Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: d-Mannose suppresses osteoarthritis development in vivo and delays IL-1β-induced degeneration in vitro by enhancing autophagy activated via the AMPK pathway.

doi: 10.1016/j.biopha.2020.111199

Figure Lengend Snippet: Fig. 1. D-mannose promotes the proliferation and reduces apoptosis of IL-1β-treated rat chondrocytes. (A) Verification of chondrocytes with toluidine blue, alcian blue, and safranin O staining and IHC for COL2A1. (B) D-mannose cytotoxicity in chondrocytes measured using the CCK-8 assay. Chondrocytes were incubated with various D-mannose concentrations (0, 5, 10, 25, and 50 mM) for 12 h and (C) with 25 mM D-mannose for various time periods (0, 6, 12, 24, and 48 h). (D) Chondrocytes were stimulated with IL-1β (10 ng/mL) for 12 h after preincubation with D-mannose (25 mM) for 12 h (DM 12 h) and 24 h (DM 24 h), followed by evaluation with the CCK-8 assay. (E) Cell proliferation evaluated with EdU staining and (F) quantified in IL-1β-treated chondrocytes after preincubation with D- mannose. The Edu + rate = Edu + cells / total cells. (G) Analysis and (H) quantification of flow cytometry results using IL-1β-treated chondrocytes following preincubation with D-mannose. Data represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. CCK-8, Cell Counting Kit-8; Ctrl, control; COL2A1, collagen, type 2, α1; IHC, immunohistochemistry; IL, interleukin; ns, not significant; SD, standard deviation.

Article Snippet: To investigate whether D-mannose regulates OA degeneration by autophagy, culture medium was replaced with a mixture of IL-1β+autophagy inhibitor [5 mM 3-MA dissolved in dimethyl sulfoxide (DMSO); 3-MA was acquired from MedChemExpress, Monmouth Junction, NJ, USA] or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h. To evaluate whether D-mannose regulates autophagy by phosphorylating AMPK, culture medium was replaced with a mixture of IL-1β+AMPK inhibitor compound C (5 μM; dissolved in DMSO; MedChemExpress) or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h.

Techniques: Staining, CCK-8 Assay, Incubation, Flow Cytometry, Cell Counting, Control, Immunohistochemistry, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: d-Mannose suppresses osteoarthritis development in vivo and delays IL-1β-induced degeneration in vitro by enhancing autophagy activated via the AMPK pathway.

doi: 10.1016/j.biopha.2020.111199

Figure Lengend Snippet: Fig. 2. D-mannose promotes autophagy and delays catabolism in IL-1β-treated rat chondrocytes. (A) Ultrastructures and (B) quantification of APs observed with TEM in chondrocytes stimulated with IL-1β (10 ng/mL) for 12 h following preincubation with D-mannose (25 mM) for 12 h (DM 12 h) or 24 h (DM 24 h). Boxes show high-magnification regions, and blue arrows show APs. (C) Chondrocytes were transfected with mRFP-GFP-LC3 adenovirus, and (D) fluorescence was quantified following incubation with D-mannose and IL-1β. Merge (yellow) showing APs, free mRFP (red) showing ALs. (E) qPCR analysis of Col2a1 and Mmp13, with β-actin used as a control, in chondrocytes incubated with D-mannose and IL-1β. (F) Immunoblot and (G) quantification of COL2A1, ADAMTS5, MMP13, and LC3 II/I, and SQSTM1, with β-actin used as a control, in chondrocytes incubated with D-mannose and IL-1β. Data represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ADAMTS, a disintegrin-like and metalloproteinase with thrombospondin-1 motifs; AL, autolysosome; AP, autophagosome; Ctrl, control; COL2A1, collagen, type 2, α1; IL, interleukin; LC, light chain; MMP, matrix metalloproteinase; ns, not significant; qPCR, quantitative polymerase chain reaction; SD, standard deviation; TEM, transmission electron microscopy.

Article Snippet: To investigate whether D-mannose regulates OA degeneration by autophagy, culture medium was replaced with a mixture of IL-1β+autophagy inhibitor [5 mM 3-MA dissolved in dimethyl sulfoxide (DMSO); 3-MA was acquired from MedChemExpress, Monmouth Junction, NJ, USA] or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h. To evaluate whether D-mannose regulates autophagy by phosphorylating AMPK, culture medium was replaced with a mixture of IL-1β+AMPK inhibitor compound C (5 μM; dissolved in DMSO; MedChemExpress) or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h.

Techniques: Transfection, Fluorescence, Incubation, Control, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Transmission Assay, Electron Microscopy

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: d-Mannose suppresses osteoarthritis development in vivo and delays IL-1β-induced degeneration in vitro by enhancing autophagy activated via the AMPK pathway.

doi: 10.1016/j.biopha.2020.111199

Figure Lengend Snippet: Fig. 3. A median dose of D-mannose suppresses OA progression in MIA-treated rats. (A) Schematic model of the time course for establishment of the MIA model of OA rats treated with D-mannose (DM) by gavage administration at different doses (low, median, and high; 5 days/week from weeks 6 to 10). (B) Weight change in MIA-treated rats from week 6 to 10 after gavage administration of D-mannose. (C) Gross images, MRI results, plain radiographs, and (D) macroscopic scores of gross imaging in MIA-treated rats following gavage administration of D-mannose. (E) Toluidine Blue staining of knee joints and (F) Makin and OARSI scores from MIA- treated rats following gavage administration of D-mannose. Boxed regions show high-magnification images. (G) Synovitis scores in MIA-treated rats following gavage administration of D-mannose. Data represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Ctrl, gavage of drinking water for 4 weeks + articular cavity injection of saline; MIA, monosodium iodoacetate; MIA, gavage of drinking water for 4 weeks + articular cavity injection of MIA; MIA&DM-H, gavage of high-dose D-mannose for 4 weeks + articular cavity injection of MIA; MIA&DM-L, gavage of low-dose D-mannose for 4 weeks + articular cavity injection of MIA; MIA&DM-M, gavage of median-dose D-mannose for 4 weeks + articular cavity injection of MIA; MRI, magnetic resonance imaging; ns, not significant; OARSI, Osteoarthritis Research Society International; OA, osteoarthritis.

Article Snippet: To investigate whether D-mannose regulates OA degeneration by autophagy, culture medium was replaced with a mixture of IL-1β+autophagy inhibitor [5 mM 3-MA dissolved in dimethyl sulfoxide (DMSO); 3-MA was acquired from MedChemExpress, Monmouth Junction, NJ, USA] or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h. To evaluate whether D-mannose regulates autophagy by phosphorylating AMPK, culture medium was replaced with a mixture of IL-1β+AMPK inhibitor compound C (5 μM; dissolved in DMSO; MedChemExpress) or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h.

Techniques: Imaging, Staining, Injection, Saline, Magnetic Resonance Imaging

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: d-Mannose suppresses osteoarthritis development in vivo and delays IL-1β-induced degeneration in vitro by enhancing autophagy activated via the AMPK pathway.

doi: 10.1016/j.biopha.2020.111199

Figure Lengend Snippet: Fig. 4. A median dose of D-mannose attenuates OA catabolism and promotes autophagy in MIA-treated rats. (A) qPCR analysis of Col2A1 and Mmp13, with β-actin used as a control, in MIA-treated OA model rats following gavage administration of D-mannose. (B) Immunoblot and (C) quantification of COL2A1, ADAMTS5, MMP13, and LC3 II/I, and SQSTM1, with β-actin used as a control. Data represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ADAMTS, a disintegrin-like and metalloproteinase with thrombospondin-1 motifs; COL2A1, collagen, type 2, α1; Ctrl, gavage of drinking water for 4 weeks + articular cavity injection of saline; LC, light chain; MIA, monosodium iodoacetate; MIA, gavage of drinking water for 4 weeks + articular cavity injection of MIA; MIA&DM-H, gavage of high-dose D-mannose for 4 weeks + articular cavity injection of MIA; MIA&DM-L, gavage of low-dose D-mannose for 4 weeks + articular cavity injection of MIA; MIA&DM-M, gavage of median-dose D-mannose for 4 weeks + articular cavity injection of MIA; ns, not significant; MMP, matrix metal loproteinase; ns, not significant; OA, osteoarthritis; qPCR, quantitative polymerase chain reaction.

Article Snippet: To investigate whether D-mannose regulates OA degeneration by autophagy, culture medium was replaced with a mixture of IL-1β+autophagy inhibitor [5 mM 3-MA dissolved in dimethyl sulfoxide (DMSO); 3-MA was acquired from MedChemExpress, Monmouth Junction, NJ, USA] or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h. To evaluate whether D-mannose regulates autophagy by phosphorylating AMPK, culture medium was replaced with a mixture of IL-1β+AMPK inhibitor compound C (5 μM; dissolved in DMSO; MedChemExpress) or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h.

Techniques: Control, Western Blot, Injection, Saline, Real-time Polymerase Chain Reaction

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: d-Mannose suppresses osteoarthritis development in vivo and delays IL-1β-induced degeneration in vitro by enhancing autophagy activated via the AMPK pathway.

doi: 10.1016/j.biopha.2020.111199

Figure Lengend Snippet: Fig. 5. D-mannose attenuates OA degeneration by activating autophagy in IL-1β-treated rat chondrocytes. (A) Chondrocytes were stimulated with IL-1β (10 ng/mL) and treated with the autophagy inhibitor 3-MA for 12 h following preincubation with D-mannose for 24 h (DM 24 h) and evaluated using the CCK-8 assay. (B) Cell proliferation was evaluated with EdU staining and (C) quantified in chondrocytes. EdU + rate = EdU + cells / total cells. (D) Analysis and (E) quantification of flow cytometry results in chondrocytes. (F) Immunoblot and (G) quantification of COL2A1, ADAMTS5, and MMP13, with β-actin used as a control. Data represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. 3-MA, 3-methyladenine; ADAMTS, a disintegrin-like and metalloproteinase with thrombospondin-1 motifs; CCK-8, Cell Counting Kit-8; COL2A1, collagen, type 2, α1; IL, interleukin; MMP, matrix metalloproteinase; ns, not significant; OA, osteoarthritis; SD, standard deviation.

Article Snippet: To investigate whether D-mannose regulates OA degeneration by autophagy, culture medium was replaced with a mixture of IL-1β+autophagy inhibitor [5 mM 3-MA dissolved in dimethyl sulfoxide (DMSO); 3-MA was acquired from MedChemExpress, Monmouth Junction, NJ, USA] or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h. To evaluate whether D-mannose regulates autophagy by phosphorylating AMPK, culture medium was replaced with a mixture of IL-1β+AMPK inhibitor compound C (5 μM; dissolved in DMSO; MedChemExpress) or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h.

Techniques: CCK-8 Assay, Staining, Flow Cytometry, Western Blot, Control, Cell Counting, Standard Deviation

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: d-Mannose suppresses osteoarthritis development in vivo and delays IL-1β-induced degeneration in vitro by enhancing autophagy activated via the AMPK pathway.

doi: 10.1016/j.biopha.2020.111199

Figure Lengend Snippet: Fig. 6. D-mannose activates autophagy by phosphorylating AMPK in IL-1β-treated rat chondrocytes. (A) Immunoblot and (B) quantification of phosphorylated (p)AMPK and AMPK, with β-actin used as a control, in chondrocytes stimulated with IL-1β (10 ng/mL) for 12 h after preincubation with 25 mM D-mannose for 12 h (DM 12 h) or 24 h (DM 24 h). (C) Immunoblot and (D) quantification of LC3 II/I, beclin1, and SQSTM1, with β-actin used as a control, in chondrocytes. Chondrocytes were stimulated with IL-1β (10 ng/mL) and the AMPK inhibitor compound C for 12 h following preincubation with D-mannose for 24 h. (E) Chondrocytes were transfected with mRFP-GFP-LC3 adenovirus, and (F) fluorescence was quantified following incubation with D-mannose, IL-1β, and compound C as mentioned above. Merge (yellow) showing APs, free mRFP (red) showing ALs. (G) Schematic model of D-mannose-mediated delay of IL-1β-induced degeneration in rat chondrocytes by enhanced autophagy activated through the AMPK pathway. Data represent mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001. AMPK, 5′ AMP-activated protein kinase; IL, interleukin; LC, light chain; OA, osteoarthritis; SD, standard deviation.

Article Snippet: To investigate whether D-mannose regulates OA degeneration by autophagy, culture medium was replaced with a mixture of IL-1β+autophagy inhibitor [5 mM 3-MA dissolved in dimethyl sulfoxide (DMSO); 3-MA was acquired from MedChemExpress, Monmouth Junction, NJ, USA] or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h. To evaluate whether D-mannose regulates autophagy by phosphorylating AMPK, culture medium was replaced with a mixture of IL-1β+AMPK inhibitor compound C (5 μM; dissolved in DMSO; MedChemExpress) or IL-1β+DMSO for 12 h following pretreatment of primary chondrocytes with D-mannose for 24 h.

Techniques: Western Blot, Control, Transfection, Fluorescence, Incubation, Standard Deviation