The Influence of Sex Steroid Hormone Fluctuations on Capsaicin-Induced Pain and TRPV1 Expression

Abstract

Sexual dimorphism among mammals includes variations in the pain threshold. These differences are influenced by hormonal fluctuations in females during the estrous and menstrual cycles of rodents and humans, respectively. These physiological conditions display various phases, including proestrus and diestrus in rodents and follicular and luteal phases in humans, distinctly characterized by varying estrogen levels. In this study, we evaluated the capsaicin responses in male and female mice at different estrous cycle phases, using two murine acute pain models. Our findings indicate that the capsaicin-induced pain threshold was lower in the proestrus phase than in the other three phases in both pain assays. We also found that male mice exhibited a higher pain threshold than females in the proestrus phase, although it was similar to females in the other cycle phases. We also assessed the mRNA and protein levels of TRPV1 in the dorsal root and trigeminal ganglia of mice. Our results showed higher TRPV1 protein levels during proestrus compared to diestrus and male mice. Unexpectedly, we observed that the diestrus phase was associated with higher TRPV1 mRNA levels than those in both proestrus and male mice. These results underscore the hormonal influence on TRPV1 expression regulation and highlight the role of sex steroids in capsaicin-induced pain.

Keywords: TRPV1; diestrus; estrogens; hormones; pain; proestrus.

Figure 1

Evaluation of capsaicin-induced acute pain in male and female mice. (A) Paw licking assay: vehicle solution injection (bars with empty symbols) induced minimal paw licking response in all experimental groups (males: n = 6, P: n = 14, E: n = 6, M: n = 3, and D: n = 12). The nociceptive response was observed following capsaicin injection in all experimental groups (bars with filled symbols, males: n = 11, P: n = 17, E: n = 9, M: n = 3, and D: n = 14). (B) Cheek injection assay: The graph presents data generated from counting the number of wiping bouts following injection of either capsaicin or vehicle solution. Nociceptive response to capsaicin application was observed in all experimental groups (bars with filled symbols, males: n = 18, P: n = 12, E: n = 6, M: n = 4, and D: n = 8). The vehicle induced minimal wiping bouts in all experimental groups (bars with empty symbols, males: n = 6, P: n = 4, E: n = 3, M: n = 6, and D: n = 7). Proestrus (P), estrus (E), metestrus (M), and diestrus (D). Data are the mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test, **, ***, and **** for p < 0.05, p < 0.001, and p < 0.0001, respectively; ns: non-statistically significant.

Figure 2

Evaluation of TRPV1 gene expression in the dorsal root ganglia (DRGs) of male and female mice in the proestrus (P) and diestrus (D) phases. (A) TRPV1 mRNA levels were assessed using qPCR. The data obtained showed similar TRPV1 mRNA levels across the three experimental groups. (B) Western Blot experiments demonstrated that TRPV1 protein levels were lower in the diestrus phase than in the proestrus phase (n = 9). Data are the mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test. *** p < 0.005; ns: non-statically significant. ADU: arbitrary densitometric unit.

Figure 3

Evaluation of TRPV1 gene expression in the trigeminal ganglia (TGs) of male and female mice in the proestrus (P) and diestrus phases (D). (A) TRPV1 mRNA levels were assessed using qPCR. The data showed that TRPV1 mRNA levels are higher in diestrus than in proestrus and male mice (n = 3). (B) Western Blot experiments demonstrated that TRPV1 protein levels are lower in the diestrus phase compared to females in the proestrus phase (n = 13). Data are the mean ± SEM. One-way ANOVA test followed by Tukey’s post hoc test. * p < 0.05; *** p < 0.005. ADU: arbitrary densitometric unit.

Figure 4

Calcium imaging recordings of TG neurons treated with low and high 17β-estradiol concentrations. (A) Summary graphs of the calcium imaging recordings performed on TG neurons in response to 500 nM capsaicin (left and middle) and high potassium (right) stimuli. The left graph shows the percentage of capsaicin-responding neurons under different experimental conditions: control condition (Ctr, n = 86), low E2 concentration (LE2, n = 125), and high E2 concentration (HE2, n = 117). The middle graph in A shows the amplitude responses to capsaicin, indicating that neurons from all groups respond similarly. The right graph in A displays the amplitude of the responses to the high potassium stimulus. (B) Representative recording of TG neuron groups, the capsaicin-positive neurons treated with HE2 show faster responses to capsaicin compared to those treated with LE2. The arrows represent the time capsaicin is added, and the arrowhead is when the high K⁺ solution is added. (C) Representative recording of the capsaicin response latency under each treatment (records taken from B) and the summary graph of the time to peak of the capsaicin responses. The bar graph represents the mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test. *, **, and **** for p < 0.05, p < 0.01, and p < 0.005, respectively.

Figure 5

Evaluation of ERα total protein in TGs from male and female mice in the proestrus and diestrus phases. (A) Representative WB for ERα (upper panel) and GAPDH (lower panel) immunodetection. The membrane was stripped for TRPV1 immunodetection (middle panel). (B) The graph shows the normalized data of the mean values for ERα (n = 3) or TRPV1 (n = 13; these data are the same as those used in Figure 3) with respect to load control (GAPDH). Two-way ANOVA with Šídák’s multiple comparison test. ** p < 0.005. ADU: arbitrary densitometric unit.