Background: Influenza A virus (IAV) is a negative-sense RNA virus of the Orthomyxoviridae family and is the etiological agent of a highly contagious acute respiratory disease that can lead to acute lung injury. Objective: To elucidate the molecular mechanisms of IAV infection, an integrative research approach combining gene expression profiling, multinetwork analysis, and in vivo experimental validations was employed. Methods: First, a series of network-based analyses were performed, including protein-protein interaction network construction, weighted gene co-expression network analysis, and subsequent gene set enrichment analysis, to identify the major underlying mechanisms of IAV infection. Following gene expression analysis, core targets, both direct and indirect regulators, were screened. An IAV (H1N1) strain A/PR/8/34-induced acute lung injury mouse model was constructed for in vivo validations. Batch one included two groups to evaluate findings from the multi-network analysis: Mock (n = 10; 5 males and 5 females) and IAV (n = 10; 5 males and 5 females). Batch two included three groups to assess the role of toll-like receptor 3 (TLR3) in IAV infection: Mock (n = 6; 3 males and 3 females), IAV (n = 6; 3 males and 3 females), and TLR3 inhibitor (n = 6; 3 males and 3 females). Body weight was measured on days 0, 3, and 5 after infection. On day 5, lung tissues were collected to assess viral load and histopathological changes. Key targets were examined using enzyme-linked immunosorbent assay, Western blotting, and immunofluorescence staining, both in sera and lung tissues. Results: IAV infection was significantly associated with dysregulation of the immune-inflammation system, such as the LTR, nucle-otide-binding oligomerization domain-(NOD) like receptor, retinoic acid-inducible gene I-like receptor, and nuclear factor kappa-B signaling pathways. Gene set enrichment analysis further indicated that the TLR and necroptosis signaling pathways played crucial roles in the progression of IAV infection (TLR signaling pathway normalized enrichment score = 2.3941, P = 1.00 × 10 ⁻¹⁰; necroptosis normalized enrichment score = 1.9421, P = 6.21 × 10 ⁻⁷). Among the core targets, TLR3 and mixed lineage kinase domain-like protein (MLKL) may regulate gene expression at the transcriptional level (all P < 0.05). In vivo validation using an IAV (PR8) infected acute lung injury mouse model demonstrated increased viral load and lung index, alveolar structural damage, and inflammatory cell infiltration. Immunofluorescence staining exhibited large gaps in Lamin B1 staining and breaches in Emerin signals following IAV-PR8 infection. Expression levels of TLR3, p-receptor-interacting serine/threonine-protein kinase 3 (RIPK3)/RIPK3, and p-mixed lineage kinase domain-like protein (MLKL)/MLKL proteins in lung tissues, as well as proinflammatory factors and mediators in sera, were significantly elevated after IAV infection. Moreover, enhanced neutrophil infiltration (myeloperoxidase) and citrullinated histone H3 (a neutrophil extracellular trap-specific marker), both established indicators of neutrophil extracellular trap formation, were observed. Notably, treatment with a TLR3 inhibitor significantly ameliorated IAV-induced acute lung injury by regulating necroptosis-related targets. Conclusion: Our study provides network-based in vivo evidence that TLR3-receptor-interacting serine/threonine-protein kinase 3-MLKL-mediated necroptosis may underlie IAV-induced acute lung injury and could serve as a potential therapeutic target in severe influenza cases.

Objective: To develop a convenient, direct, and highly sensitive method for screening trace chemical additives in complex Chinese patent medicines, thereby addressing core technological bottlenecks in pharmaceutical analysis and quality control. Methods: A brush ionization probe device was independently designed and constructed, and an efficient detection method was established through systematic optimization of key parameters. Twenty-three Chinese patent medicine samples, representing 6 dosage forms (capsules, tablets, pills, granules, powders, and liquid preparations), were analyzed using 10 common chemical additives as target analytes. Results: All samples were successfully analyzed without complex pretreatment, and 5 chemical additives were detected in 7 Chinese patent medicines. The brush ionization probe device exhibited cost-effectiveness (~0.2 USD per probe), operational simplicity, rapid analysis (~10s per sample), high efficiency, and minimal reagent consumption (~10 μL per sample). Conclusion: This advancement is expected to provide an innovative scientific tool for improving the generality and convenience of on-site quality control, while promoting technological progress in disciplines such as pharmacology and traditional Chinese medicine.

Accurate characterization of the chemical composition of complex traditional Chinese medicine (TCM) is an essential foundation for the modern scientific interpretation of TCM principles. Mass spectrometry is the most dominant technique in current research on the material basis of TCM, offering the highest sensitivity and the richest information provision. Establishing mass spectrometry databases represents the most effective approach to facilitating the structural analysis of TCM chemical components. This paper systematically searches and reviews literature published from January 2005 to January 2025 through online databases such as China National Knowledge Infrastructure, PubMed, and Web of Science, using “mass spectrometry database” and “traditional Chinese medicine” as keywords. It reviews the current status of seven TCM chemical component mass spectrometry databases and seven natural product mass spectrometry databases. The key advancements of these mass spectrometry databases for natural products are summarized, detailing their characteristics, search methodologies, included information, and data sources. Additionally, challenges related to data quality, standardization, timely updates, database interaction, retrieval functionality, and data sharing and security are discussed in depth. Furthermore, the paper explores prospective development directions for TCM mass spectrometry databases, emphasizing the importance of open data sharing, technological innovation, and data security. Through this analysis, the paper aims to offer theoretical guidance and practical recommendations for the precise identification of TCM components, as well as for the construction and application of these databases.

Accurate characterization of the chemical composition of complex traditional Chinese medicine(TCM)is an essential foundation for the modern scientific interpretation of TCM principles.Mass spectrometry is the most dominant technique in current research on the material basis of TCM,offering the highest sensitivity and the richest information provision.Establishing mass spectrometry databases represents the most effective approach to facilitating the structural analysis of TCM chemical components.This paper systematically searches and reviews literature published from January 2005 to January 2025 through online databases such as China National Knowledge Infrastructure,PubMed,and Web of Science,using"mass spectrometry database"and"traditional Chinese medicine"as keywords.It reviews the current status of seven TCM chemical component mass spectrometry databases and seven natural product mass spectrometry databases.The key advancements of these mass spectrometry databases for natural products are summarized,detailing their characteristics,search methodologies,included information,and data sources.Additionally,challenges related to data quality,standardization,timely updates,database interaction,retrieval functionality,and data sharing and security are discussed in depth.Furthermore,the paper explores prospective development directions for TCM mass spectrometry databases,emphasizing the importance of open data sharing,technological innovation,and data security.Through this analysis,the paper aims to offer theoretical guidance and practical recommendations for the precise identification of TCM components,as well as for the construction and application of these databases.

ObjectiveThe U6 promoter is an essential element for driving sgRNA expression in the clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9（CRISPR/Cas9）gene editing system in dicotyledonous plants. Endogenous U6 promoters typically exhibit higher transcriptional activity， which can significantly improve gene editing efficiency. This study aims to identify endogenous U6 promoters in Artemisia annua to optimize its CRISPR/Cas9 gene editing system， which holds significant importance for its molecular breeding. MethodsOn the basis of the highly conserved U6 snRNA sequences in Arabidopsis thaliana， endogenous U6 promoters were screened in the A. annua genome. Expression vectors were constructed with candidate AaU6 promoter driving the firefly luciferase （LUC） reporter gene， and then transiently transformed into Nicotiana benthamiana. Transcriptional activities of the promoters were measured and compared by in vivo imaging and the Dual Luciferase Reporter assay. ResultsEight endogenous U6 promoters were successfully cloned from A. annua. Sequences alignment revealed that all these promoters contained the two conserved cis-acting elements， upstream sequence element （USE） and TATA-box， which affected their transcriptional activity. Dual-luciferase activity assays indicated that the transcriptional activities of AaU6-3， AaU6-1， and AaU6-5 were significantly higher than that of the Arabidopsis AtU6-26 promoter， with AaU6-3 exhibiting the highest activity. ConclusionThis study identified three endogenous AaU6 promoters with high transcriptional activity in A. annua， providing key functional elements for establishing an efficient gene editing system in A. annua. These findings will contribute to advancing precision molecular breeding and high-quality germplasm innovation in A. annua.

Sophorae Flavescentis Radix is one of the commonly used traditional Chinese medicine in China, and a large amount of pharmaceutical residue generated during its processing and production is discarded as waste, which not only wastes resources but also pollutes the environment. Therefore, elucidating the chemical composition of the residue of Sophorae Flavescentis Radix and the differences between the residue and Sophorae Flavescentis Radix itself is of great significance for the comprehensive utilization of the residue. This study, based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) technology combined with multivariate statistical methods, provides a thorough characterization, identification, and differential analysis of the overall components of Sophorae Flavescentis Radix and its residue. Firstly, 61 compounds in Sophorae Flavescentis Radix were rapidly identified based on their precise molecular weight, fragment ions, and compound abundance, using a self-constructed compound database. Among them, 41 compounds were found in the residue, mainly alkaloids and flavonoids. Secondly, through principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA), 15 key compounds differentiating Sophorae Flavescentis Radix from its residue were identified. These included highly polar alkaloids, such as oxymatrine and oxysophocarpine, which showed significantly reduced content in the residue, and less polar flavonoids, such as kurarinone and kuraridin, which were more abundant in the residue. In summary, this paper clarifies the overall composition, structure, and content differences between Sophorae Flavescentis Radix and its residue, suggesting that the residue of Sophorae Flavescentis Radix can be used as a raw material for the extraction of its high-activity components, with promising potential for development and application in cosmetics and daily care. This research provides a scientific basis for the future comprehensive utilization of Sophorae Flavescentis Radix and its residue.
Drugs, Chinese Herbal/chemistry* ; Chromatography, High Pressure Liquid/methods* ; Mass Spectrometry/methods* ; Sophora/chemistry* ; Flavonoids/chemistry* ; Alkaloids/chemistry*

Accurate characterization of the chemical composition of complex traditional Chinese medicine(TCM)is an essential foundation for the modern scientific interpretation of TCM principles.Mass spectrometry is the most dominant technique in current research on the material basis of TCM,offering the highest sensitivity and the richest information provision.Establishing mass spectrometry databases represents the most effective approach to facilitating the structural analysis of TCM chemical components.This paper systematically searches and reviews literature published from January 2005 to January 2025 through online databases such as China National Knowledge Infrastructure,PubMed,and Web of Science,using"mass spectrometry database"and"traditional Chinese medicine"as keywords.It reviews the current status of seven TCM chemical component mass spectrometry databases and seven natural product mass spectrometry databases.The key advancements of these mass spectrometry databases for natural products are summarized,detailing their characteristics,search methodologies,included information,and data sources.Additionally,challenges related to data quality,standardization,timely updates,database interaction,retrieval functionality,and data sharing and security are discussed in depth.Furthermore,the paper explores prospective development directions for TCM mass spectrometry databases,emphasizing the importance of open data sharing,technological innovation,and data security.Through this analysis,the paper aims to offer theoretical guidance and practical recommendations for the precise identification of TCM components,as well as for the construction and application of these databases.

OBJECTIVE: In Pakistan, traditional medicines are an important component of the medical system, with numerous varieties and great demands. However, due to the scattered resources and the lack of systematic collection and collation, adulteration of traditional Pakistani medicine (TPM) is common, which severely affects the safety of their medicinal use and the import and export trades. Therefore, it is urgent to systematically organize and unify the management of TPM and establish a set of standards and operable methods for the identification of TPM. METHODS: We collected and organized the information on 128 TPMs with regard to their medicinal parts, efficacy, usage, and genetic material, based on Pakistan Hamdard Pharmacopoeia of Eastern Medicine: Pharmaceutical Codex. The genetic information of TPM is summarized from national center for biotechnology information (NCBI) and global pharmacopoeia genome database (GPGD). Furthermore, we utilized bioinformatics technology to supplement the chloroplast genome (cp-genome) data of 12 TPMs. To build the web server, we used the Linux + Apache + MySQL + PHP (LAMP) system and constructed the webpage on a PHP: Hypertext Preprocessor (PHP) model view controller (MVC) framework. RESULTS: We constructed a new genomic database, the traditional Pakistani medicine genomic database (TPMGD). This database comprises five entries, namely homepage, medicinal species, species identification, basic local alignment search tool (BLAST), and download. Currently, TPMGD contains basic profiles of 128 TPMs and genetic information of 102 TPMs, including 140 cytochrome c oxidase subunit I (COI) sequences and 119 mitochondrial genome sequences from Bombyx mori, 1 396 internal transcribed spacer 2 (ITS2) sequences and 1 074 intergenic region (psbA-trnH) sequences specific to 92 and 83 plant species, respectively. Additionally, TPMGD includes 199 cp-genome sequences of 82 TPMs. CONCLUSION TPMGD is a multifunctional database that integrates species description, functional information inquiry, genetic information storage, molecular identification of TPM, etc. The database not only provides convenience for TPM information queries but also establishes the scientific basis for the medication safety, species identification, and resource protection of TPM.

ObjectiveTo conduct a systematic comparative study on wild and cultivated Codonopsis pilosula（CP） from three aspects， including characters， microscopy， and contents of primary and secondary metabolites. MethodWild and cultivated CP samples were collected， their characters were measured using vernier caliper， tape measure and balance， the paraffin sections were stained with safranin-fixed green dyeing， and their microstructure were observed under the optical microscope. The content of alcohol-soluble extracts in wild and cultivated CP was determined according to the method for determination of extract under CP in the 2020 edition of Chinese Pharmacopoeia， the starch content was determined by anthrone colorimetry， the content of total polysaccharides was determined by kit method， Fiber analyzer was used to determine the content of fiber components， and ultra performance liquid chromatography（UPLC） was used to determine the content of monosaccharides， disaccharides and some secondary metabolites. Multivariate statistical analysis methods such as principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were employed to screen key differential components between wild and cultivated CP on the basis of variable importance in the projection（VIP） value>1 and P<0.05. ResultIn terms of morphological characteristics， the "lion's head-like" shape， longitudinal wrinkles， and circumferential wrinkles below the root cap of wild CP were more pronounced in wild CP compared to the cultivated ones. Regarding transverse sectional features， wild CP had more fissures on the outer side of the cortex and a larger duramen. Under microscopic examination， wild CP had more stone cells， a larger proportion of xylem， and the presence of cork cells arranged in rings in the xylem， while cultivated CP has a larger proportion of phloem， smaller vessel diameters， and a more loosely arranged vascular system. In terms of primary metabolites， the contents of 45% ethanol-soluble extract and total polysaccharides in cultivated CP were significantly higher than those in the wild ones（P<0.05）， the contents of lignin， hemicellulose， cellulose， fructose and glucose in wild CP were significantly higher than those in the cultivated ones（P<0.05）， while sucrose content in the cultivated CP was significantly higher than that in the wild ones（P<0.05）. Concerning secondary metabolites， the contents of tryptophan and tangshenoside Ⅰ in cultivated CP were significantly higher than those in the wild ones（P<0.05）， whereas the contents of lobetyolinin， lobetyol and atractylenolide Ⅲ in wild CP were significantly higher than those in the cultivated ones（P<0.05）. ConclusionThere are significant differences between wild and cultivated CP in terms of morphological characteristics， microscopic features and chemical composition. Glucose， fructose， sucrose， tangshenoside Ⅰ， tryptophan and cellulose components are the key differential components between wild and cultivated CP. Wild CP contains more polyacetylenes and fructose， whereas cultivated CP has higher levels of tangshenoside Ⅰ and sucrose， with noticeably lower cellulose content. These distinctions may be related to their growth conditions， growth years and cultivation techniques. Based on the results of this study， it is recommended to increase polyacetylenes and the content ratio of fructose to sucrose as an indicators to characterize different production methods of CP， in order to guide the high-quality production of CP.

ObjectiveTo compare wild and cultivated Paeoniae Radix Rubra（PRR） in three aspects， including character， microscope， determination of primary and secondary metabolites. MethodSeventeen batches of wild and nine batches of cultivated PRR were collected，their character data were measured by vernier caliper and scales， and their paraffin sections were made by safranin-fixed green dyeing for the observation of microscopic features. The content of ethanol-soluble extracts and total tannin from wild and cultivated PRR was determined by the method of general principle 2201 and 2202 in the 2020 edition of Chinese Pharmacopoeia， the content of polysaccharides was determined by phenol-sulfuric acid method. Anthrone colorimetry was used to determine the content of starch， and Van Soest method of washing fiber was used to determine the content of fiber. The contents of fructose， glucose and sucrose in wild and cultivated PRR were determined by ultra-high performance liquid chromatography evaporative light scattering detection（UPLC-ELSD）， and the secondary metabolites（gallic acid， methyl gallate， catechin， oxypaeoniflorin， albiflorin， paeoniflorin， ellagic acid， 1，3，4，6-tetragalloylglucose， galloylpaeoniflorin， 1，2，3，4，6-O-pentagalloylglucose， naringenin， benzoylpaeoniflorin and benzoylalbiflorin） were determined by UPLC. Principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were used to analyze the data of wild and cultivated PRR， the contribution of different factors to the difference was determined according to the variable importance in the projection（VIP） value>1 and P<0.05. ResultIn term of characters， wild PRR showed the traditional characteristic of Zaopi Fencha， its outer skin was loose and easy to fall off， its surface had longitudinal furrow and wrinkle， but the outer skin of cultivated PRR was not easy to fall off， and its surface was relatively smooth. The radial texture of xylem of wild PRR cross-section was more obvious， showing radial striations， vacuoles and more cracks， while the radial texture of xylem of cultivated PRR cross-section was not obvious， dense and some had cracks. Microscopically， the number of radial vessels arranged in the xylem of wild PRR was more than that of cultivated PRR， the number of calcium oxalate clusters in the phloem and xylem of wild PRR was more than that of cultivated PRR， while the number of starch grains was significantly higher in cultivated PRR. In terms of the content of primary chemical constituents， the contents of polysaccharides and starch of cultivated PRR were significantly higher than those of wild PRR（P<0.05）， while the contents of cellulose， lignin， fructose and glucose of wild PRR were significantly higher than those of cultivated PRR（P<0.05）. The results of determination of 13 secondary metabolites showed that the contents of paeoniflorin， methyl gallate， catechin and oxypaeoniflorin in wild PRR were significantly higher than those in cultivated PRR（P<0.05）， while the contents of albiflorin， gallic acid， ellagic acid， naringenin， benzoylpaeoniflorin and benzoylalbiflorin were significantly lower than those of cultivated PRR（P<0.05）. A total of 10 variables contributing to the differentiation between wild and cultivated PRR were screened， including albiflorin， cellulose， benzoylpaeoniflorin， oxypaeoniflorin， naringenin， ellagic acid， starch， lignin， paeoniflorin and total tannins. ConclusionThere are significant differences between wild and cultivated PRR in characters， microscopic characteristics， contents of primary and secondary metabolites. It is suggested that the content ratio of paeoniflorin and albiflorin， the contents of oxypaeoniflorin and cellulose can be used as indicators to characterize production methods of PRR so as to improve the quality standard of PRR. This study can provide reference for the improvement of quality standard of PRR and the guidance of high quality production of PRR.