Journal: Molecular Medicine Reports
Article Title: Exploring the role of cytochrome P450 family 1 subfamily B member 1 and quercetin in modulating neuropathic pain after spinal cord injury
doi: 10.3892/mmr.2025.13717
Figure Lengend Snippet: (A) Flow chart of the animal experiment. (B) MWT measurements for the SHAM, SCI and SCI + Que groups were recorded on day 14. (C) TWL for the SHAM, SCI and SCI + Que groups was also measured on day 14. (D) BMS scores for the SHAM, SCI and SCI + Que groups were assessed at baseline and on postoperative days 1, 3, 5, 7, 10 and 14. A statistically significant difference was observed (***P<0.001) between the SCI and SHAM groups of mice (n=6 each). Additionally, a significant difference was noted between the SCI and SCI + Que groups (*P<0.05), with n=6. (E) RT-qPCR assessment of CYP1B1 mRNA levels in spinal cord tissues from SHAM, SCI and SCI + Que groups, with expression levels normalized to GAPDH. (F) Semi-quantitative analysis of CYP1B1 protein levels in spinal cord tissues from the SHAM, SCI and SCI + Que groups, with fold-changes normalized to GAPDH. (G) Western blot analysis was conducted to assess CYP1B1 expression in spinal cord tissues from the SHAM, SCI and SCI + Que groups on day 14 post-modeling. (H) Immunofluorescence analysis was performed to assess the expression levels of CYP1B1 and the fibroblast marker PDGF-D in spinal cord tissues from the SHAM, SCI and SCI + Que groups, with n=6. Scale bar, 50 µm. (I) Quantitative fluorescence analysis of CYP1B1 expression was conducted on spinal cord tissues from the SHAM, SCI and SCI + Que groups. (J) RT-qPCR evaluation of CYP1B1 mRNA expression in fibroblasts treated under control conditions, with 10 ng/ml TGF-β and with 10 ng/ml TGF-β + 10 µmol/ml Que, with mRNA levels normalized to GAPDH. (K) Western blot analysis was performed to evaluate CYP1B1 expression in fibroblasts subjected to control conditions, 10 ng/ml TGF-β stimulation or co-treatment with 10 ng/ml TGF-β and 10 µmol/ml Que. (L) Semi-quantitative evaluation of CYP1B1 protein expression in fibroblasts subjected to control conditions, treated with 10 ng/ml TGF-β or treated with 10 ng/ml TGF-β combined with 10 µmol/ml Que, with protein levels normalized to GAPDH. (M) A semi-quantitative analysis was conducted on the Fn protein levels in fibroblasts subjected to control conditions, 10 ng/ml TGF-β treatment or treatment with a combination of 10 ng/ml TGF-β and 10 µmol/ml Que, with the resulting fold-changes normalized against GAPDH. (N) Western blot analysis was utilized to investigate Fn expression in fibroblasts under control conditions, after treatment with 10 ng/ml TGF-β or after co-treatment with 10 ng/ml TGF-β and 10 µmol/ml Que. The data are presented as the mean ± SEM, with sample sizes of n=6 per group for (B, C, D, E, F, G, H and I), n=3 per group for (J) and n=4 per group for (K, L, M and N). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. BMS, Basso Mouse Scale; Con, control; CYP1B1, cytochrome P450 family 1 subfamily B member 1; PDGF-D, platelet-derived growth factor D; dpi, days post-injury; Fn, Fibronectin; MWT, mechanical withdrawal threshold; Que, quercetin; RT-qPCR, reverse transcription-quantitative PCR; SCI, spinal cord injury; SHAM, sham surgery group; TWL, thermal withdrawal latency.
Article Snippet: The protein samples were then transferred to polyvinylidene fluoride membranes, which were subsequently blocked for 1 h at room temperature., washed with TBST solution and incubated with the following primary antibodies: CYP1B1 (1:500; cat. no. 18505-1-AP; Proteintech Group, Inc.), Fibronectin (Fn) (1:1,000; cat. no. ab2413; Abcam) and GAPDH (1:50,000; cat. no. 1E6D9; Proteintech Group, Inc.).
Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunofluorescence, Marker, Fluorescence, Control, Derivative Assay, Reverse Transcription, Real-time Polymerase Chain Reaction
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