Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Dietary Lauric Acid Suppresses Inflammation, Cholestasis, Hepatocyte Injury, and Senescence in 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine-induced Inflammatory Cholangiopathy

doi: 10.1016/j.jcmgh.2026.101731

Figure Lengend Snippet: Effect of lauric acid on expression of inflammation, senescence, and oxidative stress genes and biomarkers in hepatocytes isolated from experimental mouse groups. qRT-PCR-mRNA analysis in hepatocytes isolated from chow, DDC, LA, and DDC/LA mice. ( A ) Genes involved in inflammation and oxidative stress. ( B ) Senescence genes. ( C ) Mitochondrial fatty acid oxidation and oxidative stress genes. ( D ) β-gal staining ( yellow arrow ) as a senescence marker in overnight cultured primary hepatocytes isolated from chow, DDC, LA, and DDC/ LA mice. Bar graph showing the number of senescent (β-gal + ) cells per microscopic field in liver sections from chow, DDC, DDC/LA, and LA groups. ( E ) Immunoblotting and quantification of β-gal in fresh hepatocytes isolated from chow, DDC, LA, and DDC/LA mice. ( F ) Serum hydrogen peroxide levels and CCL2 concentrations (ELISA), and lipid peroxides, nitrite, and malondialdehyde levels from isolated hepatocytes. ( G–H ) Co-culture of human cholangiocytes H69 ( upper wells ) and Huh7 cells was conducted. H69 cells were incubated with and without DDC overnight in the presence or absence of DLPC. ( G ) mRNA expression by qPCR was measured in Huh7 cells ( bottom wells ) for CCL2, CDKN1A, NR0B2, and ABCB11 and ( H ) in H69 cells for CCL2, CDKN1A, KRT19, CTGF, and TNF . ( I ) Primary mouse hepatocytes were incubated with CCL2 in the presence or absence of DLPC overnight, and mRNA expression by qPCR was analyzed for senescence genes Cdkn1a and Cdkn1b . ( J ) β-gal staining ( yellow arrow = senescent cells) of cultured Huh7 cells incubated with CCL2 overnight in the presence or absence of DLPC. Bar graph showing the number of senescent (β-gal + ) Huh7 cells after treatment with CCL2 alone or in combination with DLPC. CCL2 treatment markedly increased cellular senescence, which was inhibited by DLPC. ( K ) Confocal fluorescence microscopy of primary cultured hepatocytes from chow, DDC, LA, and DDC/LA mice stained with senescence marker P16 ( red ), and nuclear DAPI ( blue ) from showing increased senescence in DDC mouse hepatocytes, which was prevented by LA treatment. ( L ) Confocal fluorescence microscopy of primary mouse hepatocytes from chow, DDC, LA, and DDC/LA mice stained with senescence marker P21/WAF/CIP ( red ) and nuclear DAPI ( blue ) also showing increased senescence in DDC mouse hepatocytes, which was prevented by LA treatment. For ( G–I ), data points represent replicates in 3 independent experiments. For ( D and J ), photomicrographs are shown that are representative of 3 separate experiments. For ( K and L ), immunofluorescent images are shown that are representative of 3 separate experiments. ( M ) Western analysis of pSTAT1 protein, total STAT1, and actin expression in hepatocytes isolated from the mouse groups, including quantification of integrated density values (IDVs). ( N ) ChIP assay of hepatocytes isolated from mouse groups for STAT1 binding to the promoter region of Cdkn1b using a STAT1-specific antibody. ( O ) Western analysis of pSTAT1 protein expression in cultured primary mouse hepatocytes exposed to CCL2 overnight in the presence or absence of DLPC, including quantification of IDVs of immunoblots. Statistical analysis was performed by 1-way ANOVA with Tukey’s correction for multiple comparisons. a P < .05 vs all other groups; b P < .05 vs DDC; b P < .05 vs CCL2.

Article Snippet: The mouse CCL2 detection assay kit (Cat: 6721, Chondrex, Inc) was employed as per the manufacturer’s instructions to measure serum concentrations of CCL2.

Techniques: Expressing, Isolation, Quantitative RT-PCR, Staining, Marker, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Incubation, Fluorescence, Microscopy, Binding Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Dietary Lauric Acid Suppresses Inflammation, Cholestasis, Hepatocyte Injury, and Senescence in 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine-induced Inflammatory Cholangiopathy

doi: 10.1016/j.jcmgh.2026.101731

Figure Lengend Snippet: LA treatment induces an anti-inflammatory gene polarization in IHMCs and BMDMs in DDC mice. ( A ) mRNA expression of proinflammatory genes ( Il-1b, Itgam, Adgre1, Ly6c, Cxcl1, Cxcl2, Ccl2, Cd80, and Cd86) in IHMCs demonstrating activation by DDC and suppression by LA. ( B ) mRNA expression of anti-inflammatory/repair genes in IHMCs ( Clec4f, Klf4, Msr1, Mrc1, and Il1rn ) demonstrating induction by LA in DDC mice. ( C ) Flow cytometric analysis of IHMCs shows decreased F4/80+LY6C+ and increased F4/80+CD206+ macrophages in DDC/LA mice compared with DDC mice. ( D ) Co-culture of human H69 cholangiocytes exposed overnight to DDC with and without DLPC ( upper wells ) and human monocyte/macrophage THP-1 ( lower wells ) was conducted. IL1B , KLF4, and IL1RN mRNA expression was analyzed in THP-1 cells. ( E ) Confocal microscopy of immunostaining for pSTAT6 ( green ), LRH-1 ( red ), and DAPI ( blue ) in cultured BMDMs from chow, DDC, LA, and DDC/ LA mice. Colocalization of pSTAT6 and LRH-1 was demonstrated in cells from DDC/LA mice ( yellow ). ( F ) ChIP assay examining STAT6 binding to the promoter of Arg1 and Chil3 was performed in liver homogenate from chow, DDC, LA, and DDC/ LA mice. ( G ) Confocal fluorescence microscopy of BMDMs isolated from mouse groups that were stained with CD206 ( green ), F4/80 ( red ), LY6C ( magenta ), and DAPI ( blue ). ( H ) Confocal fluorescence microscopy of BMDMs from mouse groups stained with F4/80 ( red ), CD163 ( green ), and DAPI ( blue ). ( I ). Proposed mechanism of DDC-induced hepatocyte and cholangiocyte injury, promoting inflammation and cholangiopathy (Created with BioRender.com ). Feeding mice a DDC diet leads to the accumulation of hydrophobic bile acids and induction of inflammation, causing hepatocyte and cholangiocyte injury. Injured hepatocytes secrete CCL2 and undergo senescence, characterized by pSTAT1 activation and increased Cdkn1b expression. Cholangiocyte injury further amplifies CCL2 production. Elevated CCL2 recruits monocytes and macrophages (LY6C1 + ) into the liver. The combined hepatocyte and cholangiocyte injury, along with macrophage infiltration and ductular reaction, drives persistent inflammation and progressive cholangiopathy. LA supplementation enhances LRH-1 signaling through DLPC, promoting hepatocyte recovery and normalization of bile acid homeostasis. This intervention reduces CCL2 production, macrophage infiltration, hepatocyte senescence, and ductular reaction, collectively attenuating cholangiopathy progression. Data are mean ± SEM. Data points represent individual mice. N = 3–8 animals per group. Statistical analysis was performed by 1-way ANOVA with Tukey’s correction for multiple comparisons. a P < .05 vs all other groups; b P < .05 vs DDC. c P < .05 vs chow.

Article Snippet: The mouse CCL2 detection assay kit (Cat: 6721, Chondrex, Inc) was employed as per the manufacturer’s instructions to measure serum concentrations of CCL2.

Techniques: Expressing, Activation Assay, Co-Culture Assay, Confocal Microscopy, Immunostaining, Cell Culture, Binding Assay, Fluorescence, Microscopy, Isolation, Staining

Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-C. Histones (50 μg/ml, 24 hours) induced chemokine production and release as demonstrated with a Proteome Profiler™ Antibody Array in Hepa1-6 cells. D. Knockdown of NF-κB p65 and TLR4 (but not TLR2 and RAGE) in Hepa1-6 cells inhibited histone (50 μg/ml, 24 hours)-induced CCL9/10 release as demonstrated by ELISA assay (n=3, *, p<0.05 versus control shRNA group). E. Anti-CCL9/10 neutralizing antibody (1 mg/ml) partly inhibited histone (50 μg/ml, 24 hours)-induced Hepa1-6 cell migration (n=3, *, p<0.05).

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Ab Array, Knockdown, Enzyme-linked Immunosorbent Assay, Control, shRNA, Migration

Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-B. Compared with the control group, TLR4 depletion (by using TLR4 −/− mice or TLR4 knockdown cells) or inhibition of histone release (by administration of 10 mg/kg heparin or 10 mg/kg H3 neutralizing antibody) limited the formation of lung metastasis (as shown in arrow) in mice based on tail vein injection of 3×10 6 Hepa1-6 cells (N=5 mice/group, *, p<0.05 versus control group). In contrast, control IgG (10 mg/kg) did not inhibit the formation of lung metastasis (B). C. Serum nucleosome levels were reduced after treatment with heparin in wildtype, but not in TLR4 −/− mice (N=5 mice/group, *, p<0.05 versus control group). D. Conceptual relationships between histone and tumor metastasis. Histone is a nuclear DAMP and can be released during cell injury or death. Once released, histone can promote cell migration and invasion through the TLR4-ERK-NF-κB pathway, which induces chemokine production and release.

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Control, Knockdown, Inhibition, Injection, Migration

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: Antihuman Fractalkine Reagents Used in this Study

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Control

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: Distinguishing between cleaved and membrane-tethered fractalkine, generation of specific reagents. A: Samples of Western lysates from WT CHO-K1 and CHO-K1 cells transfected with a human fractalkine expression vector 1 along with samples of supernatant taken from fractalkine-transfected CHO-K1 cells, were run on 7.5% acrylamide gels under standard reducing conditions. Samples were transferred to nitrocellulose membranes and identical membranes probed using goat anti-fractalkine polyclonal reagent (goat α-Fkn, R&D Systems) (lanes 1–3) or chicken anti-C-peptide polyclonal reagent (chicken α-C-pep, lanes 4–6). The goat α-Fkn reagent is reactive against the chemokine domain of the molecule and specifically detects twobands at the predicted size of 95 kd (lane 2, asterisk). These two bands are also detected by the chicken α-C-pep reagent (lane 5, asterisk). In addition, these reagents discriminate between cleaved and intact forms of the molecule as the goat α-Fkn detects the cleaved form of fractalkine within transfected cell supernatant (lane 3, 85 to 90 kd), whereas the chicken α-C-pep does not (lane 6). Furthermore, the goat α-Fkn detects one larger (lane 2, 100 kd) and two smaller bands (lane 2, 75 and 66 kd) within transfected CHO-K1 samples that are not detected by the chicken α-C-pep (lane 5). The larger band may be nonspecific because it has no counterpart detected by the chicken α-C-pep. The two smaller bands may indicate partially degraded forms of fractalkine, still containing the N-terminus chemokine domain. B–I: The specificity of a range of anti-fractalkine antibodies was evaluated by immunohistochemistry. Cytospins were prepared from NIH/3T3 cells transiently transfected as above, with fractalkine (3T3-Fkn) and were stained as follows. B: 3T3-Fkn stained with mouse IgG1 control as a control for C. C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb. D: 3T3-Fkn stained with no primary antibody as a control for E and F. E: 3T3-Fkn stained with goat α-Fkn. F: 3T3-Fkn stained with chicken α-C-pep. G: 3T3-Fkn stained with rabbit IgG as a control for H and I. H: 3T3-Fkn stained with rabbit α-C-peptide. I: 3T3-Fkn stained with rabbit α-N-pep polyclonal reagent. 1 Note that although there is light nonspecific staining of the nucleus within the control sections (B, D, and F) this is in marked contrast to the strong cell surface staining in sections stained with the specific reagents. Similar results were obtained using transfected CHO-K1 cells and via immunofluorescence. Original magnification, ×400.

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Membrane, Western Blot, Transfection, Expressing, Plasmid Preparation, Immunohistochemistry, Staining, Control, Immunofluorescence

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: The transmembrane form of fractalkine is expressed by the human colorectal adenocarcinoma cell line, DLD-1. A: DLD-1, cells were grown to confluence on glass coverslips and stained using indirect immunofluorescence for transmembrane-expressed fractalkine using the anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; green) mAb and rabbit anti-C-peptide reagent (α-C-pep; red). Strong double labeling (orange) occurred on a subset of cells where the intracellular epitope was most strongly expressed. Lower levels of anti-chemokine domain staining could be detected on most cells. B: Anti-chemokine domain reagent specificity was demonstrated by double labeling using an isotype control antibody for the anti-chemokine mAb (green) and α-C-pep (red). α-C-pep staining was also competed out by addition of 10× molar excess of the immunizing peptide (data not shown). C: The α-Fkn (green) but not α-C-pep staining (red) couldbe competed totally by pre-incubation with a 10× molar excess of recombinant human fractalkine chemokine domain (rhFkn; 362-CX-025; R&D Systems). D: Cells were double-labeled with α-cytokeratin (clone AE1/AE3, DAKO; green). Original magnifications, ×400 (A–D). E: Total RNA was prepared from DLD-1 and HUVECs cultured with or without 10 U/ml TNF-α. RNA was reverse-transcribed and triplicate 25 ng cDNA samples subjected to PCR reactions using primers specific for fractalkine (Fkn) or HPRT. There was no fractalkine or HPRT signal amplified in reverse transcriptase samples (data not shown). F: DLD-1 cells were permeabilized and stained using i) mouse α-Fkn (clone 51636.11) mAb or control mouse IgG1 mAb (Serotech), ii) goat α-Fkn polyclonal or 10% goat serum, iii) α-C-pep or rabbit IgG, iv) α-N-pep polyclonal 1 or rabbit IgG, and fractalkine expression analyzed by FACS. The bold trace shows the fluorescence of cells stained with the specific antibody, whereas the normal trace shows the background fluorescence of cells stained with the control reagent.

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Staining, Immunofluorescence, Labeling, Control, Incubation, Recombinant, Cell Culture, Reverse Transcription, Amplification, Expressing, Fluorescence

Journal: Brain Sciences

Article Title: Glabridin Therapy Reduces Chronic Allodynia, Spinal Microgliosis, and Dendritic Spine Generation by Inhibiting Fractalkine-CX3CR1 Signaling in a Mouse Model of Tibial Fractures

doi: 10.3390/brainsci13050739

Figure Lengend Snippet: The fractures induce a spinal increase in fractalkine and CX3CR1, and microgliosis is reduced following repetitive injections with glabridin. ( A – C ) The ELISA experiments detected the dynamic alternations of spinal fractalkine, CX3CR1, and Iba-1 proteins following the fractures and orthopedic surgeries ( n = 5). Intrathecal glabridin (50 μg) was delivered daily for 4 consecutive days on days 3, 4, 5, and 6 following the fractures. ( D – F ) The ELISA experiments revealed that the up-modulation of spinal fractalkine, CX3CR1, and Iba-1 proteins after the fractures and orthopedic surgeries was inhibited by the glabridin pretreatment ( n = 5). ( G ) The immunofluorescence staining showed representative photomicrographs of Iba-1 in the dorsal horn following the fractures and glabridin exposure (the scale bar is 50 μm). All data are expressed as means ± SEM and analyzed by a one-way or two-way ANOVA with Bonferroni post hoc comparisons.

Article Snippet: Recombinant fractalkine (Sigma-Aldrich, F2302), microglia inhibitor minocycline (ab120661, Abcam, Cambridge, UK), a neutralizing antibody against fractalkine (AF472, anti-fractalkine; R&D Systems, Minneapolis, MN, USA), and a neutralizing antibody against CX3CR1 (AF5825, anti-CX3CR1; R&D Systems) were diluted in 10% DMSO for the injections.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Brain Sciences

Article Title: Glabridin Therapy Reduces Chronic Allodynia, Spinal Microgliosis, and Dendritic Spine Generation by Inhibiting Fractalkine-CX3CR1 Signaling in a Mouse Model of Tibial Fractures

doi: 10.3390/brainsci13050739

Figure Lengend Snippet: Pharmacological inhibition of fractalkine/CX3CR1 protects against fracture-associated chronic allodynia. ( A , B ) The anti-fractalkine and anti-CX3CR1 were intrathecally applied (20 μg) on days 3, 4, 5, and 6 after the fracture surgeries. The mechanical allodynia and cold allodynia were measured. ( C , D ) Post-treatment with anti-fractalkine and anti-CX3CR1 (20 μg) via the intrathecal route two weeks following the orthopedic surgeries considerably ameliorated the existing mechanical and cold allodynia. All behavioral results are shown as means ± SEM ( n = 6) and analyzed by a two-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05 vs. group sham + vehicle; # p < 0.05 vs. group fracture + vehicle.

Article Snippet: Recombinant fractalkine (Sigma-Aldrich, F2302), microglia inhibitor minocycline (ab120661, Abcam, Cambridge, UK), a neutralizing antibody against fractalkine (AF472, anti-fractalkine; R&D Systems, Minneapolis, MN, USA), and a neutralizing antibody against CX3CR1 (AF5825, anti-CX3CR1; R&D Systems) were diluted in 10% DMSO for the injections.

Techniques: Inhibition

Journal: Brain Sciences

Article Title: Glabridin Therapy Reduces Chronic Allodynia, Spinal Microgliosis, and Dendritic Spine Generation by Inhibiting Fractalkine-CX3CR1 Signaling in a Mouse Model of Tibial Fractures

doi: 10.3390/brainsci13050739

Figure Lengend Snippet: Spinal exposure to exogenous fractalkine abolishes glabridin-induced anti-nociceptive effects on chronic allodynia. Glabridin (50 μg) and recombinant fractalkine (50 ng) were intrathecally injected on days 3, 4, 5, and 6 after the fracture surgeries. The behavioral phenotypes of mechanical allodynia ( A ) and cold allodynia ( B ) were recorded. ( C , D ) The ELISA experiments identified changes in the spinal CX3CR1 and Iba-1 proteins after the fractures and the glabridin and fractalkine interventions. ( E , F ) Representative photomicrographs and statistical results of the spine morphology following the fractures and glabridin and fractalkine treatments (the scale bar is 5 μm). All behavioral results ( n = 6) and biochemical results ( n = 3–5) are shown as means ± SEM and analyzed by a one-way or two-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05 vs. group sham + vehicle; # p < 0.05 vs. group fracture + vehicle.

Article Snippet: Recombinant fractalkine (Sigma-Aldrich, F2302), microglia inhibitor minocycline (ab120661, Abcam, Cambridge, UK), a neutralizing antibody against fractalkine (AF472, anti-fractalkine; R&D Systems, Minneapolis, MN, USA), and a neutralizing antibody against CX3CR1 (AF5825, anti-CX3CR1; R&D Systems) were diluted in 10% DMSO for the injections.

Techniques: Recombinant, Injection, Enzyme-linked Immunosorbent Assay

Journal: Brain Sciences

Article Title: Glabridin Therapy Reduces Chronic Allodynia, Spinal Microgliosis, and Dendritic Spine Generation by Inhibiting Fractalkine-CX3CR1 Signaling in a Mouse Model of Tibial Fractures

doi: 10.3390/brainsci13050739

Figure Lengend Snippet: Acute nociceptive phenotypes are initiated by exogenous fractalkine and ameliorated by co-applications of minocycline. The microglial inhibitor minocycline (i.t.; 20 μg) and anti-CX3CR1 (i.t.; 20 μg) were given one hour prior to the delivery of the recombinant fractalkine (i.t.; 50 ng). ( A , B ) The exogenous fractalkine-evoked acute nociceptive phenotypes were ameliorated following the pre-applications of minocycline and anti-CX3CR1, respectively. All behavioral results are shown as means ± SEM ( n = 6) and analyzed by a two-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05 vs. baseline; # p < 0.05 vs. group recombinant fractalkine (50 ng). The biochemical experiments were performed 3 hours following the fractalkine injections. ( C ) The ELISA experiments showed that anti-CX3CR1 decreased the overexpression of the Iba-1 proteins in the dorsal horn following the exogenous fractalkine injections. All biochemical results are expressed as means ± SEM ( n = 5) and analyzed by a one-way ANOVA with Bonferroni post hoc comparisons. * p < 0.05 vs. naïve; # p < 0.05 vs. group recombinant fractalkine.

Article Snippet: Recombinant fractalkine (Sigma-Aldrich, F2302), microglia inhibitor minocycline (ab120661, Abcam, Cambridge, UK), a neutralizing antibody against fractalkine (AF472, anti-fractalkine; R&D Systems, Minneapolis, MN, USA), and a neutralizing antibody against CX3CR1 (AF5825, anti-CX3CR1; R&D Systems) were diluted in 10% DMSO for the injections.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Over Expression