OBJECTIVE: To elucidate the molecular mechanisms underlying sepsis-induced injury in mouse intestinal organoids and investigate the possible mechanisms or potential drug targets of myeloid differentiation factor 88 inhibitor [TJ-M2010-5 (TJ5)] on this condition. METHODS: Small intestinal organoids from C57BL/6 mice aged 6-8 weeks were established and characterized using immunofluorescence for cell growth and proliferation marker nuclear antigen Ki-67, goblet cell marker mucin-2 (MUC-2), epithelial cell marker E-cadherin, and Paneth cell marker lysozyme (Lyz). Small intestinal organoids after 3 days of passaging were divided into different groups: a normal control group treated with culture medium containing 0.2% dimethyl sulfoxide (DMSO) for 10 hours, a lipopolysaccharide (LPS) group treated with culture medium containing 200 mg/L LPS and 0.2% DMSO for 10 hours, and a TJ5 group pre-treated with 10 mmol/L TJ5 for 2 hours followed by treatment with culture medium containing 200 mg/L LPS for 10 hours. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to measure the expression levels of interleukin-6 (IL-6) and zonula occludens-1 (ZO-1) in the small intestinal organoids. RNA transcriptome sequencing was performed on the small intestinal organoids from each group to analyze differentially expressed genes between groups, and significant enrichment was analyzed using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). RESULTS: By the 7th day of primary culture, mature organoids had formed, and their growth rate increased after passaging. Immunofluorescence identification showed expressions of Ki-67, MUC-2, E-cadherin, and Lyz, indicating that the mouse small intestinal organoids maintained their cellular composition and functional characteristics under in vitro culture conditions. RT-qPCR results showed that compared with the normal control group, the mRNA expression of IL-6 in the small intestinal organoids of the LPS group was significantly increased (2^-ΔΔCT: 1.83±0.16 vs. 1.02±0.28, P < 0.05), while the mRNA expression of ZO-1 was significantly decreased (2^-ΔΔCT: 0.53±0.11 vs. 1.01±0.18, P < 0.05). In contrast, the mRNA expression trends of both IL-6 and ZO-1 were reversed in the TJ5 group, showing statistically significant differences as compared with the LPS group (2^-ΔΔCT: IL-6 mRNA was 1.24±0.01 vs. 1.83±0.16, ZO-1 mRNA was 1.97±0.29 vs. 0.53±0.11, both P < 0.05). RNA transcriptome sequencing showed 49 differentially expressed genes in the LPS group compared to the normal control group, with 42 upregulated and 7 downregulated. Compared to the LPS group, the TJ5 group showed 84 differentially expressed genes, with 47 upregulated and 37 downregulated. GO enrichment analysis of these differentially expressed genes showed that the significantly enriched biological processes of the differentially expressed genes between the normal control group and the LPS group included responses to LPS, responses to molecule of bacterial origin and responses to bacterium. The significantly enriched biological processes of the differentially expressed genes between the LPS group and the TJ5 group included glutathione metabolic processes, responses to stress cellular and responses to chemical stimulus. In molecular function groups, glutathione binding and oligopeptide binding were significantly enriched by the differentially expressed genes. In cellular component classifications, the enrichment of the differentially expressed genes was mainly observed in the cytoplasm, endoplasmic reticulum, and microsomes. KEGG pathway enrichment analysis indicated that the differentially expressed genes between the normal control group and LPS group were enriched in IL-17 signaling pathways, tumor necrosis factor (TNF) signaling pathways, viral protein interactions with cytokines and cytokine receptors signaling pathways, and cytokine-cytokine receptor interaction signaling pathways. In contrast, the differentially expressed genes between the LPS and TJ5 groups were mainly enriched in atherosclerosis signaling pathways, ferroptosis signaling pathways, glutathione metabolism signaling pathways, and cytochrome P450-mediated drug metabolism signaling pathways. CONCLUSIONS Mouse small intestinal organoids were successfully extracted and cultured. TJ5 may exert its protective effects by regulating gene expression and related signaling pathways (fluid shear stress and atherosclerosis, ferroptosis, glutathione metabolism, cytochrome P450 drug metabolism, etc.) in sepsis-injured mouse small intestinal organoids. These genes and signaling pathways may be key targets for treating sepsis-induced intestinal injury.
Animals ; Mice ; Sepsis/genetics* ; Organoids/drug effects* ; Mice, Inbred C57BL ; Intestine, Small/metabolism* ; Gene Expression Profiling ; Transcriptome ; Lipopolysaccharides

Pristimerin, which is one of the compounds present in Celastraceae and Hippocrateaceae, has antitumor effects. However, its mechanism of action in esophageal squamous cell carcinoma (ESCC) remains unclear. This study aims to investigate the efficacy and mechanism of pristimerin on ESCC in vitro and in vivo. The inhibitory effect of pristimerin on cell growth was assessed using trypan blue exclusion and colony formation assays. Cell apoptosis was evaluated by flow cytometry. Gene and protein expressions were analyzed through quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry. RNA sequencing (RNA-Seq) was employed to identify significantly differentially expressed genes (DEGs). Cell transfection and RNA interference assays were utilized to examine the role of key proteins in pristimerin?s effect. Xenograft models were established to evaluate the antitumor efficiency of pristimerin in vivo. Pristimerin inhibited cell growth and induced apoptosis in ESCC cells. Upregulation of Noxa was crucial for pristimerin-induced apoptosis. Pristimerin activated the Forkhead box O3a (FoxO3a) signaling pathway and triggered FoxO3a recruitment to the Noxa promoter, leading to Noxa transcription. Blocking FoxO3a reversed pristimerin-induced Noxa upregulation and cell apoptosis. Pristimerin treatment suppressed xenograft tumors in nude mice, but these effects were largely negated in Noxa-KO tumors. Furthermore, the chemosensitization effects of pristimerin in vitro and in vivo were mediated by Noxa. This study demonstrates that pristimerin exerts an antitumor effect on ESCC by inducing AKT/FoxO3a-mediated Noxa upregulation. These findings suggest that pristimerin may serve as a potent anticancer agent for ESCC treatment.
Forkhead Box Protein O3/genetics* ; Humans ; Apoptosis/drug effects* ; Esophageal Squamous Cell Carcinoma/physiopathology* ; Esophageal Neoplasms/physiopathology* ; Pentacyclic Triterpenes ; Animals ; Cell Line, Tumor ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Mice ; Signal Transduction/drug effects* ; Mice, Nude ; Cell Proliferation/drug effects* ; Triterpenes/pharmacology* ; Xenograft Model Antitumor Assays ; Mice, Inbred BALB C ; Male ; Gene Expression Regulation, Neoplastic/drug effects*

Ophiopogon japonicus, a precious medicinal plant endemic to Zhejiang Province. Its tuberous roots are rich in bioactive components such as flavonoids, possessing anti-inflammatory, antioxidant, and immunomodulatory properties. To elucidate the impact of cadmium (Cd) stress on the accumulation and biosynthetic pathway of flavonoids in O. japonicus, this study exposed O. japonicus to different concentrations of Cd stress and explored the changes through integrated transcriptomics and metabolomics analysis. The results demonstrated that Cd stress (1 mg/L and 10 mg/L) significantly increased the content of flavonoids in O. japonicus in a concentration-dependent manner. The metabolomics analysis revealed a total of 110 flavonoids including flavones, flavanols, flavonols, flavone and flavonol derivatives, flavanones, isoflavonoids, chalcones and dihydrochalcones, and anthocyanins in O. japonicus, among which flavones, flavonols, flavone and flavonol derivatives, and anthocyanins increased under Cd stress. The transcriptomics analysis identified several key flavonoid biosynthesis-associated genes with up-regulated expression under Cd stress, including 14 genes encoding 4-coumarate CoA ligase (4CL), 2 genes encoding chalcone isomerase (CHI), and 14 genes encoding phenylalanine ammonia lyase (PAL). The gene-metabolite regulatory network indicated significant positive correlations of 4CL (Cluster-21637.5012, Cluster-21637.90648, and Cluster-21637.62637) and CHI (Cluster-21637.111909 and Cluster-21637.123300) with flavonoid metabolites, suggesting that these genes promoted the synthesis of specific flavonoid metabolites, which led to the accumulation of total flavonoids under Cd stress. These findings provide theoretical support for the cultivation and utilization of medicinal plants in Cd-contaminated environments and offered new perspectives for studying plant responses to heavy metal stress.
Cadmium/toxicity* ; Flavonoids/biosynthesis* ; Metabolomics ; Ophiopogon/drug effects* ; Stress, Physiological ; Transcriptome ; Gene Expression Profiling ; Gene Expression Regulation, Plant

Soil cadmium (Cd) pollution is one of the major environmental problems globally. Ophiopogon japonicus, a multifunctional plant extensively used in traditional Chinese medicine, has demonstrated potential in environmental remediation. This study investigated the Cd accumulation pattern of O. japonicus under cadmium stress and identified the heavy metal ATPase (HMA) family members in this plant. Our results demonstrated that O. japonicus exhibited a Cd enrichment factor (EF) of 2.75, demonstrating strong potential for soil Cd pollution remediation. Nine heavy metal ATPase (HMA) members of P1B-ATPases were successfully identified from the transcriptome data of O. japonicus, with OjHMA1-OjHMA6 classified as the Zn/Co/Cd/Pb-ATPases and OjHMA7-OjHMA9 as the Cu/Ag-ATPases. The expression levels of OjHMA1, OjHMA2, OjHMA3, and OjHMA7 were significantly up-regulated under Cd stress, highlighting their crucial roles in cadmium ion absorption and transport. The topological analysis revealed that these proteins possessed characteristic transmembrane (TM) segments of the family, along with functional A, P, and N domains involved in regulating ion absorption and release. Metal ion-binding sites (M4, M5, and M6) existed on the TM segments. Based on the number of transmembrane domains and the residues at metal ion-binding sites, the plant HMA family members were categorized into three subgroups: P1B-1 ATPases, P1B-2 ATPases, and P1B-4 ATPases. Specifically, the P1B-1 ATPase subgroup included the motifs TM4(CPC), TM5(YN[X]₄P), and TM6(M[XX]SS); the P1B-2 ATPase subgroup featured the motifs TM4(CPC), TM5(K), and TM6(DKTGT); the P1B-4 ATPase subgroup contained the motifs TM4(SPC) and TM6(HE[X]GT), all of which were critical for protein functions. Molecular docking results revealed the importance of conserved sequences such as CPC/SPC, DKTGT, and HE[X]GT in metal ion coordination and stabilization. These findings provide potential molecular targets for enhancing Cd uptake and tolerance of O. japonicus by genetic engineering and lay a theoretical foundation for developing new cultivars with high Cd accumulation capacity.
Cadmium/metabolism* ; Adenosine Triphosphatases/metabolism* ; Ophiopogon/drug effects* ; Soil Pollutants/toxicity* ; Plant Proteins/metabolism* ; Stress, Physiological ; Multigene Family ; Gene Expression Regulation, Plant

Bacillus velezensis DJ1 exhibits broad-spectrum antagonistic activity against diverse phytopathogenic fungi, while its biocontrol mechanisms against Fusarium graminearum, the causal agent of maize stalk rot, remain poorly characterized. In this study, we integrated genomics and transcriptomics to elucidate the antifungal mechanisms of strain DJ1. The results demonstrated that DJ1 inhibited F. graminearum with the efficacy of 64.4%, while its polyketide crude extract achieved the control efficacy of 55% in pot experiments against this disease. Whole-genome sequencing revealed a single circular chromosome (3 929 792 bp, GC content of 47%) harboring 12 biosynthetic gene clusters for secondary metabolites, six of which encoded known antimicrobial compounds (macrolactin H, bacillaene, difficidin, surfactin, fengycin, and bacilysin). Transcriptomic analysis identified 243 differentially expressed genes (152 upregulated and 91 downregulated, P < 0.05), which were potentially associated with the antagonistic activity against F. graminearum. KEGG enrichment analysis highlighted activation (P < 0.05) of cysteine/methionine metabolism, pentose phosphate pathway, and polyketide biosynthesis pathways, indicating that DJ1 employed synergistic strategies involving antimicrobial compound synthesis, energy metabolism enhancement, and nutrient competition to suppress pathogens. This study provides a theoretical foundation for developing novel microbial resources and application technologies to combat phytopathogenic fungi.
Fusarium/drug effects* ; Bacillus/metabolism* ; Plant Diseases/prevention & control* ; Antifungal Agents/pharmacology* ; Genomics ; Zea mays/microbiology* ; Transcriptome ; Gene Expression Profiling ; Antibiosis ; Multigene Family ; Multiomics

Numerous studies on the formation and consolidation of memory have shown that memory processes are characterized by phase-dependent and dynamic regulation. Memory retrieval, as the only representation of memory content and an active form of memory processing that induces memory reconsolidation, has attracted increasing attention in recent years. Although the molecular mechanisms specific to memory retrieval-induced reconsolidation have been gradually revealed, an understanding of the time-dependent regulatory mechanisms of this process is still lacking. In this study, we applied a transcriptome analysis of memory retrieval at different time points in the recent memory stage. Differential expression analysis and Short Time-series Expression Miner (STEM) depicting temporal gene expression patterns indicated that most differential gene expression occurred at 48 h, and the STEM cluster showing the greatest transcriptional upregulation at 48 h demonstrated the most significant difference. We then screened the differentially-expressed genes associated with that met the expression patterns of those cluster-identified genes that have been reported to be involved in learning and memory processes in addition to dipeptidyl peptidase 9 (DPP9). Further quantitative polymerase chain reaction verification and pharmacological intervention suggested that DPP9 is involved in 48-h fear memory retrieval and viral vector-mediated overexpression of DPP9 countered the 48-h retrieval-induced attenuation of fear memory. Taken together, our findings suggest that temporal gene expression patterns are induced by recent memory retrieval and provide hitherto undocumented evidence of the role of DPP9 in the retrieval-induced reconsolidation of fear memory.
Animals ; Fear/physiology* ; Male ; Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics* ; Memory Consolidation/physiology* ; Time Factors ; Mental Recall/drug effects* ; Mice ; Gene Expression Profiling

OBJECTIVE: To explore the potential apoptotic mechanisms of 3 Morchella extracts (Morchella conica, Morchella esculenta and Morchella delicosa) on breast and colon cancer cell lines using apoptotic biomarkers. METHODS: Human breast cell line (MCF-7) and colon cancer cell line (SW-480) were treated with methanol and ethanol extracts of 3 Morchella species with concentration ranging from 0.0625 to 2 mg/mL. After that their effects on gene expression of apoptosis related markers (pro-apoptotic markers including Bax, caspase-3, caspase-7, and caspase-9, and the antiapoptotic marker including Bcl-2) were determined using reverse transcription polymerase chain reaction. RESULTS: All Morchella extracts reduced breast and colon cancer cells proliferation at half inhibitory concentration (IC₅₀) of 0.02 ±0.01 to 0.68 ±0.30 mg/mL. As expected, all Morchella extracts significantly increased gene expressions of Bax, caspase-3, caspase-7, and caspase-9 and downregulated the gene expression of Bcl-2 in MCF-7 and SW-480 cell lines (P<0.05). CONCLUSIONS Morchella extracts demonstrated significant anti-proliferative activity against breast and colon cancer cell lines via an apoptosis induction mechanism. Anticancer activity of Morchella extracts and activation of apoptosis in breast and colon cancer cells suggest that it may be used to develop chemotherapeutic agents against cancer in future.
Humans ; Apoptosis/genetics* ; Colonic Neoplasms/drug therapy* ; Breast Neoplasms/drug therapy* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Plant Extracts/pharmacology* ; Gene Expression Regulation, Neoplastic/drug effects* ; MCF-7 Cells ; Ascomycota/chemistry*

OBJECTIVE: To explore the key target molecules and potential mechanisms of oridonin against non-small cell lung cancer (NSCLC). METHODS: The target molecules of oridonin were retrieved from SEA, STITCH, SuperPred and TargetPred databases; target genes associated with the treatment of NSCLC were retrieved from GeneCards, DisGeNET and TTD databases. Then, the overlapping target molecules between the drug and the disease were identified. The protein-protein interaction (PPI) was constructed using the STRING database according to overlapping targets, and Cytoscape was used to screen for key targets. Molecular docking verification were performed using AutoDockTools and PyMOL software. Using the DAVID database, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted. The impact of oridonin on the proliferation and apoptosis of NSCLC cells was assessed using cell counting kit-8, cell proliferation EdU image kit, and Annexin V-FITC/PI apoptosis kit respectively. Moreover, real-time quantitative PCR and Western blot were used to verify the potential mechanisms. RESULTS: Fifty-six target molecules and 12 key target molecules of oridonin involved in NSCLC treatment were identified, including tumor protein 53 (TP53), Caspase-3, signal transducer and activator of transcription 3 (STAT3), mitogen-activated protein kinase kinase 8 (MAPK8), and mammalian target of rapamycin (mTOR). Molecular docking showed that oridonin and its key target molecules bind spontaneously. GO and KEGG enrichment analyses revealed cancer, apoptosis, phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), and other signaling pathways. In vitro experiments showed that oridonin inhibited the proliferation, induced apoptosis, downregulated the expression of Bcl-2 and Akt, and upregulated the expression of Caspase-3. CONCLUSION Oridonin can act on multiple targets and pathways to exert its inhibitory effects on NSCLC, and its mechanism may be related to upregulating the expression of Caspase-3 and downregulating the expressions of Akt and Bcl-2.
Diterpenes, Kaurane/chemistry* ; Carcinoma, Non-Small-Cell Lung/pathology* ; Humans ; Network Pharmacology ; Lung Neoplasms/pathology* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Molecular Docking Simulation ; Protein Interaction Maps/drug effects* ; Cell Line, Tumor ; Signal Transduction/drug effects* ; Gene Expression Regulation, Neoplastic/drug effects* ; Reproducibility of Results ; Gene Ontology

OBJECTIVE: To entrap carvacrol (CAR) in bovine serum albumin nanoparticles (BSANPs) to form CAR-loaded BSANPs (CAR@BSANPs) and to explore the anti-cancer effects in breast adenocarcinoma cells (MCF-7 cells) treated with CAR and CAR@BSANPs. METHODS: A desolvation method was used to synthesize BSANPs and CAR@BSANPs. The BSANPs and CAR@BSANPs were characterized by several physicochemical methods, including visual observation, high-resolution field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and high-performance liquid chromatography. MCF-7 cells were used and analyzed after 24 h of exposure to CAR and CAR@BSANPs at half-maximal inhibitory concentration. The anti-proliferative, apoptotic, reactive oxygen species (ROS), and nitric oxide (NO) scavenging activity as well as gene expression analysis were investigated by the cell viability assay, phase-contrast microscopy, 2',7'-dichlorofluorescein-diacetate assay, Griess-Illosvoy colorimetric assay, and quantitative real-time polymerase chain reaction, respectively. RESULTS: CAR and CAR@BSANPs showed anti-proliferative, apoptotic, ROS generation, and NO scavenging effects on MCF-7 cells. Expression profile of B-cell lymphoma 2-like 11 (BCL2L11), vascular endothelial growth factor A (VEGFA), hypoxia inducible factor factor-1α (HIF1A), BCL2L11/apoptosis regulator (BAX), and BCL2L11/Bcl2 homologous antagonist/killer 1 (BAK1) ratios revealed downregulated genes; and BAX, BAK1, and CASP8 were upregulated by CAR and CAR@BSANPs treatment. In vitro anticancer assays of the CAR and CAR@BSANPs showed that CAR@BSANPs demonstrated higher therapeutic efficacy in the MCF-7 cells than CAR. CONCLUSIONS CAR and CAR@BSANPs affect gene expression and may subsequently reduce the growth and proliferation of the MCF-7 cells. Molecular targeting of regulatory genes of the MCF-7 cells with CAR and CAR@BSANPs may be an effective therapeutic strategy against breast cancer.
Humans ; Cymenes ; Nanoparticles/ultrastructure* ; MCF-7 Cells ; Breast Neoplasms/genetics* ; Apoptosis/drug effects* ; Serum Albumin, Bovine/chemistry* ; Monoterpenes/therapeutic use* ; Adenocarcinoma/genetics* ; Cell Proliferation/drug effects* ; Reactive Oxygen Species/metabolism* ; Female ; Cell Survival/drug effects* ; Animals ; Gene Expression Regulation, Neoplastic/drug effects* ; Nitric Oxide/metabolism* ; Cattle

OBJECTIVE: To investigate the effect of miR-483-5p on hepatocellular carcinoma (HCC) cells proliferation and stemness, as well as the attenuating effect of Pien Tze Huang (PZH). METHODS: Differentially expressed miRNA between HepG2 cells and hepatic cancer stem-like cells (HCSCs) were identified by a miRNA microarray assay. miR-483-5p mimics were transfected into HepG2 cells to explore the effects of miR-483-5p on cell proliferation and stemness. HepG2 cells and HCSCs were treated with PZH (0, 0.25, 0.50 and 0.75 mg/mL) to explore the effects of PZH on the proliferation and stemness, both in non-induced state and the state induced by miR-483-5p mimics. RESULTS: miR-483-5p was significantly up-regulated in HCSCs and its overexpression increased cell proliferation and stemness in HepG2 cells (P<0.05). PZH not only significantly inhibited proliferation in HepG2 cells, but also significantly suppressed the cell proliferation and self-renewal of HCSCs (P<0.05). The effects of miR-483-5p mimics on proliferation and stemness of HepG2 cells were partially abolished by PZH. CONCLUSIONS miR-483-5p promotes proliferation and enhances stemness of HepG2 cells, which were attenuated by PZH, demonstrating that miR-483-5p is a potential molecular target for the treatment of HCC and provide experimental evidence to support clinical use of PZH for patients with HCC.
Humans ; MicroRNAs/metabolism* ; Cell Proliferation/drug effects* ; Liver Neoplasms/drug therapy* ; Carcinoma, Hepatocellular/drug therapy* ; Hep G2 Cells ; Neoplastic Stem Cells/metabolism* ; Drugs, Chinese Herbal/therapeutic use* ; Gene Expression Regulation, Neoplastic/drug effects*