Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: NAD metabolism is disrupted in tendinopathic tendons in vivo . a . A schematic diagram of the harvested region of human Achilles tendons. (The mid-portion of Achilles tendon) and the gross image of healthy and AT tendons. b. NAD + /NADH ratio and quantification of NAD metabolism (including, NAD + , NADH, total NAD pool) in human Achilles tendons. c. Quantification of ATP production. d. GSEA revealing top NAD metabolism-related GO terms enriched in human Achilles tendons. e. GSEA revealing top degeneration-related GO terms enriched in human Achilles tendon. f. Images of H&E staining, AB (Alcian blue) staining and Masson staining of human normal tendons and tendinopathic tendons. Bonar scores. g. Immunohistochemical staining of COL1 and COL3. h. Immunohistochemical staining of PARP and NOX4. i. Quantitative analysis of COL1, COL3, PARP, NOX4, and ratio of COL3/COL1. j. Immunohistochemical staining and quantification of TUNEL. Scale bar: 50 μm. The results are presented as medians with 95% CIs. n = 3; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: In Vivo, Staining, Immunohistochemical staining, TUNEL Assay

Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: MB corrects NAD dysmetabolism and matrix degeneration in the rat tendinopathy model. a . A schematic illustration of the rat tendinopathy model. b. Gross image of the Rat AT model. c. Gait analysis results for the different groups. The blue dotted line represents stride length; the red dotted line represents step length. Blue print: forepaw; red print: hind paw. d. Quantification of stride length, step length, and the length of the front/rear paw prints. e. Hot plate test. f. Paw contraction thresholds were assessed utilizing von Frey fibers to gauge mechanical sensitivity (n = 6). g. Quantification of ATP in the rat AT model. h. NAD + /NADH ratio i. Quantification of NAD metabolism (including, NAD + , NADH, total NAD pool). j. H&E staining, AB staining and Masson staining. Bonar scores of rats AT model. k. Immunohistochemical staining of Col1 and Col3. l. Immunohistochemical staining of Parp and Nox4. m. Quantitative analysis of Col1, Col3, Parp, Nox4. n. TUNEL staining and quantitative analysis of apoptotic cells. Scale bar: 50 μm. The results are presented as medians with 95% CIs. n = 5; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: Hot Plate Test, Staining, Immunohistochemical staining, TUNEL Assay

Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: MB reduces TBHP-induced NAD metabolism disorder in vitro and in vivo. a . The cell viability. b. Quantitation of NAD metabolism (including NAD + /NADH ratio, NAD + , NADH, total NAD pool) in rat tenocytes. c. Quantitation of ATP in rat tenocytes. d. Representative Western blot results for Col3, Col1, MMP3, and MMP13 in rat tenocytes. e. Ratio of Col3/Col1 by d. f. Representative Western blot results for Parp, c-Parp, Nox4, Bcl2, and Bax. g, h. Immunofluorescence and quantification analysis of Col1, Col3, Parp, and Nox4 expression in rat tenocytes. The ratio of Col1/Col3. DAPI, 4′,6-diamidino-2-phenylindole. i. Fluorescence images of the DCFH-DA probe for hydrogen peroxide in rat tenocytes. j. JC-1 staining in rat tenocytes. k. Quantitative analysis of ROS. l. Quantitative analysis of JC-1. m. Annexin V and activated caspase-3 staining and quantitative analysis of apoptotic cells in rat tenocytes. The results are presented as medians with 95% CIs. n = 3, 5, ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: In Vitro, In Vivo, Quantitation Assay, Western Blot, Immunofluorescence, Expressing, Fluorescence, Staining

Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: MB alleviates TBHP-induced tendinopathy in human Achilles tendon explant. a . A schematic illustration of the human extra explant. b. Quantification of NAD metabolism in human extra explants under TBHP treatment combined with or without MB. (n = 3) c. H&E staining, AB staining, and Masson staining of human tendon explants. The Bonar scores. d. Quantification of ATP in human tendon explants. e. NFR. f. Immunohistochemical staining of COL1 and COL3. g. Immunohistochemical staining of PARP and NOX4. h. Immunohistochemical staining of AIF and NDUFB8. i. Quantification of the immunohistochemical staining data. Scale bar: 50 μm. The results are presented as medians with 95% CIs. n = 5; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: Staining, Immunohistochemical staining

Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: MB alleviates oxidative stress in a MC1-dependent manner in rat tenocytes. a. Quantification of ATP in rat tenocytes after TBHP treatment combined with/without MB or rotenone (Rot, a complex I inhibitor). b. NFR. c. Quantification of NAD metabolism. d. Representative Western blot results for Parp, c-Parp, Nox4, Aif, Col3, Col1, and Ndufb8. e. Parp, Nox4, Aif expression by immunofluorescence microscopy (n = 3). f. Col3, Col1, Ndufb8 expression by immunofluorescence microscopy. g. Quantification of the immunofluorescence data from e and f . h. JC-1 staining. i. Quantitative analysis of JC-1. j, k. Fluorescence images and quantitative analysis of ROS. Scale bar: 50 μm. The results are presented as medians with 95% CIs. n = 3; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: Western Blot, Expressing, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: Journal of Orthopaedic Translation

Article Title: Methylene blue restores NAD + /NADH homeostasis to attenuate Achilles tendinopathy via activating AIF/Mitochondrial respiratory chain complex I

doi: 10.1016/j.jot.2026.101085

Figure Lengend Snippet: MB increases the expression of Aif under Aif knockdown in tenocytes. a. The NAD metabolism after Aif was knocked down in rat tenocytes. b. Quantification of ATP after Aif knockdown. c. NFR. d. Representative Western blot results for Parp, c-Parp, Nox4, Aif, Col3, Col1, and Ndufb8. e. Quantification of the Western blot data from d. f. Quantification of the immunofluorescence staining data . g. Col3, Col1, Parp, Nox4, Ndufb8, and Aif expression by immunofluorescence microscopy; h. Fluorescence images of ROS. i. JC-1 staining. j. Quantification of DCFH-DA staining data. k. Quantification of JC-1 staining data. l. Schematic illustration of the mechanism of MB in tendinopathy. Scale bar: 50 μm. The results are presented as medians with 95% CIs. n = 3; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.

Article Snippet: Then, for antigen retrieval, the sections were incubated with 0.4% pepsin (Sigma–Aldrich) in 1 mM hydrochloric acid at 37 °C for 1 h. After blocking with 5% bovine serum albumin for 30 min at 37 °C, the sections were incubated with primary antibody against Col1(1:1000, #ab138492, Abcam, UK), Col3(1:1000, #A00788-3, Boster, China), Nox4 (1:2000, #14347-1-AP, Proteintech, China), Parp (1:1000, #13371-1-AP, Proteintech, China), Aif (1:1000, #67791-1-Ig, Proteintech, China), Ndufb8 (1:1000, #67690-1-Ig, Proteintech, China), and Gapdh (1:2000, #2118, Cell Signaling Technology, USA) overnight at 4 °C and finally with an HRP-conjugated secondary antibody (#BL003A or #BL001A, Biosharp, China).

Techniques: Expressing, Knockdown, Western Blot, Immunofluorescence, Staining, Microscopy, Fluorescence