Journal: International Journal of Molecular Medicine

Article Title: Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2

doi: 10.3892/ijmm.2026.5764

Figure Lengend Snippet: Elevated HAS2 expression and HA accumulation in murine and cellular fibrosis models. (A) Schematic of the experimental design for the time-course study. C57BL/6J mice received a single intratracheal dose of BLM or saline and were sacrificed at the indicated time points (days 0, 3, 5, 7, 10, 14 and 21) for sample collection (n=5 for per group). (B) Representative western blot images (left panel) and densitometric quantification (right panel) showing the protein expression levels of COL1A1 and HAS2 in lung tissues across the time course. β-tubulin served as the loading control (n=4 for per group). (C) ELISA quantification of HA levels in BALF at different days post-BLM injury (n=5 for per group). (D) Schematic diagram illustrating the workflow for the isolation and culture of primary mouse lung fibroblasts from BLM-treated fibrotic mice. (E) Western blotting analysis of HAS2 and COL1A1 protein expression in primary lung fibroblasts isolated from control (saline) and BLM-induced fibrotic mice. Representative blot images (left panel) and densitometric quantification of protein levels normalized to β-tubulin are shown (n=3 for each group). (F) Schematic diagram depicting TGF-β1-induced transition of NIH/3T3 fibroblasts into myofibroblasts. (G) COL1A1, ACTA2, and HAS2 mRNA expression in NIH/3T3 cells was detected using PCR after 5 ng/ml TGF-β1 administration for 12 h (n=3-4 for each group). (H) HAS2 protein expression in NIH/3T3 was detected using western blotting after 5 ng/ml TGF-β1 administration for 24 h (n=3 for each group). (I) ELISA was used to quantify HA concentrations in the culture media of myofibroblasts (n=6 for each group). * P<0.05, ** P<0.01, *** P<0.001. HAS2, hyaluronic acid synthase 2; HA, hyaluronic acid; BLM, bleomycin; BALF, bronchoalveolar lavage fluid; COL1A1, Collagen type I α 1 chain; ACTA2, actin alpha 2, smooth muscle.

Article Snippet: Antibodies for Collagen type I α 1 chain (COL1A1; cat. no. 72026; 1:1,000), Phospho-STAT6 (Tyr641; cat. no. 56554S; 1:1,000) and α-smooth muscle actin (α-SMA; cat. no. 19245; 1:1,000) were obtained from Cell Signaling Technology, Inc.

Techniques: Expressing, Saline, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Isolation

Journal: International Journal of Molecular Medicine

Article Title: Orcinol glucoside ameliorates pulmonary fibrosis by suppressing hyaluronic acid synthesis and macrophage M2 polarization via targeting hyaluronic acid synthase 2

doi: 10.3892/ijmm.2026.5764

Figure Lengend Snippet: OG exerts an anti-pulmonary fibrosis effect by impairing myofibroblast-mediated macrophage M2 polarization. (A) Schematic workflow: Fibroblast treatment with OG during TGF-β1 stimulation, followed by conditioned medium collection and macrophage culture for downstream assays. (B) TGF-β1 release from macrophages measured by ELISA (n=3 for each group). (C) Flow cytometric analysis of CD206 and CD86 expression in macrophages cultured in conditioned medium. The bar graph quantifies the proportion of CD206 + CD86 − cells (M2-like macrophage subpopulation; n=3 for each group). (D) Animal experimental protocol. (E) A BLM-induced mouse model was established to assess the anti-fibrotic effects of OG. Histopathological alterations in lung tissues from different groups were assessed using H&E and Masson's trichrome staining. Scale bars: 500 μ m; magnification, ×40. (F) Western blotting analysis to measure the expression levels of fibrotic markers, COL1A1 and α-SMA, in lung tissues (n=4 for each group). (G) The levels of HA in mouse serum were quantified by ELISA. (H) The levels of TGF-β1 in mouse serum were quantified by ELISA (n=6 for each group). (I) OG concentrations in serum and lung tissues after 14-day oral administration. Left: serum concentrations of OG across treatment groups. Right: lung tissue concentrations of OG, expressed as a percentage of tissue weight (n=3 for each group). * P<0.05, ** P<0.01, *** P<0.001. OG, glucoside; BLM, bleomycin; COL1A1, Collagen type I α 1 chain; α-SMA, α-smooth muscle actin; H&E, hematoxylin and eosin.

Article Snippet: Antibodies for Collagen type I α 1 chain (COL1A1; cat. no. 72026; 1:1,000), Phospho-STAT6 (Tyr641; cat. no. 56554S; 1:1,000) and α-smooth muscle actin (α-SMA; cat. no. 19245; 1:1,000) were obtained from Cell Signaling Technology, Inc.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Staining, Western Blot

Journal: Biomedical reports

Article Title: Octanoyl esterification of low molecular weight sulfated galactan enhances the cellular uptake and collagen expression in fibroblast cells.

doi: 10.3892/br.2023.1681

Figure Lengend Snippet: Figure 4. Effect of Oct‑LMSG on type I collagen mRNA transcription in L929 fibroblasts. The mRNA levels of Col1A1 and Col1A2 were determined by reverse transcription‑quantitative PCR assay after treatment with either Oct‑LMSG (100 µg/ml) or TGF‑β (10 ng/ml) for 6, 12 and 24 h relative to GAPDH. Values are expressed as the mean ± standard error of the mean of three independent experiments. *P<0.05 vs. control. Oct‑LMSG, low molecular weight sulfated galactan added with octanoyl moiety; Col1A1, collagen type I α1 chain.

Article Snippet: After 12 h of treatment, cells were incubated with anti‐collagen type I α1 chain (Col1A1) antibody (dilution, 1:250; cat. no. A22089; ABclonal Science, Inc.) overnight at 4 ̊C, followed by incu‐ bation with the secondary antibody, FITC‐conjugated goat anti‐rabbit IgG (dilution, 1:500; cat. no. 701078; Thermo Fisher Scientific, Inc.), for 1 h at RT.

Techniques: Control, Molecular Weight

Journal: Biomedical reports

Article Title: Octanoyl esterification of low molecular weight sulfated galactan enhances the cellular uptake and collagen expression in fibroblast cells.

doi: 10.3892/br.2023.1681

Figure Lengend Snippet: Figure 5. Effect of Oct‑LMSG on type I collagen protein expression in L929 fibroblasts. The Col1A1 and Col1A2 protein levels were detected by western blot assay after treatment with either Oct‑LMSG (100 µg/ml) or TGF‑β (10 ng/ml) for 6, 12 and 24 h relative to β‑actin. Values are expressed as the mean ± standard error of the mean of three independent experiments. *P<0.05 vs. control. Oct‑LMSG, low molecular weight sulfated galactan added with octanoyl moiety; Col1A1, collagen type I α1 chain.

Article Snippet: After 12 h of treatment, cells were incubated with anti‐collagen type I α1 chain (Col1A1) antibody (dilution, 1:250; cat. no. A22089; ABclonal Science, Inc.) overnight at 4 ̊C, followed by incu‐ bation with the secondary antibody, FITC‐conjugated goat anti‐rabbit IgG (dilution, 1:500; cat. no. 701078; Thermo Fisher Scientific, Inc.), for 1 h at RT.

Techniques: Expressing, Western Blot, Control, Molecular Weight

Journal: Biomedical reports

Article Title: Octanoyl esterification of low molecular weight sulfated galactan enhances the cellular uptake and collagen expression in fibroblast cells.

doi: 10.3892/br.2023.1681

Figure Lengend Snippet: Figure 6. Effect of Oct‑LMSG on collagen production in L929 fibroblasts. The collagen synthesis was observed by Col1A1‑immunofluorescence staining assay. Fluorescent micrographs of Col1A1‑stained fibroblasts. Cells were treated with either Oct‑LMSG (100 µg/ml) or TGF‑β (10 ng/ml) for 12 h and stained with Col1A1, followed by secondary conjugation with FITC antibodies. Cells were counterstained with DAPI (nucleus) and CellMask Deep Red (cell cytoskeleton). Collagen‑Col1A1 is represented by fluorescent green, DAPI by fluorescent blue and CellMask Deep Red by fluorescent red. The negative control was prepared by omission of the secondary antibody, resulting in the absence of fluorescent green (data not shown). Merged fluorescent micrographs are also provided (scale bars, 75 µm). Oct‑LMSG, low molecular weight sulfated galactan added with octanoyl moiety; Col1A1, collagen type I α1 chain.

Article Snippet: After 12 h of treatment, cells were incubated with anti‐collagen type I α1 chain (Col1A1) antibody (dilution, 1:250; cat. no. A22089; ABclonal Science, Inc.) overnight at 4 ̊C, followed by incu‐ bation with the secondary antibody, FITC‐conjugated goat anti‐rabbit IgG (dilution, 1:500; cat. no. 701078; Thermo Fisher Scientific, Inc.), for 1 h at RT.

Techniques: Staining, Conjugation Assay, Negative Control, Molecular Weight

Journal: Molecular Medicine Reports

Article Title: Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse

doi: 10.3892/mmr.2016.5264

Figure Lengend Snippet: Mechanical strain induces hUSLF apoptosis and senescence and suppresses collagen anabolism. The hUSLF were exposed with or without mechanical strain of 5333 µ ε. (A) Cellular apoptosis was observed by staining with Annexin V/PI and detected by flow cytometry. (B) Cell senescence was evaluated by positive senescence-associated-β-galactosidase percentage. Scale bars, 100 µ m and arrows indicate examples of positive staining. (C and D) The expression of COL1A1 were assessed by western blotting. The level of COL1A1 protein concentration was calculated and normalized to the level of GAPDH protein concentration. All experiments were performed in triplicate. * P<0.05 vs. the control group. hUSLF, human uterosacral ligament fibroblasts; PI, propidium iodide; COL1A1, procollagen type 1 α1.

Article Snippet: The following primary antibodies were used: Rabbit polyclonal Akt (1:500; cat. no. 9272), rabbit monoclonal phosphorylated (p)-Akt (1:500; cat. no. 4058), mouse monoclonal FOXO1 (1:1,000; cat. no. 97635) and rabbit polyclonal p-FOXO1 (1:1,000; cat. no. 9461) all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); mouse monoclonal Mn-superoxide dismutase (Mn-SOD; 1:500; cat. no. sc-130345) and rabbit polyclonal procollagen type I α1 (COL1A1; 1:1,000; cat. no. sc-28657) all obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); and rabbit polyclonal GPX1 (1:1,000; cat. no. ab22604) and rabbit polyclonal GAPDH (1:1,000; cat. no. ab9485) obtained from Abcam, Cambridge, UK).

Techniques: Staining, Flow Cytometry, Expressing, Western Blot, Protein Concentration, Control

Journal: Molecular Medicine Reports

Article Title: Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse

doi: 10.3892/mmr.2016.5264

Figure Lengend Snippet: Mechanical strain induces hUSLF apoptosis and senescence and suppresses collagen I production via the PI3K/Akt signaling pathway. The hUSLF were treated with or without mechanical strain of 5333 µ ε for 4 h in the presence or absence of LY294002. Cells were incubated with LY294002 (20 µ M) for 30 min prior to the application of mechanical strain. (A and B) Cellular apoptosis was detected by Annexin V/PI double staining and flow cytometer. (C and D) Cell senescence was evaluated by positive SA-β-gal percentage. Scale bars, 100 µ m. (E–G) The mRNA and protein expression levels of COL1A1 were assessed by reverse transcription-quantitative polymerase chain reaction and western blotting. The level of COL1A1 mRNA and protein expression were calculated and normalized to the level of GAPDH. All experiments were performed in triplicate. * P<0.05, ** P<0.01, *** P<0.001 vs. other groups or groups indicated. hUSLF, human uterosacral ligament fibroblasts; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PI, propidium iodide; COL1A1, procollagen type 1 α1.

Article Snippet: The following primary antibodies were used: Rabbit polyclonal Akt (1:500; cat. no. 9272), rabbit monoclonal phosphorylated (p)-Akt (1:500; cat. no. 4058), mouse monoclonal FOXO1 (1:1,000; cat. no. 97635) and rabbit polyclonal p-FOXO1 (1:1,000; cat. no. 9461) all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); mouse monoclonal Mn-superoxide dismutase (Mn-SOD; 1:500; cat. no. sc-130345) and rabbit polyclonal procollagen type I α1 (COL1A1; 1:1,000; cat. no. sc-28657) all obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); and rabbit polyclonal GPX1 (1:1,000; cat. no. ab22604) and rabbit polyclonal GAPDH (1:1,000; cat. no. ab9485) obtained from Abcam, Cambridge, UK).

Techniques: Incubation, Double Staining, Flow Cytometry, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Medicine Reports

Article Title: Role of mechanical strain-activated PI3K/Akt signaling pathway in pelvic organ prolapse

doi: 10.3892/mmr.2016.5264

Figure Lengend Snippet: Apoptosis level increased and collagen type I production was reduced in USLs of POP patients. (A) Cell apoptosis in USL tissue explants were detected using the TUNEL assay. TUNEL stain positive cells stain green and nuclei were stained blue by DAPI. Scale bars, 100 µ m. (B) Apoptosis levels were calculated by percentage of TUNEL positive cells. (C) USL RNA was collected and COL1A1 mRNA expression was detected by reverse transcription-quantitative polymerase chain reaction. GAPDH mRNA served as an internal control. (D and E) COL1A1 concentration in USL was detected by western blotting. GAPDH concentration served as an internal control. The non-POP group served as the control and all experiments performed in triplicate. *** P<0.001 vs. the non-POP group. USL, uterosacral ligament; POP, pelvic organ prolapse; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole; COL1A1, procollagen type 1 α1.

Article Snippet: The following primary antibodies were used: Rabbit polyclonal Akt (1:500; cat. no. 9272), rabbit monoclonal phosphorylated (p)-Akt (1:500; cat. no. 4058), mouse monoclonal FOXO1 (1:1,000; cat. no. 97635) and rabbit polyclonal p-FOXO1 (1:1,000; cat. no. 9461) all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA); mouse monoclonal Mn-superoxide dismutase (Mn-SOD; 1:500; cat. no. sc-130345) and rabbit polyclonal procollagen type I α1 (COL1A1; 1:1,000; cat. no. sc-28657) all obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA); and rabbit polyclonal GPX1 (1:1,000; cat. no. ab22604) and rabbit polyclonal GAPDH (1:1,000; cat. no. ab9485) obtained from Abcam, Cambridge, UK).

Techniques: TUNEL Assay, Staining, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Concentration Assay, Western Blot