Figures & data
Figure 1. Neurokinin 1 receptor (NK-1R) is expressed in human fibroblast-like synoviocytes (FLSs). We applied the human glioblastoma cell line U251 as a positive control. (a). RT-PCR results indicate that NK-1R is expressed in human FLSs at the gene level; (b). Western blot results indicate that NK-1R is expressed in human FLSs at the protein level.
![Figure 1. Neurokinin 1 receptor (NK-1R) is expressed in human fibroblast-like synoviocytes (FLSs). We applied the human glioblastoma cell line U251 as a positive control. (a). RT-PCR results indicate that NK-1R is expressed in human FLSs at the gene level; (b). Western blot results indicate that NK-1R is expressed in human FLSs at the protein level.](/cms/asset/c4afebe5-52dc-45a8-b1fc-525fd6fd8f3e/ianb_a_1573177_f0001_c.jpg)
Figure 2. Expression of neurokinin 1 receptor (NK-1R) is increased in rheumatoid arthritis (RA)-FLSs as compared to normal FLSs. (a). Real-time PCR analysis of NK-1R; (b). Western blot analysis of NK-1R (*, p < .01 vs. vehicle group).
![Figure 2. Expression of neurokinin 1 receptor (NK-1R) is increased in rheumatoid arthritis (RA)-FLSs as compared to normal FLSs. (a). Real-time PCR analysis of NK-1R; (b). Western blot analysis of NK-1R (*, p < .01 vs. vehicle group).](/cms/asset/92957fc5-8811-4a71-96c8-8faa178cc093/ianb_a_1573177_f0002_b.jpg)
Figure 3. Expression of neurokinin 1 receptor (NK-1R) is increased in normal FLSs in response to TNF-α treatment (5, 10, 20 ng/ml) in a concentration-dependent manner. (a) Expression of NK-1R at the gene level was determined by real-time PCR analysis; (b). Expression of NK-1R at the protein level was determined by Western blot analysis (*, #, $, p < .01 vs. previous column group).
![Figure 3. Expression of neurokinin 1 receptor (NK-1R) is increased in normal FLSs in response to TNF-α treatment (5, 10, 20 ng/ml) in a concentration-dependent manner. (a) Expression of NK-1R at the gene level was determined by real-time PCR analysis; (b). Expression of NK-1R at the protein level was determined by Western blot analysis (*, #, $, p < .01 vs. previous column group).](/cms/asset/cb9d0e7a-377d-4983-a423-6d5155e17266/ianb_a_1573177_f0003_b.jpg)
Figure 4. The neurokinin-1 receptor (NK-1R) antagonist aprepitant ameliorates TNF-α-induced reactive oxygen species (ROS) production and NOX-4 expression in FLSs. FLSs were incubated with 10 ng/ml TNF-α in the presence or absence of aprepitant (5, 10 μM) for 24 h. (a). ROS generation was determined by DCFH-DA assay; Scale bars, 100 μm; (b). NOX-4 expression was determined by Western blot analysis (*, #, $, p < .01 vs. previous column group).
![Figure 4. The neurokinin-1 receptor (NK-1R) antagonist aprepitant ameliorates TNF-α-induced reactive oxygen species (ROS) production and NOX-4 expression in FLSs. FLSs were incubated with 10 ng/ml TNF-α in the presence or absence of aprepitant (5, 10 μM) for 24 h. (a). ROS generation was determined by DCFH-DA assay; Scale bars, 100 μm; (b). NOX-4 expression was determined by Western blot analysis (*, #, $, p < .01 vs. previous column group).](/cms/asset/ebbd9e4b-6f4f-455c-9d2a-f615fc85aef6/ianb_a_1573177_f0004_c.jpg)
Figure 5. The neurokinin-1 receptor (NK-1R) antagonist aprepitant inhibits TNF-α (10 ng/ml)-induced expression of IL-1β, IL-6, IL-8 in FLSs. FLSs were incubated with 10 ng/ml TNF-α in the presence or absence of aprepitant (5, 10 μM) for 24 h. (a). mRNA expression of IL-1β, IL-6, and IL-8 determined by real-time PCR; (b). Secretion of IL-1β, IL-6 and IL-8 determined by ELISA (*, #, $, p<.01 vs. previous column group).
![Figure 5. The neurokinin-1 receptor (NK-1R) antagonist aprepitant inhibits TNF-α (10 ng/ml)-induced expression of IL-1β, IL-6, IL-8 in FLSs. FLSs were incubated with 10 ng/ml TNF-α in the presence or absence of aprepitant (5, 10 μM) for 24 h. (a). mRNA expression of IL-1β, IL-6, and IL-8 determined by real-time PCR; (b). Secretion of IL-1β, IL-6 and IL-8 determined by ELISA (*, #, $, p<.01 vs. previous column group).](/cms/asset/ac39a9b0-0da5-46be-a3ad-c9af8562e0be/ianb_a_1573177_f0005_b.jpg)
Figure 6. The neurokinin-1 receptor (NK-1R) antagonist aprepitant inhibits TNF-α (10 ng/ml)-induced expression of MMP-3 and MMP-13 in FLSs. FLSs were incubated with 10 ng/ml TNF-α in the presence or absence of aprepitant (5, 10 μM) for 24 h. (a). mRNA expression of MMP-3 and MMP-13 determined by real-time PCR; (b). Protein expression of MMP-3 and MMP-13 determined by ELISA (*, #, $, p < .01 vs. previous column group).
![Figure 6. The neurokinin-1 receptor (NK-1R) antagonist aprepitant inhibits TNF-α (10 ng/ml)-induced expression of MMP-3 and MMP-13 in FLSs. FLSs were incubated with 10 ng/ml TNF-α in the presence or absence of aprepitant (5, 10 μM) for 24 h. (a). mRNA expression of MMP-3 and MMP-13 determined by real-time PCR; (b). Protein expression of MMP-3 and MMP-13 determined by ELISA (*, #, $, p < .01 vs. previous column group).](/cms/asset/23664bef-e41f-4580-97d2-e1fdc642696a/ianb_a_1573177_f0006_b.jpg)
Figure 7. The neurokinin-1 receptor (NK-1R) antagonist aprepitant inhibits TNF-α (10 ng/ml)-induced expression of p-IκBα and IκBα degradation in FLSs. FLSs were incubated with 10 ng/ml TNF-α in the presence or absence of aprepitant (5, 10 μM) for 6 h. Western blot analysis showing amounts of p-IκBα and IκBα in FLSs (*, #, $, p<.01 vs. previous column group).
![Figure 7. The neurokinin-1 receptor (NK-1R) antagonist aprepitant inhibits TNF-α (10 ng/ml)-induced expression of p-IκBα and IκBα degradation in FLSs. FLSs were incubated with 10 ng/ml TNF-α in the presence or absence of aprepitant (5, 10 μM) for 6 h. Western blot analysis showing amounts of p-IκBα and IκBα in FLSs (*, #, $, p<.01 vs. previous column group).](/cms/asset/9369ff78-1d83-4b14-8ed2-76c0fd11d43f/ianb_a_1573177_f0007_b.jpg)
Figure 8. The neurokinin-1 receptor (NK-1R) antagonist aprepitant inhibits TNF-α (10 ng/ml)-induced nuclear translocation of p65 and luciferase activity of NF-κB. (a) Nuclear levels of p65 was determined by Western blot analysis with lamin B as a positive control; (b) Luciferase reporter assay results reveal that NF-κB activity was attenuated by treatment with aprepitant (*, #, $, p < .01 vs. previous column group).
![Figure 8. The neurokinin-1 receptor (NK-1R) antagonist aprepitant inhibits TNF-α (10 ng/ml)-induced nuclear translocation of p65 and luciferase activity of NF-κB. (a) Nuclear levels of p65 was determined by Western blot analysis with lamin B as a positive control; (b) Luciferase reporter assay results reveal that NF-κB activity was attenuated by treatment with aprepitant (*, #, $, p < .01 vs. previous column group).](/cms/asset/eca3618d-f792-4ed4-9beb-5d8cf0bb80f8/ianb_a_1573177_f0008_b.jpg)
Figure 9. Silencing of NK-1R ameliorates the inflammatory response in RA-FLSs. RA-FLSs were infected with NK-1R lentiviral shRNA for 24 h. (a). Western blot analysis revealed successful knockdown of NK-1R; (b). Expression of MMP-3 and MMP-13 at the gene level determined by real-time PCR analysis; (c). Nuclear translocation of NK-1R determined by Western blot analysis (*, p < .01 vs. vehicle control).
![Figure 9. Silencing of NK-1R ameliorates the inflammatory response in RA-FLSs. RA-FLSs were infected with NK-1R lentiviral shRNA for 24 h. (a). Western blot analysis revealed successful knockdown of NK-1R; (b). Expression of MMP-3 and MMP-13 at the gene level determined by real-time PCR analysis; (c). Nuclear translocation of NK-1R determined by Western blot analysis (*, p < .01 vs. vehicle control).](/cms/asset/4cb0935e-13e5-4794-b3dd-1e8358a940d3/ianb_a_1573177_f0009_b.jpg)