Objective To explore the potential mechanism of Polygonum cuspidatum in alleviating silicosis using network pharmacology, molecular docking, and experimental validation. Methods i) The targets of active components of Polygonum cuspidatum and silicosis-related targets were retrieved from multiple databases. The overlapping targets were imported into Venny2.1.0 and STRING platforms. "Polygonum cuspidatum-active components-potential therapeutic targets for silicosis" network was constructed and core targets of Polygonum cuspidatum that can alleviate silicosis were identified using the Cytoscape3.10.2 software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using the DAVID database, and molecular docking was conducted with AutoDock software. ii) Human embryonic lung fibroblast MRC-5 cells were divided into three groups. Cells in the control group were cultured in high-glucose Dulbecco's modified Eagle's medium containing 10.00% (V/V)fetal bovine serum. Cells in the silica group were treated with silica suspension at a final concentration of 150 μg·cm² (equivalent to 75 mg/L) for 48 hours. Cells in the silica + β-sitosterol group were first treated with silica suspension for 24 hours, then were cultured in medium containing β-sitosterol at a mass concentration of 50 μg/L for another 24 hours. Immunofluorescence assay and Western blotting were used to detect the effects of β-sitosterol, a core component of Polygonum cuspidatum, on the expression of phosphorylated protein kinase B (p-AKT), B-cell lymphoma-2 (BCL-2), and α-smooth muscle actin (α-SMA) in cells. Results i) A total of ten main active components of Polygonum cuspidatum, 840 targets of the active components, and 231 potential therapeutic targets for silicosis were screened through retrieval. A total of 10 core therapeutic targets were further screened, including tumor protein p53, serine/threonine protein kinase B1 (AKT1), heat shock protein 90-α, interleukin (IL)-6 , tumor necrosis factor, Src tyrosine kinase, estrogen receptor 1, epidermal growth factor receptor, BCL-2, and IL-1β. KEGG enrichment analysis yielded 178 related target pathways, among which, the phosphatidylinositol 3-kinase-protein kinase B (PI3K/AKT) signaling pathway was most relevant to silicosis. Molecular docking results showed that the top five compounds of Polygonum cuspidatum with high degree centrality, including quercetin, luteolin, physovenine, rhein, and β-sitosterol, had strong binding affinities with core targets. Among them, β-sitosterol, a key component of Polygonum cuspidatum, ranked high in binding affinity to all core targets. Molecular docking visualization indicated that β-sitosterol ranked high in binding affinitiy with the core target proteins AKT1 and BCL-2. ii) Both immunofluorescence and Western blotting results showed that, the relative expression of p-AKT, BCL-2, and α-SMA proteins increased in MRC-5 cells of the silica group compared with the control group (all P<0.05), except for p-AKT detected by Western blotting. Compared with the silica group, the relative expression levels of p-AKT, BCL-2, and α-SMA proteins in the silica+β-sitosterol group decreased (all P<0.05). Conclusion β-sitosterol, the core component of Polygonum cuspidatum, may alleviate silicosis fibrosis via the PI3K/AKT signaling pathway.

Rabdosia serra(R.serra),an important component of Chinese herbal tea,has traditionally been used to treat hepatitis,jaundice,cholecystitis,and colitis.However,the chemical composition of R.serra and its effect against colitis remain unclear.In this study,the chemical composition of the water extract of R.serra was analyzed using ultra performance liquid chromatography coupled with a hybrid linear ion trap quadrupole-orbitrap mass spectrometer(UPLC-LTQ-Orbitrap-MS).A total of 46 compounds,comprising ent-kaurane diterpenoids,flavonoids,phenolic acids,and steroids,were identified in the water extract of R.serra,and the extract could significantly alleviate dextran sulfate sodium salt-induced colitis by improving colon length,upregulating anti-inflammatory factors,downregulating proinflammatory fac-tors,and restoring the balance of T helper 17/T regulatory cells.R.serra also preserved intestinal barrier function by increasing the level of tight junction proteins(zonula occludens 1 and occludin)in mouse colonic tissue.In addition,R.serra modulated the gut microbiota composition by increasing bacterial richness and diversity,increasing the abundance of beneficial bacteria(Muribaculaceae,Bacteroides,Lactobacillus,and Prevotellaceae_UCG-O01),and decreasing the abundance of pathogenic bacteria(Turi-cibacter,Eubacterium_fissicatena_group,and Eubacterium_xylanophilum_group).Gut microbiota depletion by antibiotics further confirmed that R.serra alleviated colitis in a microbiota-dependent manner.Overall,our findings provide chemical and biological evidence for the potential application of R.serra in the management of colitis.

Long-term primary culture of mammalian cells has been always difficult due to unavoidable senescence. Conventional methods for generating immortalized cell lines usually require manipulation of genome which leads to change of important biological and genetic characteristics. Recently, conditional reprogramming (CR) emerges as a novel next generation tool for long-term culture of primary epithelium cells derived from almost all origins without alteration of genetic background of primary cells. CR co-cultures primary cells with inactivated mouse 3T3-J2 fibroblasts in the presence of RHO-related protein kinase (ROCK) inhibitor Y-27632, enabling primary cells to acquire stem-like characteristics while retain their ability to fully differentiate. With only a few years' development, CR shows broad prospects in applications in varied areas including disease modeling, regenerative medicine, drug evaluation, drug discovery as well as precision medicine. This review is thus to comprehensively summarize and assess current progress in understanding mechanism of CR and its wide applications, highlighting the value of CR in both basic and translational researches and discussing the challenges faced with CR.

Objective To establish a rapid,specific and sensitive TaqMan real-time fluorescence quantitative PCR assay for detection of murine polyomavirus ( MPyV) .Methods The specific primers and TaqMan probe were designed based on genome sequence of MPyV.The primers amplified a 69 bp fragment.After optimizing the reaction system and reaction condition, the standard curve was plotted by detecting recombinant plasmid standards.The specificity, sensitivity and reproducibility of this method were evaluated.In addition, samples of lungs, spleens and feces obtained from experimentally infected mice and 86 clinical samples were used to validate the efficacy of this real-time PCR assay.Results The specificity assay showed that this assay could specifically detect MPyV and the sensitivity for MPyV was about 100 copies/well.The coefficients of variation ( CV) of both intra-assay and inter-assay were less than 1.13%.All of the samples from experimentally infected mice were positive for MPyV and 3 out of 86 clinical samples were positive by this TaqMan-PCR detection with a positive rate of 3.5%.Conclusions The real-time fluorescence quantitative TaqMan-PCR assay established in this study has high specificity, sensitivity and stability.It can be used for clinical diagnosis, routine detection and epidemiological investigation of murine polyomavirus infections.