Background: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has ...Background: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage.Methods: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing(scRNA-seq) was then carried out on TCS-or mock-treated mice livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor(TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the cellular and molecular events after TCS exposure. To verify the TCS-induced liver fibrosis,the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson’s trichrome and Sirius red stainings. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies.Results: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control groups profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells(HSCs)were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition,other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interactionmediated cellular communication in promoting liver fibrosis.Conclusions: TCS modulates the cellular activities and fates of several specific cell types(including hepatocytes, HSCs,endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis.Overall, we provide the first comprehensive single-cell atlas of mice livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.展开更多
Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea,cacao,and coffee,but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription...Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea,cacao,and coffee,but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear.Here,we report a novel transcription factor Camellia sinensis Senescnece 40(CsS40)as a caffeine biosynthesis regulator,which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase(TCS1)promoter.Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated TCS1 promoter in yeast-one-hybrid assays,a split-luciferase complementation assay demonstrated that CsS40 acts as a transcription factor to activate the CsTCS1 gene and EMSA assay also demonstrated that CsS40 bound to the TCS1 gene promoter.Consistently,immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells.The expression pattern of CsS40 in‘Fuding Dabai’developing leaves was opposite to that of TCS1;and knockdown and overexpression of CsS40 in tea leaf calli significantly increased and decreased TCS1 expression levels,respectively.The expression levels of CsS40 were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of‘Fuding Dabai’.Furthermore,overexpression of CsS40 reduced the accumulation of xanthine and hypoxanthine in tobacco plants,meanwhile,increased their susceptibility to aging.CsS40 expression in tea leaves was also induced by senescence-promoting hormones and environmental factors.Taken together,we showed that a novel senescence-related factor CsS40 negatively regulates TCS1 and represses caffeine accumulation in tea cultivar‘Fuding Dabai’.The study provides new insights into caffeine biosynthesis regulation by a plant-specific senescence regulator in tea plants in connection to leaf senescence and hormone signaling.展开更多
基金supported by the National Key Research and Development Program of China(2020YFA0908000 and 2022YFC2303600)the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(ZYYCXTD-C-202002)+12 种基金the National Natural Science Foundation of China(82141001,82274182,82173914,82074098,81903588 and 82003814)the Science and Technology Foundation of Shenzhen(JCYJ20210324115800001)the Science and Technology Foundation of Shenzhen(Shenzhen Clinical Medical Research Center for Geriatric Diseases)the Fundamental Research Funds for the Central Public Welfare Research Institutes(ZXKT18003)the Fundamental Research Funds for the Central public welfare research institutes(ZZ14-YQ-050)the National Key R&D Program of China Key projects for international cooperation on science,technology and innovation(2020YFE0205100)the Shenzhen Governmental Sustainable Development Fund(KCXFZ20201221173612034)the Shenzhen Governmental Sustainable Development Fund(KCXFZ20201221173612034)the Shenzhen key Laboratory of Kidney Diseases(ZDSYS201504301616234)the Shenzhen Fund for Guangdong Provincial High-level Clinical Key Specialties(SZGSP001)the Shenzhen Key Laboratory of Kidney Diseases(ZDSYS201504301616234)the Shenzhen Fund for Guangdong Provincial High-level Clinical Key Specialties(SZGSP001)partially supported by a Grant from the Sanming Project of Medicine in Shenzhen(SZSM201612034).
文摘Background: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage.Methods: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing(scRNA-seq) was then carried out on TCS-or mock-treated mice livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor(TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the cellular and molecular events after TCS exposure. To verify the TCS-induced liver fibrosis,the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson’s trichrome and Sirius red stainings. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies.Results: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control groups profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells(HSCs)were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition,other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interactionmediated cellular communication in promoting liver fibrosis.Conclusions: TCS modulates the cellular activities and fates of several specific cell types(including hepatocytes, HSCs,endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis.Overall, we provide the first comprehensive single-cell atlas of mice livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.
基金supported by the National Natural Science Foundation of China(3226180451,3226180488)Guizhou Province Outstanding Young Scientific and Technological Talent Cultivation Project(Qiankehe PlatformTalent[2019]5651)Guizhou Province Science and Technology Planning Project(Qiankehe Support[2021]General 111).
文摘Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea,cacao,and coffee,but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear.Here,we report a novel transcription factor Camellia sinensis Senescnece 40(CsS40)as a caffeine biosynthesis regulator,which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase(TCS1)promoter.Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated TCS1 promoter in yeast-one-hybrid assays,a split-luciferase complementation assay demonstrated that CsS40 acts as a transcription factor to activate the CsTCS1 gene and EMSA assay also demonstrated that CsS40 bound to the TCS1 gene promoter.Consistently,immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells.The expression pattern of CsS40 in‘Fuding Dabai’developing leaves was opposite to that of TCS1;and knockdown and overexpression of CsS40 in tea leaf calli significantly increased and decreased TCS1 expression levels,respectively.The expression levels of CsS40 were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of‘Fuding Dabai’.Furthermore,overexpression of CsS40 reduced the accumulation of xanthine and hypoxanthine in tobacco plants,meanwhile,increased their susceptibility to aging.CsS40 expression in tea leaves was also induced by senescence-promoting hormones and environmental factors.Taken together,we showed that a novel senescence-related factor CsS40 negatively regulates TCS1 and represses caffeine accumulation in tea cultivar‘Fuding Dabai’.The study provides new insights into caffeine biosynthesis regulation by a plant-specific senescence regulator in tea plants in connection to leaf senescence and hormone signaling.
