Fig 1: VEGF-A isoforms alter nociception in a TRPv1 dependent manner. A. Systemic TRPV1 antagonism with SB366791 in mice resulted in inhibition of rhVEGF-A165a-induced mechanical allodynia. Arrows denote time of drug administration. B. TRPV1 knockout mice did not develop rhVEGF-A165a-induced mechanical allodynia, in contrast to wild-type strain matched controls. C. TRPV1 was co-expressed with VEGFR2 in sensory dorsal root ganglia sensory neurons (scale bar = 20 μm). D. Local administration of VEGF165a + vehicle into the plantar hindpaw resulted in a reduction in mechanical withdrawal values, which was blocked by co-administration of the TRPV1 antagonist SB366791 (TRPV1 antagonist).
Fig 2: Western blot analysis of the frontal cortex homogenates in vehicle-treated and beta-caryophyllene (BCP)-treated rats either in sham-operated or after bilateral common carotid artery occlusion followed by reperfusion (BCCAO/R). (Left) Transient receptor potential vanilloid type-1 receptor (TRPV1), brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (trkB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins. (Right) Densitometric analysis of the band gray levels expressed as a percentage of the optical density (O.D.) ratio of TRPV1-, BDNF-, trkB-positive bands to those immunostained for GAPDH. Data are reported as the mean values of 12 vehicle-treated and 10 BCP-treated rats for each experimental condition. Error bars depict the standard error of the mean (S.E.M.). Asterisks denote significant differences. Two-way ANOVA with the Tukey’s test for post-hoc analyses was applied to evaluate statistical differences between groups. BCCAO/R, significant effect of BCCAO/R; BCP, significant effect of BCP-treatment; BCP × BCCAO/R, significant BCP-treatment × BCCAO/R interaction. *** p < 0.001; **** p ≤ 0.0001 (see Table 1 for F-values and p-values relevant to effects of BCCAO/R and BCP pre-treatment and to the interaction between them).
Fig 3: Double immunofluorescence for TRPV1 (A,C,D,F,G,I) and either glial fibrillary acidic protein (GFAP) (B,C,E,F) or Iba1 (H,I) in representative coronal sections of frontal cortex of BCCAO/R + vehicle (A–C) and BCCAO/R + BCP rats (D–I); arrows point to TRPV1/GFAP double-labelled elements. *, blood vessel. Scale bars: A, B = C: 25 μm; D, E = F: 25 μm; G, H = I: 25 µm.
Fig 4: Protective effect of TRPV1 on the CCl4-induced mouse model. (a–d) The serum concentration levels of hyaluronic acid, collagen type IV, laminin, and precollagen type III in the indicated four groups were determined using radioimmunoassay kits. (e, f) ALT and AST in serums derived from mice from four different groups were determined by standard enzymatic assay kits. Data are presented as mean ± SD of three independent experiments. (g–i) The protein levels of α-SMA and COL1A1 in liver tissues from the four groups were determined using Western blot analyses. (j–n) The activity of SOD, CuZnSOD, T-AOC, GSHPX, and MDA was determined in the liver tissues of the four groups of mice. Data are presented as mean ± SD (n = 5 mice). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.
Fig 5: VEGF-A modulated TRPV1-agonist evoked responses in dorsal root ganglion neurons. A. Capsaicin stimulated a concentration-dependent increase in intracellular calcium in DRG neurons. B. This was increased by rhVEGF-A165a, and reduced by rhVEGF-A165b (mean ± SEM, n = 3–7). C. Treatment of rat DRG neurons with rhVEGF-A165a increased capsaicin-stimulated calcium influx (area under the curve of the calcium responses shown in Fig. 8B) compared with capsaicin alone or rhVEGF-A165b (2 way ANOVA main effect of drug p = 0.0051). The bell shaped concentration–response curve displays TRPV1 desensitization at higher capsaicin concentrations (5 μM). D. Example of a digitized trace of raw capsaicin-evoked current in the presence (gray) and absence of capsaicin. E. Capsaicin-evoked currents in primary DRG neurons were significantly larger in neurons incubated in VEGF-A165a overnight compared to vehicle treated neurons (box and whisker plots showing median, range, min and max). F. rhVEGF-A165b treatment enhanced TRPV1 serine phosphorylation in 50B11 immortalized DRG cells. IP of protein with TRPV1 antibody followed by IB with anti-pSer antibody showed rhVEGF-A165a, but not rhVEGF-A165b-mediated phosphorylation of TRPV1. (NGF treatment = positive control). G. Whereas 0.2 μM capsaicin alone did not alter intracellular calcium itself, overnight treatment with rhVEGF-A165a + 0.2 μM capsaicin resulted in a robust sustained increase in response to capsaicin, which was blocked by treatment with the PKC inhibitor BIM1 (2 way ANOVA main effect of drug p = 0.0003). H. Low concentration capsaicin (concentration at terminals ~ 10 nM) led to evoked activity from C fiber nociceptors in vivo. Capsaicin-evoked activity was increased by rhVEGF-A165a and blocked by rhVEGF-A165b. ‡, ‡‡, ‡‡‡, p < 0.05, 0.01, 0.001 respectively compared to baseline. *, **, *** = p < 0.05, 0.01, 0.001 respectively compared to other groups.
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