OBJECTIVE:To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤,GJDD)on alcoholic fatty live disease(AFLD)by using proteomic methods.METHODS:The male C57BL/6J mouse were randomly...OBJECTIVE:To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤,GJDD)on alcoholic fatty live disease(AFLD)by using proteomic methods.METHODS:The male C57BL/6J mouse were randomly divided into four groups:control group,model group,GJDD group and resveratrol group.After the AFLD model was successfully prepared by intragastric administration of alcohol once on the basis of the Lieber-DeCarli classical method,the GJDD group and resveratrol group were intragastrically administered with GJDD(4900 mg/kg)and resveratrol(400 mg/kg)respectively,once a day for 9 d.The fat deposition of liver tissue was observed and evaluated by oil red O(ORO)staining.4DLabel-free quantitative proteome method was used to determine and quantify the protein expression in liver tissue of each experimental group.The differentially expressed proteins were screened according to protein expression differential multiples,and then analyzed by Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway enrichment.Finally,expression validation of the differentially co-expressed proteins from control group,model group and GJDD group were verified by targeted proteomics quantification techniques.RESULTS:In semiquantitative analyses of ORO,all kinds of steatosis(ToS,MaS,and MiS)were evaluated higher in AFLD mice compared to those in GJDD or resveratroltreated mice.4DLabel-free proteomics analysis results showed that a total of 4513 proteins were identified,of which 3763 proteins were quantified and 946 differentially expressed proteins were screened.Compared with the control group,145 proteins were up-regulated and 148 proteins were down-regulated in the liver tissue of model group.In addition,compared with the model group,92 proteins were up-regulated and 135 proteins were downregulated in the liver tissue of the GJDD group.15 differentially co-expressed proteins were found between every two groups(model group vs control group,GJDD group vs model group and GJDD group vs control group),which were involved in many biological processes.Among them,11 differentially co-expressed key proteins(Aox3,H1-5,Fabp5,Ces3a,Nudt7,Serpinb1a,Fkbp11,Rpl22l1,Keg1,Acss2 and Slco1a1)were further identified by targeted proteomic quantitative technology and their expression patterns were consistent with the results of 4D label-free proteomic analysis.CONCLUSIONS:Our study provided proteomics-based evidence that GJDD alleviated AFLD by modulating liver protein expression,likely through the modulation of lipid metabolism,bile acid metabolism and with exertion of antioxidant stress.展开更多
Mass cytometry with antibodies labelled with stable metal isotopes enables both sensitive imaging and the quantification of protein expression in biological samples.Typically,these specimens are exposed to a panel of ...Mass cytometry with antibodies labelled with stable metal isotopes enables both sensitive imaging and the quantification of protein expression in biological samples.Typically,these specimens are exposed to a panel of labelled antibodies ex vivo,after sample collection.Here,we have developed a rhodium-labelled immunoconjugate of the HER2-targeted therapeutic IgG1 antibody,trastuzumab,and evaluated its in vivo biodistribution using mass cytometry techniques.A Rh^(3+)complex of a macrobicyclic sarcophagine(sar,3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane)chelator was appended with a dibromopyridazinedione(DBPD),to produce a novel disulfide bond labelling molecule,“Rh-sar-DBPD”.Rh-sar-DBPD was site-specifically conjugated to trastuzumab via its four native solvent-accessible disulfide bonds,to yield a near homogeneous,well-defined and stable pyridazinedione(PD)immunoconjugate,Rh-sar-PD-trastuzumab,in which four Rh-sar-PD groups were attached per molecule of trastuzumab.Inductively coupled plasma mass spectrometry(ICP-MS)and laser ablation inductively coupled plasma mass spectrometry(LA-ICP-MS)were then applied to measure ^(103)Rh content,as a proxy for Rh-sar-PD-trastuzumab accumulation,in in vitro and in vivo studies.ICP-MS in vitro studies indicated HER2-mediated uptake of Rh-sar-PD-trastuzumab in HER2-expressing breast cancer cells,with LA-ICP-MS images showing intercellular heterogeneity in Rh-sar-PD-trastuzumab uptake.To study the in vivo biodistribution of Rh-sar-PD-trastuzumab,female NSG mice bearing orthotopic HCC1954 breast cancer tumours were administered the immunoconjugate.Quantitative ICP-MS of ^(103)Rh signal in dissected tissues indicated receptor-specific HER2-mediated uptake in tumours,as well as accumulation in the spleen and liver.Finally,LA-ICP-MS imaging analysis of tumour and ovary tissue sections showed heterogeneous uptake in HER2-expressing HCC1954 tumour cells and follicular granulosa cells of the ovaries,which are known to express growth factor receptors.To the best of our knowledge,this is the first report in which both ICP-MS and LA-ICP-MS have been used on tissue exposed to a metal-tagged antibody in vivo,enabling quantification of the biodistribution of the novel immunoconjugate,Rh-sar-PD-trastuzumab,in a murine model of breast cancer.展开更多
There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways.By employing the SWATH-MS technique,we quantified absolute amounts of up to thousands of protei...There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways.By employing the SWATH-MS technique,we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes.Combining SWATH-MS-based network modeling and experimental validation,we found that when RIP1 level is below~1000 molecules/cell(mpc),the cell solely undergoes TRADD-dependent apoptosis.When RIP1 is above~1000 mpc,pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount,which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical obser-vations that RIP1 is required for necroptosis but its increase down-regulates necroptosis.Higher amount of RIP1(>~46,000 mpc)suppresses apoptosis,leading to necroptosis alone.The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic.Our study provides a resource for encoding the com-plexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes,enabling RIP1 to play diverse roles in governing cell fate decisions.展开更多
基金National Science Foundation-funded Project:the Study on the Changes of Energy Metabolism and Molecular Regulation Mechanism of Alcoholic Fatty Liver based on Sirtuins1-Adenosine Monophosphate-Activated Protein Kinase Signal System and the Intervention of Gehua Jiejiu dizhi decoction(No.81660752)Basic Research Project of Guizhou Provincial Science and Technology Plan:Study on the Mechanism of Sirtuins1 Mediated Deacetylation in the Regulation of Alcoholic Fatty Liver Metabolism and the Intervention of Gehua Jiejiu Dizhi Tang[QianKeHe Fundamentals-ZK[2023]General 410]。
文摘OBJECTIVE:To analyze the effect and molecular mechanism of Gehua Jiejiu Dizhi decoction(葛花解酒涤脂汤,GJDD)on alcoholic fatty live disease(AFLD)by using proteomic methods.METHODS:The male C57BL/6J mouse were randomly divided into four groups:control group,model group,GJDD group and resveratrol group.After the AFLD model was successfully prepared by intragastric administration of alcohol once on the basis of the Lieber-DeCarli classical method,the GJDD group and resveratrol group were intragastrically administered with GJDD(4900 mg/kg)and resveratrol(400 mg/kg)respectively,once a day for 9 d.The fat deposition of liver tissue was observed and evaluated by oil red O(ORO)staining.4DLabel-free quantitative proteome method was used to determine and quantify the protein expression in liver tissue of each experimental group.The differentially expressed proteins were screened according to protein expression differential multiples,and then analyzed by Gene ontology classification and Kyoto Encyclopedia of Genes and Genomes pathway enrichment.Finally,expression validation of the differentially co-expressed proteins from control group,model group and GJDD group were verified by targeted proteomics quantification techniques.RESULTS:In semiquantitative analyses of ORO,all kinds of steatosis(ToS,MaS,and MiS)were evaluated higher in AFLD mice compared to those in GJDD or resveratroltreated mice.4DLabel-free proteomics analysis results showed that a total of 4513 proteins were identified,of which 3763 proteins were quantified and 946 differentially expressed proteins were screened.Compared with the control group,145 proteins were up-regulated and 148 proteins were down-regulated in the liver tissue of model group.In addition,compared with the model group,92 proteins were up-regulated and 135 proteins were downregulated in the liver tissue of the GJDD group.15 differentially co-expressed proteins were found between every two groups(model group vs control group,GJDD group vs model group and GJDD group vs control group),which were involved in many biological processes.Among them,11 differentially co-expressed key proteins(Aox3,H1-5,Fabp5,Ces3a,Nudt7,Serpinb1a,Fkbp11,Rpl22l1,Keg1,Acss2 and Slco1a1)were further identified by targeted proteomic quantitative technology and their expression patterns were consistent with the results of 4D label-free proteomic analysis.CONCLUSIONS:Our study provided proteomics-based evidence that GJDD alleviated AFLD by modulating liver protein expression,likely through the modulation of lipid metabolism,bile acid metabolism and with exertion of antioxidant stress.
基金CRUK for a Cancer Research UK Career Establishment Award(C63178/A24959)the EPSRC(EP/S032789/1,EP/T517793/1 and EP/S022104/1)the Wellcome Trust(212885/Z/18/Z)。
文摘Mass cytometry with antibodies labelled with stable metal isotopes enables both sensitive imaging and the quantification of protein expression in biological samples.Typically,these specimens are exposed to a panel of labelled antibodies ex vivo,after sample collection.Here,we have developed a rhodium-labelled immunoconjugate of the HER2-targeted therapeutic IgG1 antibody,trastuzumab,and evaluated its in vivo biodistribution using mass cytometry techniques.A Rh^(3+)complex of a macrobicyclic sarcophagine(sar,3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane)chelator was appended with a dibromopyridazinedione(DBPD),to produce a novel disulfide bond labelling molecule,“Rh-sar-DBPD”.Rh-sar-DBPD was site-specifically conjugated to trastuzumab via its four native solvent-accessible disulfide bonds,to yield a near homogeneous,well-defined and stable pyridazinedione(PD)immunoconjugate,Rh-sar-PD-trastuzumab,in which four Rh-sar-PD groups were attached per molecule of trastuzumab.Inductively coupled plasma mass spectrometry(ICP-MS)and laser ablation inductively coupled plasma mass spectrometry(LA-ICP-MS)were then applied to measure ^(103)Rh content,as a proxy for Rh-sar-PD-trastuzumab accumulation,in in vitro and in vivo studies.ICP-MS in vitro studies indicated HER2-mediated uptake of Rh-sar-PD-trastuzumab in HER2-expressing breast cancer cells,with LA-ICP-MS images showing intercellular heterogeneity in Rh-sar-PD-trastuzumab uptake.To study the in vivo biodistribution of Rh-sar-PD-trastuzumab,female NSG mice bearing orthotopic HCC1954 breast cancer tumours were administered the immunoconjugate.Quantitative ICP-MS of ^(103)Rh signal in dissected tissues indicated receptor-specific HER2-mediated uptake in tumours,as well as accumulation in the spleen and liver.Finally,LA-ICP-MS imaging analysis of tumour and ovary tissue sections showed heterogeneous uptake in HER2-expressing HCC1954 tumour cells and follicular granulosa cells of the ovaries,which are known to express growth factor receptors.To the best of our knowledge,this is the first report in which both ICP-MS and LA-ICP-MS have been used on tissue exposed to a metal-tagged antibody in vivo,enabling quantification of the biodistribution of the novel immunoconjugate,Rh-sar-PD-trastuzumab,in a murine model of breast cancer.
基金the National Natural Science Foundation of China(Grants No.81788101,11675134,11704318,81630042 and 31420103910)the China Postdoctoral Science Foundation(Grant No.2016M602071)the 111 Project(Grant Nos.B16029 and B12001).
文摘There remains a significant gap in our quantitative understanding of crosstalk between apoptosis and necroptosis pathways.By employing the SWATH-MS technique,we quantified absolute amounts of up to thousands of proteins in dynamic assembling/de-assembling of TNF signaling complexes.Combining SWATH-MS-based network modeling and experimental validation,we found that when RIP1 level is below~1000 molecules/cell(mpc),the cell solely undergoes TRADD-dependent apoptosis.When RIP1 is above~1000 mpc,pro-caspase-8 and RIP3 are recruited to necrosome respectively with linear and nonlinear dependence on RIP1 amount,which well explains the co-occurrence of apoptosis and necroptosis and the paradoxical obser-vations that RIP1 is required for necroptosis but its increase down-regulates necroptosis.Higher amount of RIP1(>~46,000 mpc)suppresses apoptosis,leading to necroptosis alone.The relation between RIP1 level and occurrence of necroptosis or total cell death is biphasic.Our study provides a resource for encoding the com-plexity of TNF signaling and a quantitative picture how distinct dynamic interplay among proteins function as basis sets in signaling complexes,enabling RIP1 to play diverse roles in governing cell fate decisions.
