Objective: To investigate the regulatory role of miR-6824-3p in macrophage function and its molecular mechanism in inhibiting hepatitis B virus (HBV) replication, thereby providing experimental evidence to elucidate the immune regulatory mechanisms underlying persistent HBV infection. Methods: miR-6824-3p mimic and inhibitor were transfected into human THP-1-induced macrophages. Real-time quantitative PCR (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), neutral red uptake, reactive oxygen species (ROS) production, and fluorescent latex particle phagocytosis assays were employed to evaluate the effects of miR-6824-3p on macrophage phenotype and function. Through a combination of bioinformatics analysis, dual luciferase reporter assays, western blot, and siRNA interference techniques, we identified the target gene of miR-6824-3p and examined their effects on downstream signaling pathways. qRT-PCR and western blot analyses were performed to assess the impact of miR-6824-3p-regulated macrophages on HBV DNA, pgRNA, cccDNA, and HBV-associated antigen levels in HepAD38 cells. Key effector molecules were identified through neutralization assays. Results: miR-6824-3p mimic significantly promoted the expression and secretion of proinflammatory factors, such as TNF-α and IL-1β, in macrophages (P<0.001), while concurrently reducing ROS production and phagocytosis (P<0.05). Furthermore, miR-6824-3p downregulated NRAS expression in macrophages, which was accompanied by a reduction in MAPK signalling path-way activity (p-MEK, p-ERK). Compared to the control group, the medium of macrophages with overexpressed miR-6824-3p inhibited the expression of HBV DNA, pgRNA, cccDNA, and HBV-associated antigens HBsAg, HBeAg, and HBcAg in HepAD38 cells (P<0.01). Similar results were also observed in the co-culture system of macrophages with HepAD38 cells. The addition of TNF-α neutralizing antibodies markedly attenuated the aforementioned antiviral effects (P<0.001). Conclusion: miR-6824-3p targets NRAS to affect the downstream MAPK signaling pathway, regulating the immune function of macrophages. The TNF-α induced by miR-6824-3p is one of the key molecules that suppress HBV replication. This study provides evidence for further elucidating the molecular mechanisms by which miRNAs influence HBV replication via modulating the host immune microenvironment.

Pulmonary fibrosis（PF） is a progressive and life-threatening disease with limited therapeutic options， highlighting the urgent need for innovative drug discovery strategies. To address this challenge， the authors propose the formula-originated rational intelligent screening&translation（FIRST）， a systematic framework for developing anti-fibrotic monomers derived from classical traditional Chinese medicine（TCM）. The strategy integrates three key dimensions， including tissue-oriented intelligent screening of active compounds， structural optimization based on drug-target spatial interactions and plant biosynthetic pathways， and cross-scale validation of drug. We further highlight its applications in discovering tissue-oriented novel drugs from clinically validated TCM， the development and mechanistic elucidation of anti-fibrotic therapeutics， as well as the clinical translation and secondary development of candidate drugs. This strategy paves the way for first-in-class， formula-derived monomeric drugs with defined structures， clarified mechanisms， and proven safety， offering a transformative avenue to meet the urgent therapeutic needs of PF and setting a new paradigm for TCM-based drug innovation.

Pulmonary fibrosis（PF） is a progressive and life-threatening disease with limited therapeutic options， highlighting the urgent need for innovative drug discovery strategies. To address this challenge， the authors propose the formula-originated rational intelligent screening&translation（FIRST）， a systematic framework for developing anti-fibrotic monomers derived from classical traditional Chinese medicine（TCM）. The strategy integrates three key dimensions， including tissue-oriented intelligent screening of active compounds， structural optimization based on drug-target spatial interactions and plant biosynthetic pathways， and cross-scale validation of drug. We further highlight its applications in discovering tissue-oriented novel drugs from clinically validated TCM， the development and mechanistic elucidation of anti-fibrotic therapeutics， as well as the clinical translation and secondary development of candidate drugs. This strategy paves the way for first-in-class， formula-derived monomeric drugs with defined structures， clarified mechanisms， and proven safety， offering a transformative avenue to meet the urgent therapeutic needs of PF and setting a new paradigm for TCM-based drug innovation.

ObjectiveTo investigate the epidemiological characteristics of respiratory adenovirus (AdV) infection among hospitalized patients under 18 years of age with pneumonia in Yancheng City of Jiangsu Province from 2023 to 2024, and to provide a basis for the prevention and control of adenovirus infection. MethodsNasopharyngeal swab samples were collected from hospitalized patients under 18 years of age diagnosed with pneumonia at two national influenza sentinel hospitals in Yancheng City from February 2023 to June 2024. A respiratory pathogen nucleic acid detection kit was used to test for 28 pathogens, including influenza A virus, influenza B virus, respiratory syncytial virus, human rhinovirus, adenovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, human metapneumovirus, human bocavirus, enterovirus, polyomavirus K1, polyomavirus WU, parainfluenza viruses 1‒4, coronavirus 229E, HKU1, OC43, and NL63, Haemophilus influenzae, Bordetella pertussis, Group A Streptococcus, Staphylococcus aureus, Streptococcus pneumoniae, Legionella pneumophila, Klebsiella pneumoniae, and the novel coronavirus, and to analyze patients’ mixed infections. Adenovirus-positive samples were subjected to polymerase chain reaction (PCR) amplification using Hexon and Fibre as target genes, and the products were analyzed by sequencing. Whole-genome sequencing analyses were performed on positive samples with Ct values less than 22. ResultsAmong 368 hospitalized pneumonia patients under 18 years of age in Yancheng City from February 2023 to July 2024, most were male (51.63%, 190/368), with preschool-aged children (42.66%, 157/368) and primary school-aged children (40.22%, 148/368) comprising the majority. A total of 44 cases tested positive for adenovirus, yielding a positivity rate of 11.96%. Single infections, dual infections, and multiple infections accounted for 61.36% (27/44), 36.36% (16/44), and 2.27% (1/44), respectively. From November 2023, the adenovirus positivity rate rose sharply, peaking at 51.61% in December. Following PCR amplification and sequencing, valid sequences were successfully obtained for 37 strains, comprising 36 AdV-3 strains and 1 AdV-C strain. Eight adenovirus-positive samples with Ct values below 22 were inoculated into HEp-2 cell lines for virus isolation, and whole-genome sequencing analyses were performed on the five successfully isolated strains. Phylogenetic analyses revealed that the AdV-3 strains collectively belonged to the same evolutionary clade. Whole-genome sequencing results indicated that four AdV-3 strains (HAdV-B3) showed no evidence of genetic recombination, whereas one AdV-C strain (HAdV-89) might have undergone genetic recombination. ConclusionThe adenovirus positivity rate among hospitalized pneumonia patients aged under 18 years of age in Yancheng from 2023 to 2024 was slightly higher than that reported in previous studies. Adenovirus infection exhibited a pattern of high incidence in winter, with the AdV-3 type being the predominant pathogen; one AdV-C strain (HAdV-89) might have undergone genetic recombination. It is recommended to strengthen surveillance of respiratory adenovirus type AdV-3 and its variants during the winter season, promote the use of multiplex pathogen detection techniques, and explore the clinical application of an AdV-3 vaccine.

Recent studies have shown that the physiological homeostasis of oral mucosal cells is regulated by the circadian clock.Dis-ruption or dysfunction of the circadian clock is closely associated with the development of oral squamous cell carcinoma(OSCC).Research based on the circadian clock offers a novel perspective on the pathogenesis and therapeutic strategies for OSCC.However,there is current-ly limited research on this topic,and people generally have insufficient understanding and recognition of the circadian clock.Given the complexity and challenges of circadian clock which is the fourth dimension of medical research,we organize relevant experts based on summarizing the current research results of circadian clock in the pathogenesis and precision diagnosis and treatment of OSCC,combining the scientific principles of the circadian clock's role and their long-term research experience,then summarizes and recommends the con-sensus opinions for the research of circadian clock in the pathogenesis mechanism and precision diagnosis and treatment of human OSCC,with the hope of providing guidance for the basic research and clinical application of circadian clock or circadian rhythm in the pathogene-sis mechanism and precision diagnosis and treatment of oral and maxillofacial squamous cell carcinoma.

Objective:To investigate the effect and underlying mechanism of isoliquiritigenin (ISL) on chondrocyte ferroptosis in a rat model of knee osteoarthritis (KOA).Methods:Sixty male SD rats were randomly divided into a sham group, KOA group, celecoxib group, ISL low-dose group, and ISL high-dose group. Except for the sham group, KOA models were induced in the other groups using the modified Hulth method. The ISL low-dose and high-dose groups received intraperitoneal injections of 10 mg/kg and 40 mg/kg ISL, respectively; the celecoxib group was orally administered 24 mg/kg celecoxib; the sham and KOA groups received equivalent doses of saline via intraperitoneal injection. Interventions were administered once daily for 8 weeks. Behavioral changes in the open field test, histopathological observations of cartilage, inflammatory cytokine detection, ferroptosis-related indicators, and Sirt1/Nrf2/GPX4 pathway protein expression were measured to determine the optimal ISL dose. Another 60 male SD rats were randomly divided into sham, KOA, ISL high-dose, Sirt1 inhibitor (EX-527), and ISL high-dose + EX-527 groups. KOA models were established in all groups except the sham group. The ISL high-dose group received 40 mg/kg ISL, the EX-527 group received 10 mg/kg EX-527, and the combination group received both. The sham and KOA groups were given saline. Interventions lasted 8 weeks. Histopathological staining evaluated cartilage damage and scoring; ELISA measured tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels in synovial fluid; iron deposition, Fe 2+, malonaldehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) levels were assessed; Western blot analyzed Sirt1/Nrf2/GPX4 pathway proteins; immunohistochemistry detected Collagen II, Aggrecan, MMP-3, and MMP-13 expression. Results:The joint cartilage tissue damage in the ISL low-dose and high-dose rat groups was alleviated compared to the KOA group. The OARSI score, levels of TNF-α, IL-1β and IL-6 in joint fluid, iron deposition in cartilage tissue, Fe 2+, MDA and ROS levels were 8.33±1.86 and 4.50±1.52, respectively. 67.24±7.25 pg/ml, 42.06±5.12 pg/ml; 37.97±4.9 pg/ml, 23.75±4.12 pg/ml; 31.67±4.16 pg/ml, 20.91±3.28 pg/ml; 2.00±0.20, 1.53±0.14; 2.84±0.19 μmol/mg, 1.87±0.16 μmol/mg; 9.11±1.08 nmol/ml, 5.49±1.05 nmol/ml; 759.15±59.80 μmol/ml and 610.85±44.23 μmol/ml were lower than those in KOA group ( P<0.05), and the serum SOD and GSH contents were 12.12±1.52 U/ml and 16.79±2.14 U/ml, respectively. Compared with KOA group, the protein expressions of Sirt1, Nrf2, GPX4 and SLC7A11 were 0.70±0.11 and 0.96±0.13, 0.69±0.10 and 0.95±0.14, 0.51±0.06 and 0.87±0.12, 0.56±0.06 and 0.83±0.10, which were higher than those in KOA group ( P<0.05). The expressions of Acetyl-H4K16, ACSL4, MMP-3 and MMP-13 were 1.68±0.17 and 1.30±0.10, 1.39±0.12 and 0.97±0.10, 1.70±0.14 and 1.10±0.10, 1.64±0.15 and 1.28±0.10, which were lower than those of KOA group ( P<0.05). And the changes of these indexes were higher in Sirt1 inhibitor group. Compared with the ISL high-dose group, the ferroptosis-related indexes were significantly increased in the ISL high-dose+Sirt1 inhibitor group. Conclusion:ISL alleviates articular cartilage injury in KOA rats, and its mechanism is related to activating the Sirt1/Nrf2/GPX4 pathway and inhibiting ferroptosis.

Objective:To investigate the effect and underlying mechanism of isoliquiritigenin (ISL) on chondrocyte ferroptosis in a rat model of knee osteoarthritis (KOA).Methods:Sixty male SD rats were randomly divided into a sham group, KOA group, celecoxib group, ISL low-dose group, and ISL high-dose group. Except for the sham group, KOA models were induced in the other groups using the modified Hulth method. The ISL low-dose and high-dose groups received intraperitoneal injections of 10 mg/kg and 40 mg/kg ISL, respectively; the celecoxib group was orally administered 24 mg/kg celecoxib; the sham and KOA groups received equivalent doses of saline via intraperitoneal injection. Interventions were administered once daily for 8 weeks. Behavioral changes in the open field test, histopathological observations of cartilage, inflammatory cytokine detection, ferroptosis-related indicators, and Sirt1/Nrf2/GPX4 pathway protein expression were measured to determine the optimal ISL dose. Another 60 male SD rats were randomly divided into sham, KOA, ISL high-dose, Sirt1 inhibitor (EX-527), and ISL high-dose + EX-527 groups. KOA models were established in all groups except the sham group. The ISL high-dose group received 40 mg/kg ISL, the EX-527 group received 10 mg/kg EX-527, and the combination group received both. The sham and KOA groups were given saline. Interventions lasted 8 weeks. Histopathological staining evaluated cartilage damage and scoring; ELISA measured tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 levels in synovial fluid; iron deposition, Fe 2+, malonaldehyde (MDA), reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) levels were assessed; Western blot analyzed Sirt1/Nrf2/GPX4 pathway proteins; immunohistochemistry detected Collagen II, Aggrecan, MMP-3, and MMP-13 expression. Results:The joint cartilage tissue damage in the ISL low-dose and high-dose rat groups was alleviated compared to the KOA group. The OARSI score, levels of TNF-α, IL-1β and IL-6 in joint fluid, iron deposition in cartilage tissue, Fe 2+, MDA and ROS levels were 8.33±1.86 and 4.50±1.52, respectively. 67.24±7.25 pg/ml, 42.06±5.12 pg/ml; 37.97±4.9 pg/ml, 23.75±4.12 pg/ml; 31.67±4.16 pg/ml, 20.91±3.28 pg/ml; 2.00±0.20, 1.53±0.14; 2.84±0.19 μmol/mg, 1.87±0.16 μmol/mg; 9.11±1.08 nmol/ml, 5.49±1.05 nmol/ml; 759.15±59.80 μmol/ml and 610.85±44.23 μmol/ml were lower than those in KOA group ( P<0.05), and the serum SOD and GSH contents were 12.12±1.52 U/ml and 16.79±2.14 U/ml, respectively. Compared with KOA group, the protein expressions of Sirt1, Nrf2, GPX4 and SLC7A11 were 0.70±0.11 and 0.96±0.13, 0.69±0.10 and 0.95±0.14, 0.51±0.06 and 0.87±0.12, 0.56±0.06 and 0.83±0.10, which were higher than those in KOA group ( P<0.05). The expressions of Acetyl-H4K16, ACSL4, MMP-3 and MMP-13 were 1.68±0.17 and 1.30±0.10, 1.39±0.12 and 0.97±0.10, 1.70±0.14 and 1.10±0.10, 1.64±0.15 and 1.28±0.10, which were lower than those of KOA group ( P<0.05). And the changes of these indexes were higher in Sirt1 inhibitor group. Compared with the ISL high-dose group, the ferroptosis-related indexes were significantly increased in the ISL high-dose+Sirt1 inhibitor group. Conclusion:ISL alleviates articular cartilage injury in KOA rats, and its mechanism is related to activating the Sirt1/Nrf2/GPX4 pathway and inhibiting ferroptosis.

OBJECTIVE: Rhei Radix et Rhizoma has five types of products, namely, raw rhubarb (RR), wine rhubarb (WR), vinegar rhubarb (VR), cooked rhubarb (CR), and rhubarb charcoal (RC). However, Rhei Radix et Rhizoma is easily contaminated with fungi and mycotoxins if not harvested or processed properly. Here, we intend to analyze how microbiome assemblies and co-occurrence patterns are influenced by sampling locations and processing methods. METHODS: High-throughput sequencing and internal transcribed spacer 2 (ITS2) were carried out to study the diversities (α- and β-diversity), composition (dominant taxa and potential biomarkers), and network complexitity of surface fungi on RR, WR, VR, CR, and RC collected from Gansu and Sichuan provinces, China. RESULTS: The phyla Ascomycota and Basidiomycota; the genera Kazachstania, Malassezia, and Asterotremella; and the species Kazachstania exigua, Asterotremella pseudolonga, and Malassezia restricta were the dominant fungi and exhibited differences in the two provinces and the five processed products. The α-diversity and network complexity were strongly dependent on processing methods. Chao 1, the Shannon index, and network complexity and connectivity were highest in the CR group. The α-diversity and network complexity were influenced by sampling locations. Chao 1 and network complexity and connectivity were highest in the Gansu Province. CONCLUSION The assembly and network of the surface microbiome on Rhei Radix et Rhizoma were shaped by processing methods and sampling locations. This paper offers a comprehensive understanding of microorganisms, which can provide early warning for potential mycotoxins and ensure the safety of drugs and consumers.

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.

OBJECTIVE: Benzylisoquinoline alkaloids (BIAs) have pharmacological functions and clinical use. BIAs are mainly distributed in plant species across the order Ranunculales and the genus Phellodendron from Sapindales. The BIA biosynthesis has been intensively investigated in Ranunculales species. However, the accumulation mechanism of BIAs in Phellodendron is largely unknown. The aim of this study is to unravel the biosynthetic pathways of BIAs in Phellodendron amurens. METHODS: The transcriptome and metabolome data from 18 different tissues of P. amurense were meticulously sequenced and subsequently subjected to a thorough analysis. Weighted gene co-expression network analysis (WGCNA), a powerful systems biology approach that facilitates the construction and subsequent analysis of co-expression networks, was utilized to identify candidate genes involved in BIAs biosynthesis. Following this, recombinant plasmids containing candidate genes were expressed in Escherichia coli, a widely used prokaryotic expression system. The purpose of this genetic engineering endeavor was to express the candidate genes within the bacteria, thereby enabling the assessment of the resultant enzyme activity. RESULTS: The synonymous substitutions per synonymous site for paralogs indicated that at least one whole genome duplication event has occurred. The potential BIA biosynthetic pathway of P. amurense was proposed, and two PR10/Bet v1 members, 14 CYP450s, and 33 methyltransferases were selected as related to BIA biosynthesis. One PR10/Bet v1 was identified as norcoclaurine synthase, which could catalyze dopamine and 4-hydroxyphenylacetaldehyde into (S)-norcoclaurine. CONCLUSION Our studies provide important insights into the biosynthesis and evolution of BIAs in non-Ranunculales species.