Astragali Radix(AR)is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer(NK)cells.However,owing to the complexity of its com...Astragali Radix(AR)is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer(NK)cells.However,owing to the complexity of its composition,the specific active ingredients in AR that act on NK cells are not clear yet.Cell membrane chromatography(CMC)is mainly used to screen the active ingredients in a complex system of herbal medicines.In this study,a new comprehensive two-dimensional(2D)NK-92MI CMC/C18 column/time-of-flight mass spectrometry(TOFMS)system was established to screen for potential NK cell activators.To obtain a higher column efficiency,3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column.In total,nine components in AR were screened from this system,which could be washed out from the NK-92MI/CMC column after 10 min,and they showed good affinity for NK-92MI/CMC column.Two representative active compounds of AR,isoastragaloside Ⅰ and astragaloside IV,promoted the killing effect of NK cells on K562 cells in a dose-dependent manner.It can thus suggest that isoastragaloside Ⅰ and astragaloside Ⅳ are the main immunomodulatory components of AR.This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.展开更多
Cell membrane affinity chromatography has been widely applied in membrane protein(MP)-targeted drug screening and interaction analysis.However,in current methods,the MP sources are derived from cell lines or recombina...Cell membrane affinity chromatography has been widely applied in membrane protein(MP)-targeted drug screening and interaction analysis.However,in current methods,the MP sources are derived from cell lines or recombinant protein expression,which are time-consuming for cell culture or purification,and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase.In this study,a novel in situ synthesis membrane protein affinity chromatography(iSMAC)method was developed utilizing cell-free protein expression(CFE)and covalent immobilized affinity chromatography,which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase.The advantages of iSMAC are:1)There is no need to culture cells or prepare recombinant proteins;2)Specific and purified MPs with stable and controllable content can be obtained within 2 h;3)MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase;4)The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites.iSMAC was successfully applied to screen PDGFRβinhibitors from Salvia miltiorrhiza and Schisandra chinensis.Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h.Two new PDGFRβinhibitors,salvianolic acid B and gomisin D,were screened out with KD values of 13.44 and 7.39μmol/L,respectively.In vitro experiments confirmed that the two compounds decreasedα-SMA and collagen Ⅰ mRNA levels raised by TGF-βin HSC-T6 cells through regulating the phosphorylation of p38,AKT and ERK.In vivo,Sal B could also attenuate CCl_(4)-induced liver fibrosis by downregulating PDGFRβdownstream related protein levels.The iSMAC method can be applied to other general MPs,and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.:82073814,81973291,82122066,and 82003909)the Rising-Star Program of Shanghai Science and Technology Committee(Grant No.:19QA1411500).
文摘Astragali Radix(AR)is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer(NK)cells.However,owing to the complexity of its composition,the specific active ingredients in AR that act on NK cells are not clear yet.Cell membrane chromatography(CMC)is mainly used to screen the active ingredients in a complex system of herbal medicines.In this study,a new comprehensive two-dimensional(2D)NK-92MI CMC/C18 column/time-of-flight mass spectrometry(TOFMS)system was established to screen for potential NK cell activators.To obtain a higher column efficiency,3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column.In total,nine components in AR were screened from this system,which could be washed out from the NK-92MI/CMC column after 10 min,and they showed good affinity for NK-92MI/CMC column.Two representative active compounds of AR,isoastragaloside Ⅰ and astragaloside IV,promoted the killing effect of NK cells on K562 cells in a dose-dependent manner.It can thus suggest that isoastragaloside Ⅰ and astragaloside Ⅳ are the main immunomodulatory components of AR.This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.
基金supported by the National Natural Science Foundation of China (Grant Nos. 82073814, 81973275, 82003909, 81973291, 82122066, 81803815)Rising-Star Program of Shanghai Science and Technology Committee (19QA1411500)
文摘Cell membrane affinity chromatography has been widely applied in membrane protein(MP)-targeted drug screening and interaction analysis.However,in current methods,the MP sources are derived from cell lines or recombinant protein expression,which are time-consuming for cell culture or purification,and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase.In this study,a novel in situ synthesis membrane protein affinity chromatography(iSMAC)method was developed utilizing cell-free protein expression(CFE)and covalent immobilized affinity chromatography,which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase.The advantages of iSMAC are:1)There is no need to culture cells or prepare recombinant proteins;2)Specific and purified MPs with stable and controllable content can be obtained within 2 h;3)MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase;4)The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites.iSMAC was successfully applied to screen PDGFRβinhibitors from Salvia miltiorrhiza and Schisandra chinensis.Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h.Two new PDGFRβinhibitors,salvianolic acid B and gomisin D,were screened out with KD values of 13.44 and 7.39μmol/L,respectively.In vitro experiments confirmed that the two compounds decreasedα-SMA and collagen Ⅰ mRNA levels raised by TGF-βin HSC-T6 cells through regulating the phosphorylation of p38,AKT and ERK.In vivo,Sal B could also attenuate CCl_(4)-induced liver fibrosis by downregulating PDGFRβdownstream related protein levels.The iSMAC method can be applied to other general MPs,and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.
