Journal: Frontiers in Oncology

Article Title: SPAK Deficiency Attenuates Chemotherapy-Induced Intestinal Mucositis

doi: 10.3389/fonc.2021.733555

Figure Lengend Snippet: (A, B) Cell permeability in murine epithelial IEC-6 cells following treatment of 5-FU. (A) After 5-FU treatment, permeability of IEC-6 cells increases in a concentration-dependent manner (5 and 10 μM). (B) siRNA-mediated knockdown of SPAK expression in IEC-6 cells blocks this increase in cell permeability compared to control cells treated with 5-FU (10 μM). (C, D) SPAK-knockdown modulates the effects of 5-FU treatment on the proliferation and survival of IEC-6 cells. Control and SPAK-KD IEC-6 cells were treated with 10 µM 5-FU and dimethyl sulfoxide (DMSO) solvent for 3 h, respectively. Untreated and 5-FU-treated control or SPAK-KD IEC-6 cells were analyzed to determine the mean fluorescence index (MFI) of PCNA expression (C) and the percentage of annexin-V-positive cells (D) , respectively. (C) Representative flow cytometry assays are shown in the left panel, and the MFI of PCNA is shown in the right panel. (D) Representative flow cytometry assays (left panel) and the frequencies (right panel) of annexin-V-positive IEC-6 cells are presented. Data corresponding to MFIs or frequencies are representatives of at least three mice in each group, presented as the mean ± SEM. Two-tailed Student’s unpaired t -tests were used for statistical analysis. *** P < 0.0001; ** P < 0.01; * P < 0.05 n.s., not significant.

Article Snippet: Murine intestinal epithelial IEC-6 cells (Bioresource Collection and Research Center, Hsinchu, Taiwan) were cultured in Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum, 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 10 mM sodium pyruvate supplemented with 0.1 U/mL bovine insulin, as described previously ( ).

Techniques: Permeability, Concentration Assay, Knockdown, Expressing, Control, Solvent, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Gastroenterology report

Article Title: ELMO1 ameliorates intestinal epithelial cellular senescence via SIRT1/p65 signaling in inflammatory bowel disease-related fibrosis.

doi: 10.1093/gastro/goae045

Figure Lengend Snippet: Figure 1. Intestinal fibrosis was associated with cellular senescence. (A) GO analysis of DEGs in four matched tissues from strictured and non- strictured patients with IBD. (B) Heat-map visualization of cellular senescence score in four matched tissues from strictured and non-strictured patients with IBD. (C) Histogram visualization of cellular senescence score based on the mRNA expression of colonic tissues from 40 patients with IBD. (D) Representative immunofluorescence image of Lamin B1 in the strictured and non-strictured tissue. Co-staining with Lamin B1, DAPI, and E- cadherin to identify senescent intestinal epithelial cells. scale bar: 100 μm. Binary classification of the correlation between LAMINB1 expression and fibrostrictured in IBD tissues. (E) qPCR assays were used to detect the mRNA expression of p21, p16, p53, and SASP genes in strictured, non-strictured, and non-inflamed tissue. (F) Relative mRNA expressions of p53, p16, and p21 and SASP genes in chronic DSS or chronic TNBS models. (G) Distal colon tissue of unchallenged animals (n ¼ 5), mice from chronic DSS (n ¼ 5), and chronic TNBS colitis (n ¼ 5) were stained with H&E, and the width of the submucosa was measured. (Scale bar: 100 μm) The thicknesses of the mucosa, submucosa, and muscularis were quantified (n ¼ 5 per group, P < 0.05, P < 0.01, P < 0.001) from H&E staining of distal colon sections. Masson’s Trichrome staining was performed in distal colon sections of unchallenged mice and animals from the chronic DSS or chronic TNBS models, respectively. Representative images of SA-β-gal staining (scale bar, 100 μm), immunofluorescence staining images of Lamin B1 (scale bar, 200 μm). Quantitative analyses were shown in the right panel. Values were reflected as the mean ± SD of three independent experiments. GO analysis ¼ gene ontology analysis; DEGs ¼ differentially expressed genes; IBD ¼ inflammatory bowel disease; SASP ¼ senescence-associated secretory phenotype; DSS ¼ dextran sulfate sodium salt; TNBS ¼ 2,4,6-trinitro benzene sulfonic acid; SA- β-gal ¼ Senescence-associated β-galactosidase..

Article Snippet: The human intestinal epithelial cells HIEC6 (CRL-3266), human intestinal fibroblast cells CCD-18Co (CRL-1459), murine intestinal epithelial cells IEC6 (CRL-1592), murine intestinal fibroblast cells 3T3-L1 (CL-173) used in our study were purchased from the American Type Culture Collection (ATCC; Rockefeller, MD, USA) and cultured in DMEM-high glucose (C11995500BT; Gibco; Waltham, MA, USA) plus 10% fetal bovine serum (FBS; FSP500; ExCell Bio; Suzhou, Jiangsu, China) and 1% penicillinstreptomycin (15140–122; Gibco).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Gastroenterology report

Article Title: ELMO1 ameliorates intestinal epithelial cellular senescence via SIRT1/p65 signaling in inflammatory bowel disease-related fibrosis.

doi: 10.1093/gastro/goae045

Figure Lengend Snippet: Figure 4. Elmo1 deletion promotes senescence in IECs and activates fibrosis in fibroblasts. (A)Western blotting was performed to detect endogenous ELMO1 expression and senescence in the indicated cells upon LPS stimulation. Cell lysates were immunoblotted with the indicated antibodies. β-actin was used as a loading control. (B, C) IECs including IEC6, HIEC6, and primary epithelial cells were treated with or without 10 μM etoposide, 100 ng/mL LPS and PBS for 14 days. We used the etoposide as a positive control to induce senescence in IECs. Senescence activity was detected by qRCR assay (B) and Lamin B1 staining (C). (scale bar: 50 μm) (D) Fibroblasts were treated with conditioned media from normal epithelial cells (CCM), conditioned media from etoposide-induced epithelial cells (ECM), conditioned media from LPS-induced epithelial cells (LCM), and 2 ng/mL of TGF-β. TGF-β was used as a positive control for activated fibroblasts. Representative immunofluorescence images of fibronectin. (scale bar: 50 μm) (E–G) IECs including IEC6, HIEC6, and primary epithelial cells (PIEC) were transfected with sh-ELMO1 plasmids and these groups were designed to exclude the effects of ELMO1 on senescence. Senescence activity was detected by western blotting (E), QRCR assay (F), and Lamin B1 staining (G) in ELMO1-silenced and scrambled cells. (H–I) Fibroblasts were treated with conditioned media from ELMO1-KD epithelial cells (SCM#1, SCM#2), conditioned media from normal epithelial cells (CCM), and 2 ng/mL TGF-β1. Western blotting was performed to detect the markers of intestinal fibrosis (H). Representative immunofluorescence images of fibronectin (I) (scale bar: 50 μm). LPS ¼ lipopolysaccharides; IECs ¼ intestinal epithelial cells; IEC6 ¼ murine intestinal epithelial cells; HIEC6 ¼ human intestinal epithelial cells; TGF-β ¼ transforming growth factor-β; PIEC ¼ primary epithelial cells; CCM ¼ conditioned media from normal epithelial cells.

Article Snippet: The human intestinal epithelial cells HIEC6 (CRL-3266), human intestinal fibroblast cells CCD-18Co (CRL-1459), murine intestinal epithelial cells IEC6 (CRL-1592), murine intestinal fibroblast cells 3T3-L1 (CL-173) used in our study were purchased from the American Type Culture Collection (ATCC; Rockefeller, MD, USA) and cultured in DMEM-high glucose (C11995500BT; Gibco; Waltham, MA, USA) plus 10% fetal bovine serum (FBS; FSP500; ExCell Bio; Suzhou, Jiangsu, China) and 1% penicillinstreptomycin (15140–122; Gibco).

Techniques: Western Blot, Expressing, Control, Positive Control, Activity Assay, Staining, Immunofluorescence, Transfection

Journal: Gastroenterology report

Article Title: ELMO1 ameliorates intestinal epithelial cellular senescence via SIRT1/p65 signaling in inflammatory bowel disease-related fibrosis.

doi: 10.1093/gastro/goae045

Figure Lengend Snippet: Figure 7. Schematic summary of our hypotheses and results. Cellular senescence is a new mechanism for IBD-related fibrosis. ELMO1 works as a gatekeeper for cellular senescence and intestinal fibrogenesis. Under chronic inflammation, ELMO1 loss impairs SIRT1 activity, increases p65 acetylation, and consequently induces epithelial cellular senescence and intestinal fibrosis. Inhibition of p65 acetylation by irisin treatment could reduce senescence and is an efficient therapy to prevent early- stage intestinal fibrosis in ELMO1-downregulated patients.

Article Snippet: The human intestinal epithelial cells HIEC6 (CRL-3266), human intestinal fibroblast cells CCD-18Co (CRL-1459), murine intestinal epithelial cells IEC6 (CRL-1592), murine intestinal fibroblast cells 3T3-L1 (CL-173) used in our study were purchased from the American Type Culture Collection (ATCC; Rockefeller, MD, USA) and cultured in DMEM-high glucose (C11995500BT; Gibco; Waltham, MA, USA) plus 10% fetal bovine serum (FBS; FSP500; ExCell Bio; Suzhou, Jiangsu, China) and 1% penicillinstreptomycin (15140–122; Gibco).

Techniques: Activity Assay, Inhibition

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Topical Anti-Inflammatory Activity of Essential Oils of Alpinia calcarata Rosc., Its Main Constituents, and Possible Mechanism of Action

doi: 10.1155/2020/2035671

Figure Lengend Snippet: Cytotoxic effect of ACEOs (100, 50, and 5 μ g/mL) on RAW 264.7, HaCaT, HepG2, and IEC-6 cell lines. Cell viability (a) and proliferative index (b) of ACEOs (50, 5, and 0.5 μ g/mL) on RAW 264.7, HaCaT, HepG2, and IEC-6 cell lines. RO: rhizome essential oil; LO: leaf essential oil.

Article Snippet: Murine macrophages (RAW 264.7), human hepatocellular carcinoma (HepG2), human keratinocytes (HaCaT), and rat intestinal epithelial cells (IEC-6) were procured from National Centre for Cell Sciences, Pune, India.

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Topical Anti-Inflammatory Activity of Essential Oils of Alpinia calcarata Rosc., Its Main Constituents, and Possible Mechanism of Action

doi: 10.1155/2020/2035671

Figure Lengend Snippet: Effects of ACEOs on LPS-induced cytokine production in murine macrophages. TNF- α levels in supernatant (a), IL-1 β levels in supernatant, (b) and IL-6 levels in supernatant (c). N: normal; LPS: LPS-treated cells; RO: rhizome essential oil; LO: leaf essential oil; LPS + DEX: LPS along with dexamethasone at 10 μ M. The bars represent the mean ± SEM ( n = 6). Symbols “ ∗ ,” “ ∗∗ ,” and “ ∗∗∗ ” represent a significant difference p < 0.05, p < 0.01, and p < 0.001, between LPS+ and EO-treated cells. Symbol “ # ” represents a significant difference p < 0.05 between normal and LPS + cells.

Article Snippet: Murine macrophages (RAW 264.7), human hepatocellular carcinoma (HepG2), human keratinocytes (HaCaT), and rat intestinal epithelial cells (IEC-6) were procured from National Centre for Cell Sciences, Pune, India.

Techniques:

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Topical Anti-Inflammatory Activity of Essential Oils of Alpinia calcarata Rosc., Its Main Constituents, and Possible Mechanism of Action

doi: 10.1155/2020/2035671

Figure Lengend Snippet: Cytotoxic effect of ACEOs (100, 50, and 5 μ g/mL) on RAW 264.7, HaCaT, HepG2, and IEC-6 cell lines. Cell viability (a) and proliferative index (b) of ACEOs (50, 5, and 0.5 μ g/mL) on RAW 264.7, HaCaT, HepG2, and IEC-6 cell lines. RO: rhizome essential oil; LO: leaf essential oil.

Article Snippet: Murine macrophages (RAW 264.7), human hepatocellular carcinoma (HepG2), human keratinocytes (HaCaT), and rat intestinal epithelial cells (IEC-6) were procured from National Centre for Cell Sciences, Pune, India.

Techniques: