Table 1.
Specific primers in qRT‐PCR analysis.
Table 2.
Specific primary antibodies in Western blot.
Fig 1.
RD ameliorates BLM‐induced skin fibrosis in vivo.
(a) Representative skin sections stained with hematoxylin–eosin (H&E), Sirius red and Masson’s trichrome staining (20×, Scale bar = 50μm) (n = 6). Total dermal thickness of the back of each group of mice based on Hematoxylin–eosin (H&E) images. Collagen density was quantified on Masson’s trichrome and Sirius red images. (b) Immunohistochemical staining analysis and of α-SMA in the lesional skin of each group (10× and 20×, Scale bar = 50μm) (n = 6). (c) Hydroxyproline content of skin tissues in C57BL/6J mice (n = 6). (d) The mRNA levels of Col1α1 in the lesional skin (n = 6). (e) The mRNA levels of Col1α2 in the lesional skin (n = 6). (f) The mRNA levels of α‐SMA in the lesional skin (n = 6). (g) The protein levels of Col-Ⅰ and α‐SMA in the lesional skin (n = 6). The data are shown as mean ± SD (one-way ANOVA with Tukey’s post-hoc multiple comparison tests). ***, p < 0.001, ****, p < 0.0001 vs. NaCl; #, p < 0.05, ##, p < 0.01, ###, p < 0.001, ####, p < 0.0001 vs. BLM. BLM, Bleomycin; RD, Remdesivir.
Fig 2.
RD alleviates keloid xenografts-induced skin fibrosis in vivo.
(a) Macrographic images and weight of xenografted tissues on the back of nude BALB/c mice after intralesional injection of RD (n = 3). (b) The mRNA levels of Col1α1 in xenografted keloid tissues (n = 3). (c) The mRNA levels of Col3α1 in xenografted keloid tissues (n = 3). (d) The mRNA levels of α‐SMA in xenografted keloid tissues (n = 3). The data are shown as mean ± SD (one-way ANOVA with Tukey’s post-hoc multiple comparison tests). ***, p < 0.001, ****, p < 0.0001 vs. NaCl. BLM, Bleomycin; RD, Remdesivir.
Fig 3.
RD alleviates mouse primary skin fibroblasts and xenografted keloid fibroblasts autophagy in vivo.
(a) Immunofluorescence staining of p62 in skin frozen sections of BLM‐induced model (10× and 20×, Scale bar = 50μm) (n = 6). (b) The protein levels of p62 and LC3 in the lesional skin (n = 6). (c) The mRNA levels of p62 in xenografted keloid tissues (n = 3). (d) The mRNA levels of LC3 in xenografted keloid tissues (n = 3). The data are shown as mean ± SD (one-way ANOVA with Tukey’s post-hoc multiple comparison tests). *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 vs. NaCl; ##, p < 0.01, ###, p < 0.001, ####, p < 0.0001 vs. BLM. BLM, Bleomycin; RD, Remdesivir.
Fig 4.
RD attenuates TGF-β1-induced mouse primary skin fibroblasts activation in vitro.
(a) CCK-8 assays of PSFs toxic (n = 6). Cells were exposed to the indicated doses of RD (0 to 80μM) for 24 h, IC50 = 74.49μM. (b) CCK-8 assays of PSFs proliferation (n = 6). Cells were performed to test the effect of RD on cell proliferation of TGF-β1-stimulated PSFs. (c) Representative images and quantitative results of EdU incorporation assay in PSFs (n = 6). The ratio of EdU‐positive cells to DAPI‐labeled cells in each group was determined (×40, Scale bar = 100μm). (d) Representative images and quantitative results of migration in PSFs (n = 6). Representative images were captured and counted under a fluorescence microscope at ×20 (scale bar = 100μm). (e) Representative images and quantitative analysis of wound healing assay in PSFs (n = 3). The wound closure was captured at 0, 12, 24, 36, and 48h after scratch generation. (f) The protein levels of Col-Ⅰ and α-SMA in PSFs (n = 3). PSFs were treated with RD (12.5, 25μM) and TGF-β1 (5ng/ml) for 24h. The data are shown as mean ± SD (one-way ANOVA with Tukey’s post-hoc multiple comparison tests). *, p < 0.05, ***, p < 0.001, ****, p < 0.0001 vs. Control; #, p < 0.05, ##, p < 0.01, ###, p < 0.001, ####, p < 0.0001 vs. TGF-β1. RD, Remdesivir.
Fig 5.
RD attenuates keloid fibroblasts activation in vitro.
(a) CCK-8 assays of KFs toxic (n = 6). Cells were exposed to the indicated doses of RD (0 to 80μM) for 24h, IC50 = 81.7μM. (b) CCK-8 assays of KFs proliferation (n = 6). Cells were performed to test the effect of Remdesivir on cell proliferation of TGF-β1-stimulated KFs. (c) Representative images and quantitative results of migration in KFs (n = 6). Representative images were captured and counted under a fluorescence microscope at ×20 (Scale bar = 100μm). (d) Representative images of tissue explants of KFs (n = 3). KFs were cultured with RD (12.5, 25μM) at day 9 (×40, scale bar = 200μm). The cell numbers that migrated out of the tissue explants were quantified at day 9. (e) Representative images and quantitative analysis of wound healing assay in KFs (n = 3). The wound closure was captured at 0, 12, 24 and 48h after scratch generation. (f) The protein levels of Col-Ⅰ and α-SMA in KFs (n = 3). KFs were treated with RD (12.5, 25μM) for 24 h. The data are shown as mean ± SD (one-way ANOVA with Tukey’s post-hoc multiple comparison tests). *, p < 0.05, ***, p < 0.001, ****, p < 0.0001 vs. Control. RD, Remdesivir.
Fig 6.
RD suppresses TGF-β1/Smad signaling pathway both in vivo and in vitro.
(a) The protein levels of phosphorylated Smad3 in PSFs (n = 3). (b) The protein levels of phosphorylated Smad3 in KFs (n = 3). (c) The protein levels of phosphorylated Smad3 in mice skin tissues of each group (n = 6). PSFs were treated with TGF-β1 (5ng/mL) and RD (12.5, 25μM) for 24h. KFs were treated with RD (12.5, 25μM) for 24h. The data are shown as mean ± SD (one-way ANOVA with Tukey’s post-hoc multiple comparison tests). *, p < 0.05, **, p < 0.01, ****, p < 0.0001 vs. Control or NaCl. #, p < 0.05, ##, p < 0.01, ###, p < 0.001, ####, p < 0.0001 vs. TGF-β1 or BLM. BLM, Bleomycin; RD, Remdesivir.
Fig 7.
RD attenuates mouse primary skin fibroblasts and keloid fibroblasts autophagy in vitro.
(a) The protein levels of p62 and LC3 in PSFs (n = 3). (b) Immunofluorescence staining of p62 in PSFs (n = 3). (c) The protein levels of p62 and LC3 in KFs (n = 3). (d) Immunofluorescence staining of p62 in KFs (n = 3). PSFs were treated with TGF-β1 (5ng/mL) and RD (12.5, 25μM). KFs were treated with RD (12.5, 25 μM) for 24h. The data are shown as mean ± SD (one-way ANOVA with Tukey’s post-hoc multiple comparison tests). *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 vs. Control; ####, p < 0.0001 vs. TGF-β1. RD, Remdesivir.
Fig 8.
RD suppresses autophagy by PI3K/Akt/mTOR signaling pathway both in vivo and in vitro.
(a) The protein levels of phosphorylated Akt, PI3K and mTOR in PSFs (n = 3). (b) The protein levels of phosphorylated Akt, PI3K and mTOR in KFs (n = 3). (c) The protein levels of phosphorylated Akt, PI3K and mTOR in mice pathogenic skin tissues of each group (n = 6). PSFs were treated with TGF-β1 (5 ng/mL) and RD (12.5, 25μM) for 24h. KFs were treated with RD (12.5, 25 μM) for 24h. The data are shown as mean ± SD (one-way ANOVA with Tukey’s post-hoc multiple comparison tests). *, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 vs. Control or NaCl. ##, p < 0.01, ####, p < 0.0001 vs. TGF-β1 or BLM. BLM, Bleomycin; RD, Remdesivir.
Fig 9.
Mechanism for the anti-skin fibrosis effect of RD.