance-associate protein 3,MRP3)和核受体孕烷X受体(pregnane X receptor,PXR)、CYP7A启动子结合因子(CYP7A promoter-binding factor,CPF)表达的影响...目的体外HepG2细胞培养观察芒果苷(mangiferin)对多耐药相关蛋白3(multidrug resistance-associate protein 3,MRP3)和核受体孕烷X受体(pregnane X receptor,PXR)、CYP7A启动子结合因子(CYP7A promoter-binding factor,CPF)表达的影响。方法用芒果苷刺激HepG2细胞72 h后,分别抽提细胞RNA、膜蛋白及核蛋白,采用半定量RT-PCR和蛋白免疫印迹检测膜转运蛋白MRP3和核受体PXR、CPF在转录与蛋白水平的表达变化。熊脱氧胆酸(ursode-oxycholic acid,UDCA)处理的HepG2细胞作为阳性对照、DMSO处理细胞为阴性对照。结果芒果苷可显著刺激HepG2细胞膜转运蛋白MRP3的mRNA(比阴性对照组高3.0倍,P<0.05)和蛋白(比阴性对照组高3.3倍,P<0.05)表达,其作用强于UDCA。芒果苷也可明显上调核受体PXR[mRNA水平增高1.7倍(P<0.05),蛋白水平增高3.7倍(P<0.01)]、CPF[mRNA水平增高2.1倍(P<0.05),蛋白水平增高4.9倍(P<0.05)]的表达。结论芒果苷刺激肝癌细胞HepG2细胞膜转运蛋白MRP3的表达上调可能与核受体PXR、CPF途径相关。展开更多
目的通过诱导剂刺激体外培养的人肝癌细胞HepG2,观察多耐药相关蛋白MRP3(mu ltidrug resistance-assoc iatedprote in 3,MRP3)和核受体RXRα(retinoid-X receptor alpha,RXRα)蛋白的表达变化。方法用鹅去氧胆酸(chenodeoxycholic ac id...目的通过诱导剂刺激体外培养的人肝癌细胞HepG2,观察多耐药相关蛋白MRP3(mu ltidrug resistance-assoc iatedprote in 3,MRP3)和核受体RXRα(retinoid-X receptor alpha,RXRα)蛋白的表达变化。方法用鹅去氧胆酸(chenodeoxycholic ac id,CDCA)或苯巴比妥(phenobarb ital,PB)分别刺激培养的肝癌细胞HepG2后,抽提HepG2细胞总蛋白和核蛋白,W estern b lot方法检测MRP3和RXRα蛋白表达水平的变化。结果CDCA和PB可诱导HepG2细胞膜MRP3蛋白表达增高,并抑制细胞核受体RXRα蛋白表达。结论HepG2细胞中膜转运蛋白MRP3表达的上调可能与核受体RXRα表达抑制相关。展开更多
目的:探讨multidrug resistance-related protein 3(MRP3)在胆囊癌中的表达及其与病理指标及预后的关系。方法:应用免疫组化SP法检测50例胆囊癌中MRP3的表达。结果:MRP3呈散在分布的淡黄色或棕褐色颗粒,主要定位于细胞浆中,散见于细胞核...目的:探讨multidrug resistance-related protein 3(MRP3)在胆囊癌中的表达及其与病理指标及预后的关系。方法:应用免疫组化SP法检测50例胆囊癌中MRP3的表达。结果:MRP3呈散在分布的淡黄色或棕褐色颗粒,主要定位于细胞浆中,散见于细胞核中,强阳性表达率为72%。随着转移程度、Nevin分级以及分化程度的增加,强阳性表达率增加,且具有统计学差异(P<0.05)。本组病人总生存期为16.42个月,强阳性表达组病人的总生存期为13.25个月,弱阳性表达组病人的总生存期为22.64个月,两者具有显著性差异(P<0.05)。单因素分析显示总生存期与MRP3的表达、Nevin分级、转移及分化程度有关(P<0.05),多因素分析显示MRP3的表达强弱以及转移是总生存期的独立预后因子(P<0.05)。结论:高表达的MRP3参与了胆囊癌的发生发展,MRP3与胆囊癌的分化、转移有关,可作为判断预后的指标。展开更多
目的:研究黄芪甲苷IV(AS-IV)对人肝癌细胞HepG2中多药耐药相关蛋白-3(MRP3)的调节作用,为AS-IV可能引发的药物相互作用阐明机制。方法:培养HepG2细胞,用不同浓度AS-IV进行干预,Western blotting方法检测细胞中MRP3的蛋白水平,流式细胞...目的:研究黄芪甲苷IV(AS-IV)对人肝癌细胞HepG2中多药耐药相关蛋白-3(MRP3)的调节作用,为AS-IV可能引发的药物相互作用阐明机制。方法:培养HepG2细胞,用不同浓度AS-IV进行干预,Western blotting方法检测细胞中MRP3的蛋白水平,流式细胞术检测胞内5-羧基荧光素(5-CF)的荧光强度,反映MRP的功能;给予MRP3 si RNA干扰,观察AS-IV诱导的MRP3蛋白表达是否增加其外排转运功能;提取胞核、总蛋白,检测胞核核因子E2相关因子2(Nrf2)、总Nrf2蛋白水平;给予Nrf2 si RNA干扰,观察AS-IV诱导的MRP3蛋白表达是否依赖于Nrf2。结果:200μmol/L AS-IV作用48 h后,细胞MRP3的蛋白水平显著增加(P<0.05),胞内5-CF的平均荧光强度(MFI)明显低于对照组(P<0.05),与单独给予AS-IV组相比,给予MRP3 si RNA干扰显著增加了胞内5-CF的MFI(P<0.05)。AS-IV增加了胞核内Nrf2的蛋白水平(P<0.01),亦增加了总Nrf2的蛋白表达(P<0.01)。给予Nrf2 si RNA干扰后,AS-IV诱导上调的MRP3被显著抑制(P<0.05)。结论:AS-IV可通过上调MRP3的蛋白表达,增加其外排转运功能,Nrf2的激活在AS-IV诱导MRP3蛋白表达中起着重要的作用。本研究提示,AS-IV与MRP3底物合用时,可能会产生药物-药物相互作用。展开更多
文摘目的通过体外细胞培养和建立大鼠胆管结扎(b ile duct ligation,BDL)所致阻塞性胆汁淤积动物模型,在蛋白水平观察多耐药相关蛋白MRP3(mu ltidrug resistance-assoc iated prote in 3,MRP3)和核受体RXRα(retinoid-X receptor al-pha,RXRα)表达的关系。方法用鹅去氧胆酸(chenodeoxycholic ac id,CDCA)或苯巴比妥(phenobarb ital,PB)分别刺激培养的肝癌细胞HepG2并建立DBL阻塞性胆汁淤积大鼠模型后,分别抽提HepG2细胞总蛋白、核蛋白和大鼠肝脏细胞膜蛋白和核蛋白,W estern b lot方法检测MRP3和RXRα蛋白表达水平的变化。结果在细胞水平,CDCA和PB可诱导HepG2细胞膜MRP3蛋白表达增高,并抑制细胞核RXRα蛋白表达;在动物体内,BDL大鼠肝脏MRP3明显增加,同时RXRα表达明显下降。结论肝细胞膜蛋白MRP3表达的上调可能与核受体RXRα表达抑制有关。
基金supported by the the National Key Research and Development Program, Specialized Research on Modernization of TCM (Nos. 2018YFC1708006 and 2018YFC1708003)National Major New Drug Creation Special Project “National Drug New Varieties Research and Development and Its Key Innovative Technology Topics” (No. 2017ZX09301060-005)+3 种基金the National Natural Science Foundation of China (No. 81320108029)Tianshan Cedar Plan (No. 2018XS21)National “Major Scientific and Technological Special Project for Significant New Drugs Creation” Project (No. 2015ZX09501004-002-004)Specific Fund for Pub-lic Interest Research of Traditional Chinese Medicine, Ministry of Finance of China (No. 201507004-002)。
文摘Fructus Psoraleae,which is commonly consumed for the treatment of osteoporosis,bone fracture,and leucoderma,induces liver injury.This study investigated the pathogenesis of the ethanol extract of Fructus Psoraleae(EEFP)-induced liver injury in rats.EEFP(1.35,1.80,and 2.25 g·kg^–1)was administrated to Sprague Dawley(SD)rats for 30 d.We measured liver chemistries,histopathology,and quantitative isobaric tags for relative and absolute quantitation(iTRAQ)-based protein profiling.EEFP demonstrated parameters suggestive of liver injury with changes in bile secretion,bile flow rate,and liver histopathology.iTRAQ analysis showed that a total of 4042 proteins were expressed in liver tissues of EEFP-treated and untreated rats.Among these proteins,81 were upregulated and 32 were downregulated in the treatment group.KEGG pathway analysis showed that the drug metabolic pathways of cytochrome P450,glutathione metabolism,glycerolipid metabolism,and bile secretion were enriched with differentially expressed proteins.The expression of key proteins related to the farnesoid X receptor(FXR),i.e.,the peroxisome proliferators-activated receptor alpha(PPAR-α),were downregulated,and multidrug resistance-associated protein 3(MRP3)was upregulated in the EEFP-treated rats.Our results provide evidence that EEFP may induce hepatotoxicity through various pathways.Furthermore,our study demonstrates changes in protein regulation using iTRAQ quantitative proteomics analysis.
文摘目的体外HepG2细胞培养观察芒果苷(mangiferin)对多耐药相关蛋白3(multidrug resistance-associate protein 3,MRP3)和核受体孕烷X受体(pregnane X receptor,PXR)、CYP7A启动子结合因子(CYP7A promoter-binding factor,CPF)表达的影响。方法用芒果苷刺激HepG2细胞72 h后,分别抽提细胞RNA、膜蛋白及核蛋白,采用半定量RT-PCR和蛋白免疫印迹检测膜转运蛋白MRP3和核受体PXR、CPF在转录与蛋白水平的表达变化。熊脱氧胆酸(ursode-oxycholic acid,UDCA)处理的HepG2细胞作为阳性对照、DMSO处理细胞为阴性对照。结果芒果苷可显著刺激HepG2细胞膜转运蛋白MRP3的mRNA(比阴性对照组高3.0倍,P<0.05)和蛋白(比阴性对照组高3.3倍,P<0.05)表达,其作用强于UDCA。芒果苷也可明显上调核受体PXR[mRNA水平增高1.7倍(P<0.05),蛋白水平增高3.7倍(P<0.01)]、CPF[mRNA水平增高2.1倍(P<0.05),蛋白水平增高4.9倍(P<0.05)]的表达。结论芒果苷刺激肝癌细胞HepG2细胞膜转运蛋白MRP3的表达上调可能与核受体PXR、CPF途径相关。
文摘目的通过诱导剂刺激体外培养的人肝癌细胞HepG2,观察多耐药相关蛋白MRP3(mu ltidrug resistance-assoc iatedprote in 3,MRP3)和核受体RXRα(retinoid-X receptor alpha,RXRα)蛋白的表达变化。方法用鹅去氧胆酸(chenodeoxycholic ac id,CDCA)或苯巴比妥(phenobarb ital,PB)分别刺激培养的肝癌细胞HepG2后,抽提HepG2细胞总蛋白和核蛋白,W estern b lot方法检测MRP3和RXRα蛋白表达水平的变化。结果CDCA和PB可诱导HepG2细胞膜MRP3蛋白表达增高,并抑制细胞核受体RXRα蛋白表达。结论HepG2细胞中膜转运蛋白MRP3表达的上调可能与核受体RXRα表达抑制相关。
文摘目的:探讨multidrug resistance-related protein 3(MRP3)在胆囊癌中的表达及其与病理指标及预后的关系。方法:应用免疫组化SP法检测50例胆囊癌中MRP3的表达。结果:MRP3呈散在分布的淡黄色或棕褐色颗粒,主要定位于细胞浆中,散见于细胞核中,强阳性表达率为72%。随着转移程度、Nevin分级以及分化程度的增加,强阳性表达率增加,且具有统计学差异(P<0.05)。本组病人总生存期为16.42个月,强阳性表达组病人的总生存期为13.25个月,弱阳性表达组病人的总生存期为22.64个月,两者具有显著性差异(P<0.05)。单因素分析显示总生存期与MRP3的表达、Nevin分级、转移及分化程度有关(P<0.05),多因素分析显示MRP3的表达强弱以及转移是总生存期的独立预后因子(P<0.05)。结论:高表达的MRP3参与了胆囊癌的发生发展,MRP3与胆囊癌的分化、转移有关,可作为判断预后的指标。
文摘目的:研究黄芪甲苷IV(AS-IV)对人肝癌细胞HepG2中多药耐药相关蛋白-3(MRP3)的调节作用,为AS-IV可能引发的药物相互作用阐明机制。方法:培养HepG2细胞,用不同浓度AS-IV进行干预,Western blotting方法检测细胞中MRP3的蛋白水平,流式细胞术检测胞内5-羧基荧光素(5-CF)的荧光强度,反映MRP的功能;给予MRP3 si RNA干扰,观察AS-IV诱导的MRP3蛋白表达是否增加其外排转运功能;提取胞核、总蛋白,检测胞核核因子E2相关因子2(Nrf2)、总Nrf2蛋白水平;给予Nrf2 si RNA干扰,观察AS-IV诱导的MRP3蛋白表达是否依赖于Nrf2。结果:200μmol/L AS-IV作用48 h后,细胞MRP3的蛋白水平显著增加(P<0.05),胞内5-CF的平均荧光强度(MFI)明显低于对照组(P<0.05),与单独给予AS-IV组相比,给予MRP3 si RNA干扰显著增加了胞内5-CF的MFI(P<0.05)。AS-IV增加了胞核内Nrf2的蛋白水平(P<0.01),亦增加了总Nrf2的蛋白表达(P<0.01)。给予Nrf2 si RNA干扰后,AS-IV诱导上调的MRP3被显著抑制(P<0.05)。结论:AS-IV可通过上调MRP3的蛋白表达,增加其外排转运功能,Nrf2的激活在AS-IV诱导MRP3蛋白表达中起着重要的作用。本研究提示,AS-IV与MRP3底物合用时,可能会产生药物-药物相互作用。
