BACKGROUND: Cervical squamous cell carcinoma (CESC), the most common subtype of cervical cancer, is primarily caused by the high-risk human papillomavirus (HPV) infection and genetic susceptibility. Single-cell RNA sequencing (scRNA-seq) has been widely used in CESC research to uncover the diversity of cell types and states within tumor tissues, enabling a detailed study of the tumor microenvironment (TME). This technology allows precise mapping of HPV infection in cervical tissues, providing valuable insights into the initiation and progression of HPV-mediated malignant transformation. METHODS: We performed the scRNA-seq to characterize gene expression in tumor tissues and paired adjacent para-cancerous tissues from four patients with early-stage CESC using the 10× Genomics platform. The HPV infection and its subtypes were identified using the scRNA data and viral sequence mapping, and trajectory analyses were performed using HPV+ or HPV- cells. Interactions between different types of keratinized cells and their interactions with other cell types were identified, and pathways and specificity markers were screened for proliferating keratinized cells. The Cancer Genome Atlas (TCGA) dataset was used to verify the prognostic correlation between tumor-specific PI3+S100A7+ keratinocyte infiltration and CESC, and the localization relationship between PI3+S100A7+ keratinocytes and macrophages was verified by immunofluorescence staining. RESULTS: Various types of keratinocytes and fibroblasts were the two cell types with the most significant differences in percentage between the tumor tissue samples and paired adjacent non-cancerous tissue samples in the early stages of CESC. We found that PI3+S100A7+ keratinocytes were associated with early HPV-positive CESC, and PI3+S100A7+ keratinocytes were more abundant in tumors than in adjacent normal tissues in the TCGA-CESC dataset. Analysis of clinical information revealed that the infiltration of PI3+S100A7+ keratinocytes was notably higher in tumors with poor prognosis than in those with good prognosis. Additionally, multiplex immunofluorescence analysis showed a specific increase in PI3+S100A7+ expression within tumor tissues, with PI3+S100A7+ keratinocytes and CD163+ macrophages being spatially very close to each other. In the analysis of cell-cell interactions, macrophages exhibited strong crosstalk with PI3+S100A7+ proliferating keratinocytes in HPV-positive CESC tumors, mediated by tumor necrosis factor (TNF), CCL2, CXCL8, and IL10, highlighting the dynamic and tumor-specific enhancement of macrophage-keratinocyte interactions, which are associated with poor prognosis and immune modulation. Using CIBERSORTx, we discovered that patients with high infiltration of both PI3+S100A7+ proliferating keratinocytes and macrophages had the shortest overall survival. In the analysis of cell-cell interactions, PI3+S100A7+ proliferating keratinocytes and macrophages were found to be involved in highly active pathways that promote differentiation and structure formation, including cytokine receptor interactions, the Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway, and TNF signaling pathway regulation. Further subtyping of fibroblast populations identified four subtypes. The C1 group, characterized by its predominance in tumor tissues, is a subtype enriched with cancer-associated fibroblasts (CAFs), whereas the C3 group is primarily enriched in adjacent non-cancerous tissues and consists of undifferentiated cells. Moreover, the distinct molecular and cellular differences between HPV16- and HPV66-associated tumors were demonstrated, emphasizing the unique tumor-promoting mechanisms and microenvironmental influences driven by each HPV subtype. CONCLUSIONS We discovered a heterogeneous population of keratinocytes between tumor and adjacent non-cancerous tissues caused by HPV infection and identified macrophages and specific CAFs that play a crucial role during the early stage in promoting the inflammatory response and remodeling the cancer-promoting TME. Our findings provide new insights into the transcriptional landscape of early-stage CESC to understand the mechanism of HPV-mediated malignant transformation in cervical cancer.
Humans ; Female ; Papillomavirus Infections/genetics* ; Uterine Cervical Neoplasms/genetics* ; Carcinoma, Squamous Cell/pathology* ; Keratinocytes/metabolism* ; Single-Cell Analysis/methods* ; Tumor Microenvironment/genetics*

N,N-Dimethylglycine (DMG) is a glycine derivative, and its sodium salt (DMG-Na) has been demonstrated to possess various biological activities, including immunomodulation, free radical scavenging, and antioxidation, collectively contributing to the stability of tissue and cellular functions. However, its direct effects and underlying mechanisms in wound healing remain unclear. In this study, a full-thickness excisional wound model was established on the dorsal skin of mice, and wounds were treated locally with DMG-Na. Wound healing progression was assessed by calculating wound closure rates. Histopathological analysis was conducted using hematoxylin-eosin (HE) staining, and keratinocyte proliferation, migration, and differentiation were evaluated using CCK-8 assays, scratch wound assays, and quantitative reverse transcription PCR (qRT-PCR). Inflammation-related cytokine expression in keratinocytes was analyzed via ELISA and qRT-PCR. Results revealed that DMG-Na treatment significantly accelerated wound healing in mice and improved overall wound closure quality. The wound healing rates on days 3, 6, and 9 were 49.18%, 68.87%, and 90.55%, respectively, with statistically significant differences compared to the control group ( P<0.05). DMG-Na treatment downregulated the mRNA levels of keratinocyte differentiation markers while enhancing cell proliferation and migration ( P<0.05). Furthermore, DMG-Na decreased the secretion of LPS-induced keratinocyte inflammatory cytokines, including IL-1β, IL-6, IL-8, TNF-α, and CXCL10 ( P<0.05). These findings indicate that DMG-Na regulates inflammatory responses and promotes keratinocyte proliferation and migration, thereby facilitating the healing of skin wounds.
Animals ; Wound Healing/drug effects* ; Mice ; Cell Proliferation/drug effects* ; Keratinocytes/drug effects* ; Cell Movement/drug effects* ; Cell Differentiation/drug effects* ; Glycine/pharmacology* ; Skin/injuries* ; Male

OBJECTIVE: To explore the anti-photoaging properties of salvianolic acid B (Sal B). METHODS: The optimal photoaging model of human immortalized keratinocytes (HaCaT cells) were constructed by expose to ultraviolet B (UVB) radiation. The cells were divided into control, model and different concentrations of Sal B groups. Cell viability was measured via cell counting kit-8. Subsequently, the levels of oxidative stress, including reactive oxygen species (ROS), hydroxyproline (Hyp), catalase (CAT), and glutathione peroxidase (GSH-Px) were detected using the relevant kits. Silent information regulator 1 (SIRT1) protein level was detected using Western blot. The binding pattern of Sal B and SIRT1 was determined via molecular docking. RESULTS: Sal B significantly increased the viability of UVB-irradiated HaCaT cells (P<0.05 or P<0.01). Sal B effectively scavenged the accumulation of ROS induced by UVB (P<0.05 or P<0.01). In addition, Sal B modulated oxidative stress by increasing the intracellular concentrations of Hyp and CAT and the activity of GSH-Px (P<0.05 or P<0.01). The Western blot results revealed a substantial increase in SIRT1 protein levels following Sal B administration (P<0.05). Moreover, Sal B exhibited good binding affinity toward SIRT1, with a docking energy of -7.5 kCal/mol. CONCLUSION Sal B could improve the repair of photodamaged cells by alleviating cellular oxidative stress and regulating the expression of SIRT1 protein.
Humans ; Sirtuin 1/metabolism* ; Ultraviolet Rays ; Oxidative Stress/radiation effects* ; Keratinocytes/metabolism* ; Molecular Docking Simulation ; Benzofurans/pharmacology* ; Skin Aging/radiation effects* ; Reactive Oxygen Species/metabolism* ; Cell Survival/radiation effects* ; HaCaT Cells ; Hydroxyproline/metabolism* ; Glutathione Peroxidase/metabolism* ; Catalase/metabolism* ; Depsides

Understanding microbial-host interactions in the oral cavity is essential for elucidating oral disease pathogenesis and its systemic implications. In vitro bacteria-host cell coculture models have enabled fundamental studies to characterize bacterial infection and host responses in a reductionist yet reproducible manner. However, existing in vitro coculture models fail to establish conditions that are suitable for the growth of both mammalian cells and anaerobes, thereby hindering a comprehensive understanding of their interactions. Here, we present an asymmetric gas coculture system that simulates the oral microenvironment by maintaining distinct normoxic and anaerobic conditions for gingival epithelial cells and anaerobic bacteria, respectively. Using a key oral pathobiont, Fusobacterium nucleatum, as the primary test bed, we demonstrate that the system preserves bacterial viability and supports the integrity of telomerase-immortalized gingival keratinocytes. Compared to conventional models, this system enhanced bacterial invasion, elevated intracellular bacterial loads, and elicited more robust host pro-inflammatory responses, including increased secretion of CXCL10, IL-6, and IL-8. In addition, the model enabled precise evaluation of antibiotic efficacy against intracellular pathogens. Finally, we validate the ability of the asymmetric system to support the proliferation of a more oxygen-sensitive oral pathobiont, Porphyromonas gingivalis. These results underscore the utility of this coculture platform for studying oral microbial pathogenesis and screening therapeutics, offering a physiologically relevant approach to advance oral and systemic health research.
Coculture Techniques/methods* ; Humans ; Fusobacterium nucleatum/physiology* ; Gingiva/microbiology* ; Keratinocytes/microbiology* ; Host Microbial Interactions ; Mouth/microbiology* ; Host-Pathogen Interactions ; Epithelial Cells/microbiology* ; Cells, Cultured ; Porphyromonas gingivalis

Atopic dermatitis (AD) is a prevalent inflammatory skin disorder in which patients experience recurrent eczematous lesions and intense itching. The colonization of Staphylococcus aureus (S. aureus) is correlated with the severity of the disease, but its role in AD development remains elusive. Using single-cell RNA sequencing, we uncovered that keratinocytes activate a distinct immune response characterized by induction of Il24 when exposed to methicillin-resistant S. aureus (MRSA). Further experiments using animal models showed that the administration of recombinant IL-24 protein worsened AD-like pathology. Genetic ablation of Il24 or the receptor Il20rb in keratinocytes alleviated allergic inflammation and atopic march. Mechanistically, IL-24 acted through its heterodimeric receptors on keratinocytes and augmented the production of IL-33, which in turn aggravated type 2 immunity and AD-like skin conditions. Overall, these findings establish IL-24 as a critical factor for onset and progression of AD and a compelling therapeutic target.
Dermatitis, Atopic/genetics* ; Interleukins/metabolism* ; Animals ; Methicillin-Resistant Staphylococcus aureus/immunology* ; Mice ; Keratinocytes/microbiology* ; Humans ; Interleukin-33/immunology* ; Inflammation/microbiology* ; Staphylococcal Infections/microbiology* ; Disease Models, Animal ; Hypersensitivity/microbiology* ; Mice, Inbred C57BL

OBJECTIVE: To assess the safety and topical efficacy of prim-O-glucosylcimifugin (POG) and investigate the molecular mechanisms of its therapeutic effects in atopic dermatitis (AD). METHODS: The effects of POG on human keratinocyte cell viability and its anti-inflammatory properties were evaluated using cell counting kit-8 assay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Subsequently, the impact of POG on the differentiation of cluster of differentiation (CD) 4⁺ T cell subsets, including T-helper type (Th) 1, Th2, Th17, and regulatory T (Treg), was examined through in vitro experiments. Network pharmacology analysis was used to elucidate POG's therapeutic mechanisms. Furthermore, the therapeutic potential of topically applied POG was further evaluated in a calcipotriol-induced mouse model of AD. The protein and transcript levels of inflammatory markers, including cytokines, lymphocyte-specific protein tyrosine kinase (Lck) mRNA, and LCK phosphorylation (p-LCK), were quantified using immunohistochemistry, RT-qPCR, and Western blot analysis. RESULTS: POG was able to suppress cell proliferation and downregulate the transcription of interleukin 4 (Il4) and Il13 mRNA. In vitro experiments indicated that POG significantly inhibited the differentiation of Th2 cells, whereas it exerted negligible influence on the differentiation of Th1, Th17 and Treg cells. Network pharmacology identified LCK as a key therapeutic target of POG. Moreover, the topical application of POG effectively alleviated skin lesions in the calcipotriol-induced AD mouse models without causing pathological changes in the liver, kidney or spleen tissues. POG significantly reduced the levels of Il4, Il5, Il13, and thymic stromal lymphopoietin (Tslp) mRNA in the AD mice. Concurrently, POG enhanced the expression of p-LCK protein and Lck mRNA. CONCLUSION Our research revealed that POG inhibits Th2 cell differentiation by promoting p-LCK protein expression and hence effectively alleviates AD-related skin inflammation. Please cite this article as: Zhao H, Ma X, Wang H, Ding XJ, Kuai L, Song JK, Zhang Z, Yang D, Gao CJ, Li B, Zhou M. Prim-O-glucosylcimifugin mitigates atopic dermatitis by inhibiting Th2 differentiation through LCK phosphorylation modulation. J Integr Med. 2025; 23(3): 309-319.
Dermatitis, Atopic/drug therapy* ; Animals ; Humans ; Cell Differentiation/drug effects* ; Phosphorylation/drug effects* ; Mice ; Th2 Cells/drug effects* ; Keratinocytes/drug effects* ; Disease Models, Animal ; Mice, Inbred BALB C ; Calcitriol/analogs & derivatives*

OBJECTIVES: Calcium silicate (CSO) is modified to give it photothermal antibacterial properties. Its application potential in tooth mineralization and oral antibacterial is evaluated. METHODS: Based on defect-engineering modification strategy, a series of CSO-T samples (CSO-300, CSO-400, CSO-500, CSO-600) was obtained by introducing oxygen vacancy into CSO through thermal reduction using sodium borohydride. The samples were tested using scanning electron microscopy (SEM), X-ray diffraction, X-ray photoelectron spectroscopy, ultraviolet near-infrared absorption spectroscopy, and infrared thermography. The powder samples with the best photothermal performance and the most suitable material concentration (CSO-500, 500 μg/mL) were selected for subsequent experiments. High resolution transmission electron microscopy was used to analyze the microstructure and morphology of the sample, and MTT assay and Calcein AM/PI live/dead cell staining were used to evaluate the toxicity and compatibility of the sample to human oral keratinocytes. Escherichia coli and Staphylococcus aureus were selected for photothermal antibacterial experiments to evaluate their in vitro antibacterial performance. SEM, energy dispersive spectrometer, and micro Vickers hardness tester were used to evaluate the ability of materials to induce in vitro remineralization of detached teeth. RESULTS: Oxygen vacancies changed the crystal type and lattice spacing of CaSiO₃, broadened the light-absorption range, and gave it a good photothermal conversion ability in response to near infrared. Invitro experiments showed that the modified CaSiO₃ could promote the formation of hydroxyapatite on the tooth surface, thereby promoting the remineralization of teeth and improving the teeth hardness. Moreover, it had photothermal antibacterial properties and no cytotoxicity. CONCLUSIONS Defect-modified black calcium silicate has multiple functions, such as promoting tooth remineralization and photothermal bacteriostatic. When combined with the infrared luminescent toothbrush, it can simply and effectively treat tooth enamel erosion and oral bacteriostatic diseases caused by the excessive consumption of carbonated beverages and other daily bad living habits. This combination is expected to achieve the synergic treatment effect of tooth remineralization and oral bacteriostatic through daily cleaning is expected.
Calcium Compounds/pharmacology* ; Silicates/pharmacology* ; Humans ; Staphylococcus aureus/drug effects* ; Tooth Remineralization ; Escherichia coli/drug effects* ; Anti-Bacterial Agents/pharmacology* ; Keratinocytes/drug effects* ; Microscopy, Electron, Scanning

OBJECTIVE: To determine the role of Tripterygium wilfordii multiglycoside (TGW) in the treatment of psoriatic dermatitis from a cellular immunological perspective. METHODS: Mouse models of psoriatic dermatitis were established by imiquimod (IMQ). Twelve male BALB/c mice were assigned to IMQ or IMQ₊TGW groups according to a random number table. Histopathological changes in vivo were assessed by hematoxylin and eosin staining. Ratios of immune cells and cytokines in mice, as well as PAM212 cell proliferation in vitro were assessed by flow cytometry. Pro-inflammatory cytokine expression was determined using reverse transcription quantitative polymerase chain reaction. RESULTS: TGW significantly ameliorated the severity of IMQ-induced psoriasis-like mouse skin lesions and restrained the activation of CD45⁺ cells, neutrophils and T lymphocytes (all P<0.01). Moreover, TGW significantly attenuated keratinocytes (KCs) proliferation and downregulated the mRNA levels of inflammatory cytokines including interleukin (IL)-17A, IL-23, tumor necrosis factor α, and chemokine (C-X-C motif) ligand 1 (P<0.01 or P<0.05). Furthermore, it reduced the number of γ δ T17 cells in skin lesion of mice and draining lymph nodes (P<0.01). CONCLUSIONS TGW improved psoriasis-like inflammation by inhibiting KCs proliferation, as well as the associated immune cells and cytokine expression. It inhibited IL-17 secretion from γ δ T cells, which improved the immune-inflammatory microenvironment of psoriasis.
Male ; Animals ; Mice ; Tripterygium ; Psoriasis/drug therapy* ; Keratinocytes ; Skin Diseases/metabolism* ; Cytokines/metabolism* ; Imiquimod/metabolism* ; Dermatitis/pathology* ; Disease Models, Animal ; Mice, Inbred BALB C ; Skin/metabolism*

Objective: To fabricate TiO₂ nanotube material functionalized by antimicrobial peptide LL-37, and to explore its effects on biological behaviors such as adhesion and migration of human keratinocytes (HaCaT) and its antibacterial properties. Methods: The TiO₂ nanotube array (NT) was constructed on the surface of polished titanium (PT) by anodization, and the antimicrobial peptide LL-37 was loaded on the surface of TiO₂ nanotube (LL-37/NT) by physical adsorption. Three samples were selected by simple random sampling in each group. Surface morphology, roughness, hydrophilicity and release characteristics of LL-37 of the samples were analyzed with a field emission scanning electron microscope, an atomic force microscope, a contact angle measuring device and a microplate absorbance reader. HaCaT cells were respectively cultured on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of cell was observed by field emission scanning electron microscope. The number of cell adhesion was observed by cellular immunofluorescence staining. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Wound scratch assay was used to observe the migration of HaCaT. The above experiments were used to evaluate the effect of each group on the biological behavior of HaCaT cells. To evaluate their antibacterial effects, Porphyromonas gingivalis (Pg) was respectively inoculated on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of bacteria was observed by field emission scanning electron microscope. Bacterial viability was determined by live/dead bacterial staining. Results: A uniform array of nanotubes could be seen on the surface of titanium samples in LL-37/NT group, and the top of the tube was covered with granular LL-37. Compared with PT group [the roughness was (2.30±0.18) nm, the contact angle was 71.8°±1.7°], the roughness [(20.40±3.10) and (19.10±4.11) nm] and hydrophilicity (the contact angles were 22.4°±3.1° and 25.3°±2.2°, respectively) of titanium samples increased in NT and LL-37/NT group (P<0.001). The results of in vitro release test showed that the release of antimicrobial peptide LL-37 was characterized by early sudden release (1-4 h) and long-term (1-7 d) slow release. With the immunofluorescence, more cell attachment was found on NT and LL-37/NT than that on PT at the first 0.5 and 2.0 h of culture (P<0.05). The results of CCK-8 showed that there was no significant difference in the proliferation of cells among groups at 1, 3 and 5 days after culture. Wound scratch assay showed that compared with PT and NT group, the cell moved fastest on the surface of titanium samples in LL-37/NT group at 24 h of culture [(96.4±4.9)%] (F=35.55, P<0.001). A monolayer cells could be formed and filled with the scratch in 24 h at LL-37/NT group. The results of bacterial test in vitro showed that compared with the PT group, the bacterial morphology in the NT and LL-37/NT groups was significantly wrinkled, and obvious bacterial rupture could be seen on the surface of titanium samples in LL-37/NT group. The results of bacteria staining showed that the green fluorescence intensity of titanium samples in LL-37/NT group was the lowest in all groups (F=66.54,P<0.001). Conclusions: LL-37/NT is beneficial to the adhesion and migration of HaCaT cells and has excellent antibacterial properties, this provides a new strategy for the optimal design of implant neck materials.
Humans ; Titanium/chemistry* ; Antimicrobial Peptides ; Cathelicidins ; Sincalide ; Anti-Bacterial Agents/pharmacology* ; Nanotubes/chemistry* ; Dental Materials ; Bacteria ; Keratinocytes ; Surface Properties

OBJECTIVE: To explore whether the ethanol extract of Herpetospermum caudigerum Wall (EHC), a Xizang medicinal plant traditionally used for treating liver diseases, can improve imiquimod-induced psoriasis-like skin inflammation. METHODS: Immunohistochemistry and immunofluorescence staining were used to determine the effects of topical EHC use in vivo on the skin pathology of imiquimod-induced psoriasis in mice. The protein levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-17A (IL-17A) in mouse skin samples were examined using immunohistochemical staining. In vitro, IFN-γ-induced HaCaT cells with or without EHC treatment were used to evaluate the expression of keratinocyte-derived intercellular cell adhesion molecule-1 (ICAM-1) and chemokine CXC ligand 9 (CXCL9) using Western blotting and reverse transcription-quantitative polymerase chain reaction. The protein synthesis inhibitor cycloheximide and proteasome inhibitor MG132 were utilized to validate the EHC-mediated mechanism underlying degradation of ICAM-1 and CXCL9. RESULTS: EHC improved inflammation in the imiquimod-induced psoriasis mouse model and reduced the levels of IFN-γ, TNF-α, and IL-17A in psoriatic lesions. Treatment with EHC also suppressed ICAM-1 and CXCL9 in epidermal keratinocytes. Further mechanistic studies revealed that EHC suppressed keratinocyte-derived ICAM-1 and CXCL9 by promoting ubiquitin-proteasome-mediated protein degradation rather than transcriptional repression. Seven primary compounds including ehletianol C, dehydrodiconiferyl alcohol, herpetrione, herpetin, herpetotriol, herpetetrone and herpetetrol were identified from the EHC using ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry. CONCLUSION Topical application of EHC ameliorates psoriasis-like skin symptoms and improves the inflammation at the lesion sites. Please cite this article as: Zhong Y, Zhang BW, Li JT, Zeng X, Pei JX, Zhang YM, Yang YX, Li FL, Deng Y, Zhao Q. Ethanol extract of Herpetospermum caudigerum Wall ameliorates psoriasis-like skin inflammation and promotes degradation of keratinocyte-derived ICAM-1 and CXCL9. J Integr Med. 2023; 21(6): 584-592.
Animals ; Mice ; Interleukin-17/metabolism* ; Intercellular Adhesion Molecule-1 ; Imiquimod/adverse effects* ; Tumor Necrosis Factor-alpha/metabolism* ; Ligands ; Psoriasis/chemically induced* ; Keratinocytes ; Inflammation/drug therapy* ; Chemokines/metabolism* ; Interferon-gamma/metabolism* ; Disease Models, Animal ; Mice, Inbred BALB C