[Proteomics analysis of Astragalus polysaccharide on TLR4-activated lung cancer cell-derived exosomes].

Astragalus polysaccharide(APS), one of the main active components of Astragali Radix, plays an anti-tumor effect by regulating the inflammatory microenvironment of tumors. Exosomes are small extracellular vesicles with a diameter ranging from 50 to 200 nm and carry several biological components from parental cells such as nucleic acids and proteins. When combined with recipient cells, they play an important role in intercellular communication and immune response. In this study, exosomes released from H460 cells at the inflammatory state or with APS addition activated by Toll-like receptor 4(TLR4) were extracted by ultracentrifugation and characterized by Western blot, transmission electron microscopy, and nanoparticle tracking analysis. The exosomal proteins derived from H460 cells in the three groups were further analyzed by label-free proteomics, and 897, 800, and 911 proteins were identified in the three groups(Con, LPS, and APS groups), 88% of which belonged to the ExoCarta exosome protein database. Difference statistical analysis showed that the expression of 111 proteins was changed in the LPS group and the APS group(P<0.05). The biological information analysis of the differential proteins was carried out. The molecular functions, biological processes, and signaling pathways related to the differential proteins mainly involved viral processes, protein binding, and bacterial invasion of proteasome and epithelial cells. Key differential proteins mainly included plasminogen activator inhibitor-1, laminin α5, laminin α1, and CD44, indicating that tumor cells underwent systemic changes in different states and were reflected in exosomes in the inflammatory microenvironment. The analysis results also suggested that APS might affect the inflammatory microenvironment through the TLR4/MyD88/NF-κB signaling pathway or the regulation of the extracellular matrix. This study is conducive to a better understanding of the mechanism of tumor development in the inflammatory state and the exploration of the anti-inflammatory effect of APS at the exosome level.

[Novel submicron nonporous silica material modification with high carbon content and its application in reversed-phase pressurized capillary electrochromatography].

Submicron nonporous silica (NPS) materials feature small particle sizes, smooth surfaces, and regular shapes. They also exhibit excellent performance as a stationary phase; however, their use is limited by their low specific surface area and low phase ratio. Therefore, a novel surface modification strategy tailored for NPS microspheres was designed, involving a multi-step reaction. 3-Glycidyloxypropyltrimethoxysilane (GPTS) was first grafted onto NPS particles as a silane coupling agent. Polyethyleneimine (PEI), a high-molecular-weight polymer, was then coated onto the particles, providing numerous amino reaction sites. In the final step, an acylation reaction was initiated between stearoyl chloride and the amino groups to obtain the final product, designated as C18-NH2-GPTS-SiO2. Elemental analysis, FT-IR spectroscopy, Zeta potential analysis, thermogravimetric analysis (TGA), and scanning electron microscopy (SEM) were employed to investigate the success of the chemical modifications at each step. The carbon content increased from 0.55% to higher than 8.29%. Thus, it solved the low carbon loading capacity problem when modifying NPS microspheres with traditional C18 reversed phase (e. g., octadecyl chlorosilane modification). Meanwhile, the reasons for the considerable differences between NPS and porous silica (PS) microspheres in terms of the reactivity to surface modification were investigated in detail. The BET method was employed to compare the pore structures. FT-IR and 29Si solid-state NMR spectroscopy were employed to analyze the differences in the structure and quantity of silanol groups on the surfaces of the NPS and PS microspheres. Differences were observed not only in the pore size and surface area, but also in the types of silanol groups. FT-IR analysis indicated that the NPS and PS microspheres had different υSi-OH band positions, which shifted from 955 to 975 cm-1, respectively. 29Si solid-state NMR analysis further highlighted the differences in structural information for Si atom environments. Results revealed that 16% of silicon atoms in the PS microspheres had one hydroxyl group (isolated silanols, Q3, δ 100), while 19% had two hydroxyl groups (geminal silanols, Q2, δ 90). On the other hand, the NPS microspheres possessed no geminal silanols, and only 30% of the Si atoms were in the Q3 state. Therefore, the NPS microspheres had a lower density of silanol groups and lacked geminal silanol groups, compared to the PS microspheres. Geminal silanol groups have already been confirmed in previous studies to offer far higher reactivity than isolated silanols. These factors together explained the low reactivity of NPS microspheres toward surface modification. Further, the low specific surface area of the microspheres arising from their nonporous nature made it challenging to obtain a high carbon content through a simple one-step reaction. Hydrophobic substances such as hydrocarbons from the benzene series and polycyclic aromatic hydrocarbons (PAHs) were selected to study the chromatographic performance. The hydrophobic mechanism was revealed by the separation of PAHs using different ratios of acetonitrile. Separation was achieved with a C18-NH2-GPTS-SiO2 column, following which a hydrophobic phenomenon occurred. The presence of the amino coating led to the inversion of the electroosmotic flow (EOF) of the silica microspheres on the pressurized capillary electrochromatography (pCEC) platform. It also enhanced the linear velocity in the pCEC platform when the pH was selected to be low. The effects of the applied voltage on the separation ability of the 720 nm C18-NH2-GPTS-SiO2 column were examined to determine optimal conditions. Rapid and effective separation was achieved in a relatively short time. Therefore, the C18-NH2-GPTS-SiO2 stationary phase is promising for practical use with a higher phase ratio, demonstrating superiority for use in reversed-phase pCEC separation, and thus, providing a new strategy and valuable reference for the future application of submicron NPS microspheres.

[High-efficiency separation and analysis of monosaccharides in Pueraria polysaccharides by pressurized capillary electrochromatography].

Pueraria polysaccharides have been proven to possess biological activities such as bacteriostasis, anti-oxidative, anti-tumor, and immunity boosting activities. The variation in the structure, composition, and amount of monosaccharides in these polysaccharides may lead to different spatial structures and biological activities. Therefore, extraction of Pueraria polysaccharides and determination of the monosaccharide composition are of great significance for activity analysis and quality control. Direct detection of saccharides is difficult because they are strongly polar and do not show absorption in the ultraviolet region. At present, the commonly used methods for saccharide detection are liquid chromatography-ultraviolet detection, gas chromatography-ultraviolet detection, and mass spectrometry. Pressurized capillary electrochromatography is a high-efficiency microseparation technology. In this study, two kinds of Pueraria polysaccharides were extracted by an ultrasonic-assisted method, and response surface methodology was performed to explore the conditions for ultrasonic-assisted extraction of polysaccharides from Pueraria. The interaction effects of four factors, the liquid-solid ratio, ultrasonic extraction time, ultrasonic extraction temperature, and ultrasonic power, on the extraction rate of the polysaccharides were analyzed. By combining the optimal conditions predicted by the software and the actual equipment conditions, the optimal extraction conditions for Pueraria polysaccharides were chosen as follows:ultrasonic extraction temperature, 90℃; liquid-solid ratio of Pueraria thomsonii Benth, 20 mL/g; liquid-solid ratio of Pueraria lobata Ohwi, 40 mL/g; ultrasonic extraction time, 30 min; ultrasonic power, 180 W. Through data fitting, the multiple quadratic regression equation of the four factors on the extraction rate of Pueraria polysaccharides was established. A novel method based on pressurized capillary electrochromatography for the separation and analysis of eight neutral monosaccharides has been established. The monosaccharides were derivatized by the 1-phenyl-3-methyl-5-pyrazolone pre-column derivatization method. The separation conditions for these monosaccharides were explored, and the buffer concentration, buffer pH, applied voltage, type of chromatographic column, and mobile phase ratio were optimized. Finally, the established pressurized capillary electrochromatography-ultraviolet detection method was applied to the detection and identification of two kinds of actual Pueraria polysaccharide samples. The results of response surface analysis showed that among the four experimental factors, ultrasonic extraction temperature had the greatest influence on the extraction rate of polysaccharides from the two kinds of Pueraria, followed by the liquid-solid ratio; the influence of the ultrasonic extraction time and ultrasonic power was relatively weak. The experimental conditions were determined as follows:the separation of eight neutral monosaccharide derivatives could be realized within 24 min on a Halo-2.7 µm core-shell C18 capillary column with acetonitrile-50 mmol/L ammonium acetate aqueous solution (18:82, v/v, pH 4.1) as the mobile phase, by detection at 250 nm under an applied voltage of-20 kV. The separation and detection speeds and the column efficiency achieved with this method were much better than those obtained with the traditional liquid chromatography method. The results show that the proposed method has a good linear relationship and good repeatability. The separation and identification results for the actual samples showed that the polysaccharides of Pueraria thomsonii Benth were mainly composed of glucose, mannose, rhamnose, and fucose in the molar ratio 1.00:0.16:0.14:0.07. The polysaccharides of Pueraria lobata Ohwi were mainly composed of glucose and mannose in the molar ratio 1.00:0.70. This study provides a novel method for the rapid and efficient separation and detection of neutral monosaccharides, and serves as a reference for analyzing the monosaccharide composition of Pueraria polysaccharides.