Journal: Military Medical Research

Article Title: High-salt-driven gut microbiota dysfunction aggravates prostatitis by promoting AHR/SGK1/FOXO1 axis-mediated Th17 cell differentiation

doi: 10.1186/s40779-025-00607-1

Figure Lengend Snippet: HSD promoted Th17 cell development and worsened prostate inflammation in EAP mice. a Simple schematic diagram of the implementation plan of the experiment. b HE staining and inflammation scores of prostate tissues (scale bar = 100 μm, n = 4). c Tactile allodynia development in the 4 groups of mice ( n = 4). d The levels of IL-1β, TNF-α, IL-17A, and IFN-γ in the serum of the mice in the 4 groups ( n = 4). e Proportion of Th1 and Th17 cells of CD4 + T cells detected by flow cytometry in the splenic lymphocytes of vaccinated mice from those 4 groups ( n = 4). f The infiltration of Th17 cells in prostate tissues from mice was determined by immunofluorescence (white arrowheads, scale bar = 100 μm, n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns non-significant. BF breeding feed, EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, IL-1β interleukin-1β, TNF-α tumor necrosis factor-α, IFN-γ interferon-γ, IL-17A interleukin-17A, DAPI 4',6-diamidino-2-phenylindole, Th T helper

Article Snippet: The following reagents and antibodies were used: CFA (#F5881, Sigma, USA); commercial enzyme-linked immunosorbent assay (ELISA) kits for IFN-γ (#E-EL-M0048c), IL-1β (#E-EL-M0037), IL-17A (#E-EL-M0047c), tumor necrosis factor-α (TNF-α, #E-EL-M3063), and 5-HIAA (#E-EL-0075c) (Elabscience, China); anti-mouse CD4-fluorescein isothiocyanate (FITC, #553047), CD3e-allophycocyanin (APC, #561826), IFN-γ-phycoerythrin (PE, #562020), IL-17-PE (#559502), anti-human CD8-FITC (#555634), CD3-APC (#555342), IL-17-PE (#560436) (BD, USA); anti-CD3e (#BE0001-1), anti-CD28 (#BE0015-1), anti-IFN-γ (#BE0055), anti-IL-4 (#BE0045) (Bio X Cell, USA); IL-23 (#CS31), transforming growth factor-β1 (TGF-β1, #CA59), IL-6 (#CG39) (Novoprotein, China); anti-AHR (1:1000, #NB100-128, Novus, USA); anti-SGK1 (1:1000, #AF6789), anti-FOXO1 (1:1000, #AF6416), anti-p-FOXO1 (1:1000, #AF3417), anti-retinoic acid receptor-related orphan receptor gamma-t (RORγt; 1:1000, #DF3196), and anti-GAPDH (1:5000, #AF7021) (Affinity, USA).

Techniques: Staining, Flow Cytometry, Immunofluorescence

Journal: Military Medical Research

Article Title: High-salt-driven gut microbiota dysfunction aggravates prostatitis by promoting AHR/SGK1/FOXO1 axis-mediated Th17 cell differentiation

doi: 10.1186/s40779-025-00607-1

Figure Lengend Snippet: Transplantation of fecal microbiota from EAP mice with HSD elicited more severe prostate inflammation in psuedogerm-free mice. a Simple schematic diagram of the experimental workflow. b HE staining and inflammation scores of prostate tissues (scale bar = 100 μm, n = 4). c Development of tactile allodynia in mice belonging to the 4 groups ( n = 4). d The concentrations of IL-1β, TNF-α, IL-17A, and 5-HIAA in the serum of NOD mice in the 4 groups were measured ( n = 4). e Proportion of Th17 of CD4 + T cells, as determined by flow cytometry, among the splenic lymphocytes of immunized mice in the 4 groups ( n = 4). f The infiltration of Th17 cells in prostate tissues from mice was determined by immunofluorescence (white arrowheads, scale bar = 100 μm, n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns non-significant. NOD nonobese male diabetic/LtJ, EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, FMT fecal microbiota transplantation, HE hematoxylin–eosin, IL-1β interleukin-1β, TNF-α tumor necrosis factor-α, IL-17A interleukin-17A, 5-HIAA 5-hydroxyindole acetic acid, DAPI 4',6-diamidino-2-phenylindole

Article Snippet: The following reagents and antibodies were used: CFA (#F5881, Sigma, USA); commercial enzyme-linked immunosorbent assay (ELISA) kits for IFN-γ (#E-EL-M0048c), IL-1β (#E-EL-M0037), IL-17A (#E-EL-M0047c), tumor necrosis factor-α (TNF-α, #E-EL-M3063), and 5-HIAA (#E-EL-0075c) (Elabscience, China); anti-mouse CD4-fluorescein isothiocyanate (FITC, #553047), CD3e-allophycocyanin (APC, #561826), IFN-γ-phycoerythrin (PE, #562020), IL-17-PE (#559502), anti-human CD8-FITC (#555634), CD3-APC (#555342), IL-17-PE (#560436) (BD, USA); anti-CD3e (#BE0001-1), anti-CD28 (#BE0015-1), anti-IFN-γ (#BE0055), anti-IL-4 (#BE0045) (Bio X Cell, USA); IL-23 (#CS31), transforming growth factor-β1 (TGF-β1, #CA59), IL-6 (#CG39) (Novoprotein, China); anti-AHR (1:1000, #NB100-128, Novus, USA); anti-SGK1 (1:1000, #AF6789), anti-FOXO1 (1:1000, #AF6416), anti-p-FOXO1 (1:1000, #AF3417), anti-retinoic acid receptor-related orphan receptor gamma-t (RORγt; 1:1000, #DF3196), and anti-GAPDH (1:5000, #AF7021) (Affinity, USA).

Techniques: Transplantation Assay, Staining, Flow Cytometry, Immunofluorescence

Journal: Military Medical Research

Article Title: High-salt-driven gut microbiota dysfunction aggravates prostatitis by promoting AHR/SGK1/FOXO1 axis-mediated Th17 cell differentiation

doi: 10.1186/s40779-025-00607-1

Figure Lengend Snippet: 5-HIAA supplementation ameliorated the symptoms of EAP caused by HSD. a The levels of 5-HIAA secreted into the serum of the mice were measured in 4 groups: Ctrl + NSD, Ctrl + HSD, EAP + NSD, and EAP + HSD groups ( n = 4). b Simple schematic diagram of the experimental workflow. c HE staining and inflammation scores of prostate tissues (scale bar = 100 μm, n = 4). d Tactile allodynia development in NOD mice in the 4 groups ( n = 4). e Secretion levels of IL-1β, TNF-α, and IL-17A in the serum of mice in the 5 groups ( n = 4). f Flow cytometry was used to determine the proportion of Th17 of CD4 + T cells among the splenic lymphocytes of immunized mice in the 5 groups ( n = 4). g The infiltration of Th17 cells in prostate tissues from mice was determined by immunofluorescence (white arrowheads, scale bar = 100 μm, n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns non-significant. NOD nonobese male diabetic/LtJ, BF breeding feed, EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, HE hematoxylin–eosin, IL-1β interleukin-1β, TNF-α tumor necrosis factor-α, IL-17A interleukin-17A, 5-HIAA 5-hydroxyindole acetic acid, DAPI 4',6-diamidino-2-phenylindole

Article Snippet: The following reagents and antibodies were used: CFA (#F5881, Sigma, USA); commercial enzyme-linked immunosorbent assay (ELISA) kits for IFN-γ (#E-EL-M0048c), IL-1β (#E-EL-M0037), IL-17A (#E-EL-M0047c), tumor necrosis factor-α (TNF-α, #E-EL-M3063), and 5-HIAA (#E-EL-0075c) (Elabscience, China); anti-mouse CD4-fluorescein isothiocyanate (FITC, #553047), CD3e-allophycocyanin (APC, #561826), IFN-γ-phycoerythrin (PE, #562020), IL-17-PE (#559502), anti-human CD8-FITC (#555634), CD3-APC (#555342), IL-17-PE (#560436) (BD, USA); anti-CD3e (#BE0001-1), anti-CD28 (#BE0015-1), anti-IFN-γ (#BE0055), anti-IL-4 (#BE0045) (Bio X Cell, USA); IL-23 (#CS31), transforming growth factor-β1 (TGF-β1, #CA59), IL-6 (#CG39) (Novoprotein, China); anti-AHR (1:1000, #NB100-128, Novus, USA); anti-SGK1 (1:1000, #AF6789), anti-FOXO1 (1:1000, #AF6416), anti-p-FOXO1 (1:1000, #AF3417), anti-retinoic acid receptor-related orphan receptor gamma-t (RORγt; 1:1000, #DF3196), and anti-GAPDH (1:5000, #AF7021) (Affinity, USA).

Techniques: Staining, Flow Cytometry, Immunofluorescence

Journal: Military Medical Research

Article Title: High-salt-driven gut microbiota dysfunction aggravates prostatitis by promoting AHR/SGK1/FOXO1 axis-mediated Th17 cell differentiation

doi: 10.1186/s40779-025-00607-1

Figure Lengend Snippet: Inhibition of AHR abrogated the protective effects of 5-HIAA supplementation on EAP mice fed an HSD. a AHR levels in prostate tissues of mice among the Ctrl + NSD, Ctrl + HSD, EAP + NSD, EAP + HSD, and EAP + HSD + 5-HIAA groups ( n = 4). b A simple schematic diagram of the experimental workflow. c HE staining and inflammation scores of prostate tissues (scale bar = 100 μm, n = 4). d Tactile allodynia development in the mice from the Ctrl + NSD, Ctrl + HSD, EAP + NSD, EAP + HSD, EAP + HSD + 5-HIAA, and EAP + HSD + 5-HIAA + CH groups ( n = 4). e The levels of IL-1β, TNF-α, and IL-17A in the serum of the mice in the 6 groups ( n = 4). f Proportion of Th17 of CD4 + T cells, as determined by flow cytometry, among the splenic lymphocytes of immunized mice in the 6 groups (white arrowheads, n = 4). g The infiltration of Th17 cells in prostate tissues from mice was evaluated by immunofluorescence (scale bar = 100 μm, n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns non-significant. NOD nonobese male diabetic/LtJ, BF breeding feed, EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, CH CH223191 (an AHR inhibitor), HE hematoxylin–eosin, IL-1β interleukin-1β, TNF-α tumor necrosis factor-α, IL-17A interleukin-17A, 5-HIAA 5-hydroxyindole acetic acid, AHR aryl hydrocarbon receptor, DAPI 4',6-diamidino-2-phenylindole

Article Snippet: The following reagents and antibodies were used: CFA (#F5881, Sigma, USA); commercial enzyme-linked immunosorbent assay (ELISA) kits for IFN-γ (#E-EL-M0048c), IL-1β (#E-EL-M0037), IL-17A (#E-EL-M0047c), tumor necrosis factor-α (TNF-α, #E-EL-M3063), and 5-HIAA (#E-EL-0075c) (Elabscience, China); anti-mouse CD4-fluorescein isothiocyanate (FITC, #553047), CD3e-allophycocyanin (APC, #561826), IFN-γ-phycoerythrin (PE, #562020), IL-17-PE (#559502), anti-human CD8-FITC (#555634), CD3-APC (#555342), IL-17-PE (#560436) (BD, USA); anti-CD3e (#BE0001-1), anti-CD28 (#BE0015-1), anti-IFN-γ (#BE0055), anti-IL-4 (#BE0045) (Bio X Cell, USA); IL-23 (#CS31), transforming growth factor-β1 (TGF-β1, #CA59), IL-6 (#CG39) (Novoprotein, China); anti-AHR (1:1000, #NB100-128, Novus, USA); anti-SGK1 (1:1000, #AF6789), anti-FOXO1 (1:1000, #AF6416), anti-p-FOXO1 (1:1000, #AF3417), anti-retinoic acid receptor-related orphan receptor gamma-t (RORγt; 1:1000, #DF3196), and anti-GAPDH (1:5000, #AF7021) (Affinity, USA).

Techniques: Inhibition, Staining, Flow Cytometry, Immunofluorescence

Journal: Military Medical Research

Article Title: High-salt-driven gut microbiota dysfunction aggravates prostatitis by promoting AHR/SGK1/FOXO1 axis-mediated Th17 cell differentiation

doi: 10.1186/s40779-025-00607-1

Figure Lengend Snippet: HSD promoted the differentiation of Th17 cells by activating SGK1/FOXO1 signaling pathway. a The in vitro Th17 cell differentiation ratio was determined by flow cytometry ( n = 3). b Th17 cell differentiation ratio was determined in an in vitro differentiation experiment using immunofluorescence (scale bar = 100 μm, n = 3). c SGK1, p-FOXO1, and FOXO1 levels in prostate tissues from Ctrl + NSD, Ctrl + HSD, EAP + NSD, EAP + HSD, and EAP + HSD + 5-HIAA mice ( n = 4). d AHR, SGK1, p-FOXO1, and FOXO1 levels in prostate tissues of mice in the EAP + HSD + 5-HIAA and EAP + HSD + 5-HIAA + CH groups ( n = 4). e AHR, SGK1, p-FOXO1, and FOXO1 levels in naïve CD4 + T cells of Ctrl, Sti, Sti + HSD, Sti + HSD + 5-HIAA, Sti + HSD + 5-HIAA + CH groups ( n = 3). f Transcriptional levels of Sgk1 in naïve CD4 + T cells of Ctrl, Sti, Sti + HSD, Sti + HSD + 5-HIAA, Sti + HSD + 5-HIAA + CH groups (n = 3). g For the ChIP-qPCR assay, 3 potential enhancer segments of the mouse promoter region of Sgk1 were created. h qPCR was used to assess the immunoprecipitated DNA by a ChIP assay ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns non-significant. EAP experimental autoimmune prostatitis, HSD high-salt diet, NSD normal-salt diet, IL-17A interleukin-17A, 5-HIAA 5-hydroxyindole acetic acid, AHR aryl hydrocarbon receptor, SGK1 serum and glucocorticoid-regulated kinase 1, FOXO1 forkhead box protein O1, CH CH223191 (an AHR inhibitor), ChIP chromatin immunoprecipitation, qPCR quantitative polymerase chain reaction, DAPI 4’,6-diamidino-2-phenylindole, Sti stimulation with IL-6, IL-23, TGF-β, anti-IFN-γ, and anti-IL-4

Article Snippet: The following reagents and antibodies were used: CFA (#F5881, Sigma, USA); commercial enzyme-linked immunosorbent assay (ELISA) kits for IFN-γ (#E-EL-M0048c), IL-1β (#E-EL-M0037), IL-17A (#E-EL-M0047c), tumor necrosis factor-α (TNF-α, #E-EL-M3063), and 5-HIAA (#E-EL-0075c) (Elabscience, China); anti-mouse CD4-fluorescein isothiocyanate (FITC, #553047), CD3e-allophycocyanin (APC, #561826), IFN-γ-phycoerythrin (PE, #562020), IL-17-PE (#559502), anti-human CD8-FITC (#555634), CD3-APC (#555342), IL-17-PE (#560436) (BD, USA); anti-CD3e (#BE0001-1), anti-CD28 (#BE0015-1), anti-IFN-γ (#BE0055), anti-IL-4 (#BE0045) (Bio X Cell, USA); IL-23 (#CS31), transforming growth factor-β1 (TGF-β1, #CA59), IL-6 (#CG39) (Novoprotein, China); anti-AHR (1:1000, #NB100-128, Novus, USA); anti-SGK1 (1:1000, #AF6789), anti-FOXO1 (1:1000, #AF6416), anti-p-FOXO1 (1:1000, #AF3417), anti-retinoic acid receptor-related orphan receptor gamma-t (RORγt; 1:1000, #DF3196), and anti-GAPDH (1:5000, #AF7021) (Affinity, USA).

Techniques: In Vitro, Cell Differentiation, Flow Cytometry, Immunofluorescence, ChIP-qPCR, Immunoprecipitation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Mediators of Inflammation

Article Title: Notch Signaling Pathway Promotes Th17 Cell Differentiation and Participates in Thyroid Autoimmune Injury in Experimental Autoimmune Thyroiditis Mice

doi: 10.1155/2023/1195149

Figure Lengend Snippet: List of primers for qRT-PCR.

Article Snippet: SMC culture supernatants were collected, and the concentrations of IL-17A were measured using the mouse IL-17A ELISA kit (R&D Systems, USA) according to the manufacturer's instructions.

Techniques: Sequencing

Journal:

Article Title:

doi:

Figure Lengend Snippet: Biochemical and cellular on-target activities of SHR168442. ( a ) Principle of the LanthaScreen TR-FRET human RORγ LBD coactivator assay. The terbium (Tb)-anti-GST antibody indirectly labels the RORγ LBD by binding to the GST tag of the protein. Binding of the compound to RORγ LBD causes a conformational change that results in a decrease in the affinity of the receptor for a labeled coactivator peptide, fluorescein-D22. ( b ) Dose–response curve for SHR168442 as an antagonist in LanthaScreen TR-FRET human RORγ LBD coactivator assay. ( c-d ) SHR168442 inhibits IL-17 secretion from human PBMCs ( c ) and mouse splenocytes ( d ). ( e–f ) Effects of SHR168442 on RORγ ( e ) and RORα ( f ) luciferase reporter expression. Ursolic acid and ATRA were used as positive control in the assays, respectively. RLU = relative light units. Non-linear regression analyses were performed and IC 50 values determined using GraphPad Prism software. Representative curves are shown. Average of the measurement and its standard deviation are shown.

Article Snippet: On day 5, compounds were added and then incubated for 72 h, and IL-17 in the supernatant was measured using IL-17A homodimer (mouse) AlphaLISA detection kit (Perkin Elmer) according to the manufacturer’s protocol.

Techniques: Binding Assay, Protein Binding, Labeling, Luciferase, Expressing, Positive Control, Software, Standard Deviation