OBJECTIVES: To study the therapeutic mechanism of <i>Tuihuangi> Mixture against cholestasis. METHODS: Forty-eight Wistar rats were randomized equally into blank group, model group, ursodeoxycholic acid group and <i>Tuihuangi> Mixture group. Except for those in the blank group, all the rats were given α‑naphthylisothiocyanate (ANIT) to establish rat models of cholestasis, followed by treatments with indicated drugs or distilled water. Serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL of the rats were determined, and hepatic expressions IL-1β, IL-18, FXR, NLRP3, ASC, Caspase-1 and GSDMD were detected using q-PCR, ELISA or Western blotting. Histopathological changes of the liver tissues were observed using HE staining. RESULTS: The rat models of cholestasis had significantly increased serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL with increased mRNA and protein expressions of IL-1β and IL-18, decreased protein and mRNA expressions of FXR, and increased protein expressions of NLRP3 and Caspase-1 and mRNA expressions of NLRP3, ASC, Caspase-1 and GSDMD in the liver tissue, showing also irregular arrangement of liver cells, proliferation of bile duct epithelial cells and inflammatory cells infiltration. Treatment of the rat models with <i>Tuihuangi> Mixture significantly decreased serum levels of ALT, AST, ALP, γ-GT, TBA and TBIL, lowered IL-1β and IL-18 and increased FXR protein and mRNA expressions, and reduced NLRP3, ASC, Caspase-1 and GSDMD proteins and NLRP3, ASC and Caspase-1 mRNA expressions in the liver tissue. <i>Tuihuangi> Mixture also significantly alleviated hepatocyte injury, bile duct epithelial cell proliferation and inflammatory cell infiltration in the liver of the rat models. CONCLUSIONS <i>Tuihuangi> Mixture can effectively improve cholestasis in rats possibly by inhibiting NLRP3 inflammatosome-mediated pyroptosis via regulating FXR.
Animals ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rats ; Receptors, Cytoplasmic and Nuclear/metabolism* ; Cholestasis/drug therapy* ; Rats, Wistar ; Inflammasomes/metabolism* ; 1-Naphthylisothiocyanate ; Drugs, Chinese Herbal/therapeutic use* ; Male ; Interleukin-18/metabolism* ; Caspase 1/metabolism* ; Interleukin-1beta/metabolism* ; Liver/metabolism*

Taste buds relay taste sensory information to the primary taste neurons but depend on those same neurons for essential components to maintain function. While denervation-induced taste bud degeneration and subsequent regeneration were discovered decades ago, the mechanisms underlying these phenomena (e.g., heterogenous cellular responses to nerve injury and the signaling pathways involved) remain poorly understood. Here, using mouse genetics, nerve injury models, pharmacologic manipulation, and taste bud organoid models, we identify a specific subpopulation of taste cells, predominantly c-Kit-expressing sweet cells, that exhibit superior resistance to nerve injury. We found the c-Kit inhibitor imatinib selectively reduced the number of residual c-Kit-expressing sweet cells at post-operation week 2, subsequently attenuating the re-emergence of other type II cells by post-operation week 4. In taste bud organoids, c-Kit-expressing cells were resistant to R-spondin withdrawal but susceptible to imatinib, while other taste cell types showed the opposite behavior. We also observed a distinct population of residual taste cells that acquired stem-like properties, generating clonal descendent cells among suprabasal keratinocytes independent of c-Kit signaling. Together, our findings reveal that c-Kit signaling confers resilience on c-Kit-expressing sweet cells and supports the broader reconstruction of taste buds during the later regenerative stage following nerve injury.
Animals ; Taste Buds/metabolism* ; Proto-Oncogene Proteins c-kit/metabolism* ; Mice ; Signal Transduction ; Imatinib Mesylate/pharmacology* ; Mice, Inbred C57BL

BACKGROUND: Increasing evidence indicates that oxidative stress and interferon γ (IFNγ)-driven cellular immune responses are responsible for the pathogenesis of vitiligo. However, the connection between oxidative stress and the local production of IFNγ in early vitiligo remains unexplored. The aim of this study was to identify the mechanism underlying the production of IFNγ by mast cells and its impact on vitiligo pathogenesis. METHODS: Skin specimens from the central, marginal, and perilesional skin areas of active vitiligo lesions were collected to characterize changes of mast cells, CD8 + T cells, and IFNγ-producing cells. Cell supernatants from hydrogen peroxide (H 2 O 2 )-treated keratinocytes (KCs) were harvested to measure levels of soluble stem cell factor (sSCF) and matrix metalloproteinase (MMP)-9. A murine vitiligo model was established using Mas-related G protein-coupled receptor-B2 (MrgB2, mouse ortholog of human MrgX2) conditional knockout (MrgB2 -/- ) mice to investigate IFNγ production and inflammatory cell infiltrations in tail skin following the challenge with tyrosinase-related protein (Tyrp)-2 180 peptide. Potential interactions between the Tyrp-2 180 peptide and MrgX2 were predicted using molecular docking. The siRNAs targeting MrgX2 and the calcineurin inhibitor FK506 were also used to examine the signaling pathways involved in mast cell activation. RESULTS: IFNγ-producing mast cells were closely aligned with the recruitment of CD8 + T cells in the early phase of vitiligo skin. sSCF released by KCs through stress-enhanced MMP9-dependent proteolytic cleavage recruited mast cells into sites of inflamed skin (Perilesion vs . lesion, 13.00 ± 4.00/high-power fields [HPF] vs . 26.60 ± 5.72/HPF, P <0.05). Moreover, IFNγ-producing mast cells were also observed in mouse tail skin following challenge with Tyrp-2 180 (0 h vs . 48 h post-recall, 0/HPF vs . 3.80 ± 1.92/HPF, P <0.05). The IFNγ + mast cell and CD8 + T cell counts were lower in the skin of MrgB2 -/- mice than in those of wild-type mice (WT vs . KO 48 h post-recall, 4.20 ± 0.84/HPF vs . 0.80 ± 0.84/HPF, P <0.05). CONCLUSION Mast cells activated by MrgX2 serve as a local IFNγ producer that bridges between innate and adaptive immune responses against MCs in early vitiligo. Targeting MrgX2-mediated mast cell activation may represent a new strategy for treating vitiligo.
Vitiligo/metabolism* ; Mast Cells/immunology* ; Animals ; Interferon-gamma/metabolism* ; Mice ; Humans ; Melanocytes/metabolism* ; Receptors, G-Protein-Coupled/genetics* ; Mice, Knockout ; Mice, Inbred C57BL ; Male ; Female ; Matrix Metalloproteinase 9/metabolism* ; Stem Cell Factor/metabolism*

Objective To explore the effect of Evodiamine (Evo) on the immune function of allergic rhinitis (AR) rats and the regulatory mechanism on C-C motif chemokine ligand 2 (CCL2)/ C-C motif chemokine receptor 2 (CCR2) pathway. Methods The related targets of Evo-AR-immune function were screened by network pharmacology, and the protein interaction network diagram of intersecting targets was constructed. The AR rat model was established by ovalbumin (OVA) combined with aluminium hydroxide, and the rats were divided into six groups: a normal control (NC) group, a model group, a Loratadine (LOR) group, an Evodiamine low dose (Evo-L) group, a Evodiamine high dose (Evo-H) groups, and an Evo-H combined with CCL2 group. After the last administration, the symptoms of rats in each group were scored; ELISA was applied to detect the levels of histamine, immunoglobulin E (IgE), interleukin 4 (IL-4), IL-13 and interferon γ (IFN-γ); Diff-Quick staining solution was applied to detecte the number of cells in the nasal lavage fluid (NALF); hematoxylin eosin (HE) staining was applied to observe the pathological changes of nasal mucosa tissue; real-time quantitative PCR was applied to detect the levels of CCL2 and CCR2 mRNA in tissue; Western blot was applied to detect the expression levels of CCL2, CCR2 and CXC motif chemokine ligand 8 (CXCL8) proteins in nasal mucosa. Results There were eight intersection targets of EVo-AR-immune function, and protein interaction network diagram showed that CXCL8 was the core target. Compared with the NC group, the score of nasal symptoms, the levels of histamine, IgE, IL-4 and IL-13, the numbers of eosinophil, macrophages, neutrophils, lymphocytes and total cells, the mRNA and protein expression levels of CCL2 and CCR2, and the expression of CXCL8 protein in the model group were increased, while the level of IFN-γ was decreased. Compared with the model group, the score of nasal symptoms, the levels of histamine, IgE, IL-4 and IL-13, the numbers of eosinophil, macrophages, neutrophils, lymphocytes and total cells, the mRNA and protein expression levels of CCL2 and CCR2, and the expression of CXCL8 protein in LOR and Evo groups were decreased, while the level of IFN-γ was increased. Further use of CCL2 recombinant protein for compensatory experiments revealed that the improvement effect of Evo on immune function in AR rats was reversed by CCL2. Conclusion Evo can improve the immune function of AR rats, and its mechanism may be related to the inhibition of the CCL2/CCR2 pathway.
Animals ; Receptors, CCR2/immunology* ; Signal Transduction/drug effects* ; Chemokine CCL2/immunology* ; Rats ; Rhinitis, Allergic/metabolism* ; Immunoglobulin E/blood* ; Quinazolines/pharmacology* ; Male ; Interferon-gamma ; Rats, Sprague-Dawley ; Interleukin-13 ; Histamine ; Interleukin-4/immunology* ; Disease Models, Animal

Objective To explore the characteristics of the inhibitory effect of Evodiamine on the proliferation of activated T cells. Methods Mononuclear cells from peripheral blood (PBMCs) were obtained from healthy donors through density gradient centrifugation, and T cells were subsequently purified by using immunomagnetic bead separation. T cell activation was induced by employing anti-human CD3 and anti-human CD28 antibodies. T cells were treated with different concentrations of EVO (0.37, 1.11, 3.33, and 10)μmol/L. Flow cytometry was applied to evaluate the proliferation index, apoptosis rate, viability, CD25 expression levels, and cell cycle distribution of T cells. The expression levels of cytokines IL-2, IL-17A, IL-4, and IL-10 were quantified by using ELISA. Results 1.11, 3.33 and 10 μmol/L EVO effectively inhibited the proliferation of activated T cells, with an IC₅₀ of (1.5±0.3)μmol/L. EVO did not induce apoptosis in activated T cells and affect the survival rate of resting T cells. EVO did not affect the expression of CD25 and the secretion of IL-2 in activated T cells. EVO arrested the T cell cycle at the G2/M phase, resulting in an increase in G2/M phase cells, and exhibited a concentration-dependent effect. EVO did not affect the secretion of IL-4, IL-10 by activated T cells, but significantly inhibited the secretion of IL-17A. Conclusion EVO did not significantly affect the activation process of T cells but inhibited T cell proliferation by arresting the cell cycle at the G2/M phase and significantly suppressed the secretion of the pro-inflammatory cytokine IL-17A, which suggests that EVO has the potential to serve as a lead compound for the development of low-toxicity and high-efficiency immunosuppressants and elucidates the mechanisms underlying the anti-inflammatory and immunomodulatory effects of the traditional Chinese medicine Evodia rutaecarpa.
Humans ; Cell Proliferation/drug effects* ; Quinazolines/pharmacology* ; T-Lymphocytes/metabolism* ; Lymphocyte Activation/drug effects* ; Apoptosis/drug effects* ; Interleukin-4/metabolism* ; Interleukin-10/metabolism* ; Interleukin-2 Receptor alpha Subunit/metabolism* ; Interleukin-17/metabolism* ; Interleukin-2/metabolism* ; Cell Cycle/drug effects* ; Cells, Cultured

Objective To investigate the effect of ATP synthase inhibitory factor 1 (ATPIF1) on the antitumor activity of chimeric antigen receptor (CAR)-NK92 cells. Methods HER2-targeted CAR-NK92 cells with ATPIF1 overexpression or knockdown were constructed. CAR-positive expression rate was detected by flow cytometry. Cell proliferation capacity was measured using CCK-8 assay. Glycolytic capacity was analyzed by Seahorse metabolic analyzer. Mitochondrial membrane potential levels were detected using JC-1 probe. Target cell lysis rate was evaluated by firefly luciferase reporter assay. Expression levels of CD107a, natural-killer group 2 member D (NKG2D), granzyme B (GzmB), perforin, and interleukin 2 (IL-2) were detected via flow cytometry. Quantitative real-time PCR was used to measure the expression of interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), tumor necrosis factor α (TNF-α), ATPIF1, and hexokinase 1 (HK1). The impact of glycolytic inhibition by 2-Deoxy-D-glucose (2-DG) on CAR-NK92 antitumor capacity was examined. Results Successfully generated HER2-targeting control CAR-NK92 cells, as well as ATPIF1-overexpressing and ATPIF1 knockdown CAR-NK92 cells. The ATPIF1-overexpressing CAR-NK92 cells showed significantly enhanced target cell lysis rate, elevated expression levels of NKG2D and CD107a, increased secretion capacities of Granzyme B (GzmB) and IL-2, and upregulated mRNA expression levels of IFIT1 and TNF-α, while ATPIF1-knockdown cells exhibited opposite effects. ATPIF1 overexpression induced metabolic reprogramming in CAR-NK92 cells, manifested by significantly decreased mitochondrial membrane potential (δpsim), markedly upregulated HK1 mRNA expression, and enhanced basal glycolysis and glycolytic capacity. After glycolysis inhibition with 2-DG (5 μmol/L), both ATPIF1-overexpressing and knockdown CAR-NK92 cells showed no significant differences in NKG2D and CD107a expression levels compared to control cells. Conclusion ATPIF1 regulates the antitumor activity of CAR-NK92 cells through modulating glycolytic metabolism. Overexpression of ATPIF1 can enhance the antitumor efficacy of CAR-NK92 cells.
Humans ; Glycolysis ; Killer Cells, Natural/metabolism* ; Receptors, Chimeric Antigen/immunology* ; Granzymes/genetics* ; Hexokinase/metabolism* ; Cell Line, Tumor ; Interleukin-2/genetics* ; Cell Proliferation ; NK Cell Lectin-Like Receptor Subfamily K/genetics* ; Membrane Potential, Mitochondrial

OBJECTIVE: To analyze the gene mutation characteristics and survival time of patients with newly diagnosed acute myeloid leukemia (AML) based on next-generation sequencing(NGS) gene detection. METHODS: A retrospective analysis was conducted on the clinical data of 92 patients with AML (non APL) admitted to our hospital from January 2018 to May 2022. AML related genes tested were using NGS, the mutation characteristics and survival time of AML patients were analyzed. RESULTS: Among the 92 patients, 41 were males and 51 were females. A total of 38 types of gene mutations were detected. Six-two patients carried at least one gere mutation, while no gene mutations were detected in 30 patients. In the group with favourable prognosis (<i>ni> =14), the frequencies of higher gene mutations were <i>NRAS, KITi> (21.43%, <i>ni> =3), <i>KRASi> (14.29%, <i>ni> =2). In the group with intermediate prognosis (<i>ni> =64), the gene mutation frequencies from high to low were <i>DNMT3Ai> (18.75%, <i>ni> =12), <i>NPM1i> (17.19%, <i>ni> =11), <i>IDH2, FLT3-ITD, CEBPAi> (12.50%, <i>ni> =8), <i>TET2i> (10.94%, <i>ni> =7). In the poor prognosis group (<i>ni> =14), <i>ASXL1, TP53, EZH2, NRASi> had higher gene mutation frequency than others(14.29 %, <i>ni> =2 ). Statistical analysis revealed that <i>KITi> had a relative hotspot of mutations in the intermediate-risk group, and <i>DNMT3Ai> had a relative hotspot of mutations in the high-risk group (<i>Pi> < 0.05). The correlation analysis of genes with high mutation rates in different prognostic groups, such as <i>NRAS, KIT, IDH2, DNMT3A, NPM1i>, and <i>FLT3-ITDi>, with prognosis found that <i>KITi> was a factor affecting OS (<i>Pi> < 0.05), while no significant differences were observed for the others(<i>Pi> >0.05). CONCLUSION The frequency of gene mutations is high in AML patients, 67.4% of the patients carried at least one gene mutation. The mutation frequency varies among different genes in patients with different karyotypes, and there are obvious dominant mutations. <i>KITi> and <i>DNMT3Ai> can be used as factors for evaluating the prognosis of AML.
Humans ; Leukemia, Myeloid, Acute/genetics* ; Nucleophosmin ; Mutation ; Prognosis ; Retrospective Studies ; Male ; Female ; High-Throughput Nucleotide Sequencing ; Middle Aged ; DNA Methyltransferase 3A ; Adult ; Aged ; Survival Analysis ; Proto-Oncogene Proteins c-kit/genetics*

OBJECTIVE: To investigate the predictive role of the modified Endothelial Activation and Stress Index (mEASIX) in the efficacy of chimeric antigen receptor T-cell (CAR-T) therapy and cytokine release syndrome (CRS). METHODS: The clinical data of 70 relapsed and refractory (R/R) B-cell tumor patients who were treated with CAR-T therapy from September 1, 2018 to February 28, 2023 in the Department of Hematology, Affiliated Hospital of Xuzhou Medical University, were retrospectively analyzed. The value of log-2 mEASIX before conditioning (-7 d) was calculated, and the patients were divided into a low-mEASIX group (42 patients) and a high-mEASIX group (28 patients) based on the cut-off value of 5.443 determined by the receiver operating characteristic (ROC) curve. Eventually, the predictive role of mEASIX before conditioning on the efficacy of CAR-T cell therapy and CRS was analyzed. RESULTS: The high-mEASIX group exhibited significantly worse median overall survival (OS) and median progression-free survival (PFS) in comparison to the low mEASIX group (OS: 3.2 months <i>vsi> not reached, <i>Pi> < 0.01; PFS: 1.3 months <i>vsi> 6.0 months, <i>Pi> =0.009). The incidence of grade ≥2 CRS in the high-mEASIX group was substantially higher than that in the low-mEASIX group (57.1% <i>vsi> 19.0%, <i>Pi> =0.007). The degree of remission after CAR-T therapy (<i>Pi> =0.001), whether CRS occurs or not (<i>Pi> =0.041), the lactate dehydrogenase (LDH) level before conditioning (<i>Pi> =0.046), and the mEASIX score before conditioning (<i>Pi> =0.047) were independent influencing factors for the OS of patients receiving CAR-T cell therapy. CONCLUSION The mEASIX score before conditioning can predict OS and the incidence of grade ≥2 CRS in patients with relapsed and refractory B-cell tumors who receive CAR-T cell therapy.
Cytokine Release Syndrome/therapy* ; Immunotherapy, Adoptive/methods* ; Humans ; Lymphoma, B-Cell/therapy* ; Retrospective Studies ; Hematology ; China ; Receptors, Chimeric Antigen/blood* ; Predictive Value of Tests

OBJECTIVE: To screen anti-tumor drugs that improve antigen processing and presentation in acute myeloid leukemia (AML) cells. METHODS: A TCR-like or TCR mimic antibody that can specifically recognize HLA-A*0201:WT1_126-134 ( <i>RMFPNAPYLi>) complex (hereafter referred to as HLA-A2:WT1) was synthesized to evaluate the function of antigen processing and presentation machinery (APM) in AML cells. AML cell line THP1 was incubated with increasing concentrations of IFN-γ, hypomethylating agents (HMA), immunomodulatory drugs (IMiD), proteasome inhibitors (PI) and γ-secretase inhibitors (GSI), followed by measuring of HLA-ABC, HLA-A2 and HLA-A2:WT1 levels by flow cytometry at consecutive time points. RESULTS: The TCR-like antibody we generated only binds to HLA-A*0201⁺WT1⁺ cells, indicating the specificity of the antibody. HLA-A2:WT1 level of THP-1 cells detected with the TCR-like antibody was increased significantly after co-incubation with IFN-γ, showing that the HLA-A2:WT1 TCR like antibody could evaluate the function of APM. Among the anti-tumor agents screened in this study, GSI (LY-411575) and HMA (decitabine and azacitidine) could significantly increase the HLA-A2:WT1 level. The IMiD lenalidomide and pomalidomide could aslo upregulate the expression of HLA-A2:WT1 complex under certain concentrations of the drugs and incubation time. As proteasome inhibitors, carfilzomib could significantly decreased the expression of HLA-A2:WT1, while bortezomib had no significant effect on HLA-A2:WT1 expression. CONCLUSION HLA-A2:WT1 TCR-like antibody can effectively reflect the APM function. Some of the anti-tumor drugs can affect the APM function and immunogenicity of tumor cells.
Humans ; Leukemia, Myeloid, Acute/immunology* ; Antineoplastic Agents/pharmacology* ; Antigen Presentation/drug effects* ; HLA-A2 Antigen/immunology* ; Receptors, Antigen, T-Cell/immunology* ; Cell Line, Tumor ; Interferon-gamma

BACKGROUND: Depression among older adults is an important public health issue, and air and noise pollution have been found to contribute to exacerbation of depressive symptoms. This study examined the association of exposure to air and noise pollutants with clinically-newly-diagnosed depressive disorder. The mediating role of individual pro-inflammatory markers was explored. METHODS: We linked National Health Insurance claim data with 2998 healthy community-dwellers aged 55 and above who participated in the Healthy Aging Longitudinal Study between 2009 and 2013. Newly diagnosed depressive disorder was identified using diagnostic codes from the medical claim data. Pollutants were estimated using nationwide land use regression, including PM_2.5 and PM₁₀, carbon monoxide, ozone, nitrogen dioxide, sulfur dioxide, and road traffic noise. Cox proportional hazard models were employed to examine the association between pollutants and newly developed depressive disorders. The mediating effect of serum pro-inflammatory biomarkers on the relationship was examined. RESULTS: Among the 2998 participants, 209 had newly diagnosed depressive disorders. In adjusted Cox proportional hazard models, one interquartile range increase in PM_2.5 (8.53 µg/m³) was associated with a 17.5% increased hazard of developing depressive disorders. Other air pollutants and road traffic noise were not linearly associated with depressive disorder incidence. Levels of serum tumor necrosis factor receptor 1 mediated the relationship between PM_2.5 and survival time to newly onset depressive disorder. CONCLUSION PM_2.5 is related to an increased risk of newly developed depressive disorder among middle-aged and older adults, and the association is partially mediated by the pro-inflammatory marker TNF-R1.
Humans ; Particulate Matter/analysis* ; Male ; Female ; Middle Aged ; Longitudinal Studies ; Aged ; Incidence ; Air Pollutants/analysis* ; Environmental Exposure/adverse effects* ; Taiwan/epidemiology* ; Receptors, Tumor Necrosis Factor, Type I/blood* ; Proportional Hazards Models ; Biomarkers/blood* ; Depression/epidemiology* ; Aged, 80 and over ; Depressive Disorder/chemically induced* ; Risk Factors ; Air Pollution/adverse effects*