Fig 2: Cyclopamine suppresses WISP-1-induced fibrogenesis in fibroblasts.a Effect of WISP-1 on Gli1 and Smo expression in LF cells with or without cyclopamine (5 µM, n = 3). b Cyclopamine suppressed WISP-1-induced collagen expression (n = 3). c EdU staining demonstrated the effect of WISP-1 on LF cell proliferation with or without cyclopamine. Scale bar, 100 µm. d Effect of WISP-1 on the cell cycle of LF cells with or without cyclopamine (n = 3). e Flow cytometry was used to analyze the effect of Hedgehog signaling on WISP-1-induced apoptosis (n = 3). *P < 0.05, $P < 0.05, **P < 0.01, ##P < 0.01 compared with the control group. LF ligamentum flavum.

Fig 3: WISP-1 activates Hedgehog-Gli1 signaling in LF cells.a, b RT-PCR (a) and western blot (b) analyses of the effect of WISP-1 on the expression of Hedgehog-Gli1-related proteins in LF cells (n = 3). c Quantification of Gli1 protein expression in human LF cells treated with different doses of recombinant human WISP-1 (n = 3). d Effect of WISP-1 on the nuclear translocation of Gli1 in human LF cells as measured by immunofluorescence; scale bar, 100 μm. *P < 0.05, **P < 0.01 compared with the control group. LF ligamentum flavum.

Fig 4: Hedgehog-Gli1 signaling is activated in hypertrophic LF and is important for cell activity and collagen expression.a–c Expression levels of Gli1 and Shh in LF tissues as detected by RT-PCR (a) and western blotting (b, c) (n = 21). d Representative images of immunohistochemical staining of Gli1 and Shh in LF specimens from the two groups (n = 3). e Correlation analysis of Gli1 expression with the fibrosis score and LF thickness. f Phenotype identification of LF cells; scale bar, 100 μm. g Gli1 overexpression plasmids and Gli1-silencing shRNAs were transfected into human LF fibroblasts, and cloning was performed. The efficiency of Gli1 expression was tested by western blotting. h MTT was used to detect the effect of Gli1 expression on LF cell proliferation. i, j The effect of Gli1 expression on LF cell apoptosis was assessed by flow cytometry (n = 3). k, l Effect of Gli1 on collagen expression in LF cells (n = 3); scale bar, 100 μm. The results shown are the means ± SDs; *P < 0.05, **P < 0.01, ##P < 0.001 compared with the control group. LF ligamentum flavum, LSCS lumbar spinal canal stenosis, LDH lumbar disc herniation.

Fig 5: WISP-1 induces α-SMA expression via Hedgehog-Gli1 signaling.a Immunofluorescence analysis of α-SMA in human LF specimens. b–d Effect of WISP-1 on α-SMA expression in human LF cells as detected by RT-PCT (b), western blotting (c) and immunofluorescence (d) (n = 3). e, f Effect of cyclopamine on WISP-1-induced α-SMA expression as measured by western blot (e) and immunofluorescence staining (f) (n = 3). Scale bar, 100 µm. **P < 0.01, $$P < 0.05, ##P < 0.01, ***P < 0.001, compared with the control group. LF ligamentum flavum, LSCS lumbar spinal canal stenosis, LDH lumbar disc herniation.