Kinin B2 and B1 Receptors Activation Sensitize the TRPA1 Channel Contributing to Anastrozole-Induced Pain Symptoms

Aromatase inhibitors (AIs) cause symptoms of musculoskeletal pain, and some mechanisms have been proposed to explain them. However, signaling pathways downstream from kinin B2 (B2R) and B1 (B1R) receptor activation and their possible sensitizing of the Transient Receptor Potential Ankyrin 1 (TRPA1) remain unknown. The interaction between the kinin receptor and the TRPA1 channel in male C57BL/6 mice treated with anastrozole (an AI) was evaluated. PLC/PKC and PKA inhibitors were used to evaluate the signaling pathways downstream from B2R and B1R activation and their effect on TRPA1 sensitization. Anastrozole caused mechanical allodynia and muscle strength loss in mice. B2R (Bradykinin), B1R (DABk), or TRPA1 (AITC) agonists induced overt nociceptive behavior and enhanced and prolonged the painful parameters in anastrozole-treated mice. All painful symptoms were reduced by B2R (Icatibant), B1R (DALBk), or TRPA1 (A967079) antagonists. We observed the interaction between B2R, B1R, and the TRPA1 channel in anastrozole-induced musculoskeletal pain, which was dependent on the activation of the PLC/PKC and PKA signaling pathways. TRPA1 seems to be sensitized by mechanisms dependent on the activation of PLC/PKC, and PKA due to kinin receptors stimulation in anastrozole-treated animals. Thus, regulating this signaling pathway could contribute to alleviating AIs-related pain symptoms, patients’ adherence to therapy, and disease control.


Introduction
Cancer incidence is increasing each year, causing high morbidity and mortality rates. Breast cancer is the most commonly diagnosed cancer, with an estimated more than 2.3 million new cases (11.7%) per year [1]. Most breast cancer survivors carry a burden of sequelae after diagnosis and treatment, with pain being one of the most common symptoms [1,2]. The causes of pain are varied and might be related to the adjuvant endocrine treatment prescribed after the cancer treatment [2,3].
The third-generation aromatase inhibitors (AIs) are considered first-line drugs in the adjuvant endocrine treatment for postmenopausal women diagnosed with hormone receptor-positive breast cancer, and the most prescribed is anastrozole, followed by letrozole and exemestane [4][5][6]. The AIs are recommended for at least five years after diagnosis or may be used after therapy with tamoxifen (subsequent 2-3 years) [3, 5,7]. One-third of AIs users have reported adverse effects from treatment onset [6,8,9]. Painful musculoskeletal symptoms, characterized by morning stiffness, joint pain, myalgia, and decreased strength, seem to be preponderant daily symptoms in these patients [3,8]. Furthermore, this painful condition can include diffuse (22%), mixed (11%), or neuropathic (9%) pain [8][9][10]. The

Animals
Male C57BL/6 mice (25-30 g) were used and maintained temperature-controlled (22 ± 1 • C) under a 12-h light/dark cycle and with standard laboratory chow and water ad libitum. The animals were habituated to the experimental room at least 1 h before the experiments. Experimental protocols followed ethical guidelines established for investigations of experimental pain in conscious animals [39]. The experiments also were performed following the national and international legislation (guidelines of the Brazilian Council of Animal Experimentation Control-CONCEA-and of the U.S. Public Health Service's Policy on Humane Care and Use of Laboratory Animals-PHS Policy). The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Federal University of Santa Maria (CEUA, process numbers 2304280220/2020 and 3026220520/2020).
Once AIs caused incapacitating pain symptoms, an animal model was used. Using an intact organism is important to obtain an adequate response from these experimental models. In this context, male C57BL/6 mice were used based on their low oestrogen concentrations and previous studies involving AIs-associated pain [13,40]. The number of animals and intensities of noxious stimuli used were the minimum necessary to demonstrate the consistent effects of the treatments. The group size used for each experiment was based on studies with protocols similar to ours [37,38,41], which were confirmed by power calculations (G*Power version 3.1.9.7).
Allocation concealment was performed using a randomization procedure [http:// www.randomizer.org/ (accessed on 12 December 2021)] and according to the baseline thresholds before and after the treatment administrations. All experiments were also performed by experimenters blinded to the drug administration or the group to be tested.

Anastrozole-Induced Pain Model
The dose of anastrozole used to induce painful behaviors in mice was based on the human dose of 1 mg recommended by the Food and Drug Administration to treat breast cancer in postmenopausal women [4]. Thus, from the conversion factor from human to mouse indicated by the National Institute of Health [42] and previous studies [43,44], we have used the dose of 0.2 mg/kg to induce a pain model in mice. The experimental protocol consisted of administering anastrozole (0.2 mg/kg) by oral route (p.o.) or vehicle (control group, 10 mL/kg, p.o.; 0.5% CMC). The animals were subjected to behavior assessments after acute treatment (a single anastrozole administration) or prolonged treatment (anastrozole administration for 15 consecutive days). Treatments for pain are usually therapeutic; that is, they are administered after the onset of pain symptoms. Thus, all the experimental protocols were conducted according to this schedule: we first induced the painful symptoms with anastrozole administration, and then we performed antagonist or inhibitor treatments.

Study Design for Behavioral Assessment
Mechanical allodynia (von Frey test) and muscle strength (Grip test) were evaluated before (baseline, BL) and after the anastrozole or vehicle administration (time 0). Next, the animals were submitted to several treatment protocols (see more details below), and these behavioral parameters were evaluated several times (h) after treatments. In most behavioral protocols, overt nociceptive behavior was also evaluated.

Pain Induction by Anastrozole
We investigated whether anastrozole systemic treatment evokes mechanical allodynia and muscle strength loss on a repeated treatment protocol. For this, after evaluation of the baseline paw withdrawal threshold (PWT) and muscle strength of the animals, they were randomized into treatment groups. Mice received anastrozole (0.2 mg/kg, p.o.) or its vehicle (0.5% CMC, p.o.) once a day for 15 consecutive days, which corresponds to a 1-year time in humans [13]. The development of mechanical allodynia and muscle strength loss was evaluated at days 1, 3, 7, 10, 12, and 15 from 0.5 h up to 24 h after each treatment day (following the protocol described in items "2.5.2 Mechanical threshold" and "2.5.3 Muscle strength"). The experimental design is represented in Figure 1A.

Assessment of B 2 R, B 1 R, and TRPA1 Involvement in Anastrozole-Induced Pain
To confirm the contribution of B 2 R, B 1 R, and TRPA1 channels on mechanical allodynia and muscle strength loss induced by anastrozole, the mice received a single administration of Icatibant (100 nmol/kg, intraperitoneal, i.p., B 2 R antagonist) or DALBk (150 nmol/kg, i.p., B 1 R antagonist) or A967079 (100 mg/kg, p.o., TRPA1 channel antagonist) or their vehicles (10 mL/kg, i.p. or p.o.) at 3 h after anastrozole administration. After treatments, the PWT and muscle strength were evaluated at different time points (from 0.5 h to 5 h). The experimental design is represented in Supplementary Figure S1.
For the first time, it was investigated whether low doses of B 2 R, B 1 R, and TRPA1 channel agonists could enhance painful behaviors in anastrozole-treated mice. For this, the animals were previously treated with anastrozole or vehicles, and after 3 h, they received intraplantar (i.pl.) injections of Bk (1 nmol/paw, a B 2 R agonist), DABk (3 nmol/paw, a B 1 R agonist), or AITC (0.3 nmol/paw, a TRPA1 agonist), all in sub-nociceptive doses, or their vehicles (20 µL/paw, i.pl.). The overt nociceptive behavior was immediately evaluated following the protocol described in item "2.5.1 Overt nociceptive behavior". PWT and muscle strength were evaluated from 0.5 to 24 h after the stimuli. The experimental design is represented in Figure 2A.
To validate the involvement of B 2 R, B 1 R, and TRPA1 channels on the behavioral parameters evaluated above, other animal groups received Icatibant (100 nmol/kg, intraperitoneal, i.p., B 2 R antagonist), DALBk (150 nmol/kg, i.p., B 1 R antagonist), or A967079 (100 mg/kg, p.o., TRPA1 channel antagonist) at 3 h after anastrozole. After 0.5 h, the same animals were treated with sub-nociceptive doses of their respective agonists, Bk, DABk, or AITC, by the intraplantar route, and the overt nociceptive behavior was immediately evaluated for 10 min. In sequence, PWT and muscle strength were assessed until treatments with the antagonists showed an effect. The experimental design is represented in Figure 3A We explored whether the interaction between both kinin receptors and TRPA1 might be essential to mediate the anastrozole-induced pain. The animals were previously treated with anastrozole or vehicle, and after 3 h, they received Icatibant, DALBk, or A967079. After 0.5 h, the B 2 R (Icatibant) or B 1 R (DALBk) antagonist-treated animals received the intraplantar injection of the TRPA1 agonist, AITC (0.3 nmol/paw, i.pl.). The animal groups treated with the systemic TRPA1 (A967076) antagonist received the intraplantar injection of either B 2 R or B 1 R agonists (Bk or DABK, 1 or 3 nmol/paw, respectively). The overt nociceptive behavior was evaluated immediately after intraplantar agonist administration for 10 min. In sequence, PWT and muscle strength were assessed until treatments with the antagonists showed an effect. The experimental designs are represented in Figures 4A and 5A.
To confirm the results about the interaction between B 2 R and B 1 R and TRPA1 in the pain symptoms induced by anastrozole, we use an in vivo desensitization protocol (following the protocol described in item "2.5.4 Desensitization protocol"). After 0.5 h of desensitization protocol, the animals received anastrozole or vehicle, and after 3 h, the desensitized animals received an intraplantar injection of AITC (0.3 nmol/paw, i.pl.), and the overt nociceptive behavior was immediately evaluated. After that, PWT and muscle strength were assessed several times (h). The experimental design is represented in Figure 6A.

Intracellular Pathways Dependent on Kinin Receptor Activation and TRPA1 Sensitization
We also examined the participation of both intracellular signaling pathways, mediated by PLC/PKC or PKA, in the anastrozole-induced pain. For this, animals previously treated (3 h before) with anastrozole or vehicle received intraplantar co-injection containing either an inhibitor of PLC (U73122, 30 pmol/paw, i.pl.), PKC (GF109203X, 1 or 3 nmol/paw, i.pl.) or PKA (H89, 1 nmol/paw, i.pl.) plus Bk (1 nmol/paw, i.pl.) or DABK (3 nmol/paw, i.pl.). Immediately after intraplantar injection of agonists, overt nociceptive behavior was evaluated. Next, PWT and muscle strength were assessed until treatments with the inhibitors showed an effect. The experimental design is represented in Figure 7A.
Next, we evaluated whether signaling pathways downstream from B 2 R and B 1 R activation could be contributing to TRPA1 sensitization in this model. At 3 h after treatments with anastrozole or vehicle, the animals received intraplantar co-injection containing either inhibitors of PLC, PKC, or PKA plus AITC (0.3 nmol/paw, i.pl.), and the overt nociceptive behavior was immediately evaluated. Next, PWT and muscle strength were assessed until treatments with the inhibitors showed an effect. The experimental design is represented in Figure 8A.

Overt Nociceptive Behavior
We evaluated the capacities of B 2 R (Bk), B 1 R (DABk), and TRPA1 (AITC) channel agonists in increasing the nociceptive response induced by anastrozole (all at sub-nociceptive doses). For this, the animals were individually placed in chambers (transparent glass cylinders of 20 cm diameter) and adapted for 10 min. Immediately after the intraplantar injection of agonists in the right hind paw, the animals were observed for 10 min. The amount of time spent (in seconds, s) licking and lifting the injected paw was considered indicative of overt nociceptive behavior [13,44].

Mechanical Allodynia
The assessment of mechanical allodynia was carried out using flexible nylon filaments (von Frey) of increasing stiffness (0.02-10 g) by the up-and-down method [45,46]. The von Frey test is widely used in preclinical and clinical settings for measuring pain [46,47]. The PWT was calculated from the resulting scores, as previously described [48]. The PWT was expressed in grams (g), and a significant decrease in the PWT compared with the baseline values was considered as mechanical allodynia.

Muscle Strength
The muscle strength test in mice was evaluated using a grip strength meter (Grip Strength meter EFF 305-IInsight, Ribeirão Preto, SP, Brazil) [49], a test used in preclinical and clinical settings for the study of musculoskeletal pain [50,51]. The test consists of the experimenter gently holding the mouse by the base of the tail, allowing the animal to grab the metal bar with the forelimbs and hindlimbs before being gently pulled until it releases its grip. The forelimbs and hindlimbs muscle strength, expressed in grams (g), was measured three times per mouse with at least 1 min between measurements.

Desensitization Protocol
Although the presence of B 2 R on nociceptive primary afferent neurons is well established, evidence also demonstrates that B 1 R is expressed in some small and medium diameter neurons and peptidergic and non-peptidergic C fibers [26,29,52,53]. In this sense, the desensitization protocol was performed on both kinin B 2 R and B 1 R, based on and adapted from Ferreira et al. [35]. The mice received two repeated intraplantar injections of Bk (10 nmol/paw, i.pl.) or DABK (10 nmol/paw, i.pl.) with a 0.5 h interval between the injections.

Statistical Analysis
The results were expressed as the mean + Standard Error of the Mean (SEM), which were expressed as geometric means accompanied by their respective 95% confidence limits. The Kolmogorov-Smirnov test was used to assess the normality of the data. To meet parametric assumptions, data of mechanical threshold were log-transformed before analyses. Parametric data were analyzed by Student's t-test or one-or two-way ANOVA followed by Bonferroni's post hoc test, using the GraphPad Prism 8.0 software (San Diego, CA, USA). Post hoc tests were performed only when the F-value achieved the necessary level of statistical significance (p < 0.05) and when there was no significant variance in homogeneity [54]. The percentages of maximum inhibition (Imax) were calculated for the maximally developed responses compared to baseline values or the control group.

Systemic Anastrozole Induces Prolonged Pain Symptoms in Mice
Mice daily treated with anastrozole developed mechanical allodynia (from 2 h up to 6 h) ( Figure 1B) and presented reduced muscle strength (from 0.5 h up to 6 h) ( Figure 1C) at days 1, 3, 7, 10, 12, and 15 after anastrozole treatment when compared to the vehicle group. The development of maximum mechanical allodynia and muscle strength loss occurred 3 h after oral anastrozole treatment, returning to basal levels 8 h after anastrozole. Based on this, the time of 3 h on the first day after anastrozole administration was chosen for subsequent experiments.

Statistical Analysis
The results were expressed as the mean + Standard Error of the Mean (SEM), which were expressed as geometric means accompanied by their respective 95% confidence limits. The Kolmogorov-Smirnov test was used to assess the normality of the data. To meet parametric assumptions, data of mechanical threshold were log-transformed before analyses. Parametric data were analyzed by Student's t-test or one-or two-way ANOVA followed by Bonferroni's post hoc test, using the GraphPad Prism 8.0 software (San Diego, CA, USA). Post hoc tests were performed only when the F-value achieved the necessary level of statistical significance (p < 0.05) and when there was no significant variance in homogeneity [54]. The percentages of maximum inhibition (Imax) were calculated for the maximally developed responses compared to baseline values or the control group.

Systemic Anastrozole Induces Prolonged Pain Symptoms in Mice
Mice daily treated with anastrozole developed mechanical allodynia (from 2 h up to 6 h) ( Figure 1B) and presented reduced muscle strength (from 0.5 h up to 6 h) ( Figure 1C) at days 1, 3, 7, 10, 12, and 15 after anastrozole treatment when compared to the vehicle group. The development of maximum mechanical allodynia and muscle strength loss occurred 3 h after oral anastrozole treatment, returning to basal levels 8 h after anastrozole. Based on this, the time of 3 h on the first day after anastrozole administration was chosen for subsequent experiments.  ) with anastrozole (0.2 mg/kg) or its vehicle (0.5% CMC) once daily for fifteen days to mimic a 1-year time in humans. On the 1st, 3rd, 7th, 10th, 12th, and 15th days after treatments, a time curve (from 0.5 h up to 24 h) was performed to evaluate mechanical allodynia development and muscle strength loss (B,C). Baseline (BL) values were measured before anastrozole or vehicle administration. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. vehicle group. Data were expressed as the mean + SEM (n = 6/group) and analyzed by a two-way ANOVA followed by the Bonferroni post hoc test. The arrows indicate the administration of the anastrozole or vehicle. Veh: vehicle oral administration; ANS: anastrozole oral administration. The arrows indicate the administration of the anastrozole or vehicle.
The intraplantar (i.pl.) injection of Bk (1 nmol/paw) ( Figure 2B), DABk (3 nmol/paw) ( Figure 2E), or AITC (0.3 nmol/paw) ( Figure 2H), all in sub-nociceptive doses, increased paw licking and lifting time in anastrozole-treated compared to vehicle-treated animals, an indication of overt nociceptive behavior. Anastrozole (0.2 mg/kg, p.o.) induced mechanical allodynia (Time 0, Figure 2C,F,I) and muscle strength loss (Time 0, Figure 2D,G,J) in mice at 3 h after its administration when compared to BL values. Intraplantar Bk, DABk, or AITC enhanced and prolonged the mechanical allodynia ( Figure 2C,F,I) and muscle strength loss ( Figure 2D,G,J) from 0.5 up to 7 h after its injection in anastrozole-treated animals when compared to the anastrozole plus vehicle group.
loss (from 1 h up to 2 h) ( Figure 3D) with inhibitions of 55 ± 23% (at 1 h) and 89 ± 2% (at 1 h), respectively, after its administration. DALBk reduced the mechanical allodynia ( Figure  3F) and muscle strength loss ( Figure 3G) from 1 h up to 2 h (inhibition of 52 ± 13% at 1 h) and from 1 h up to 3 h (inhibition of 79 ± 6% at 2 h), respectively, after its administration. A967079 reduced the mechanical allodynia ( Figure 3I) and muscle strength loss ( Figure  3J) from 1 h up to 2 h (inhibition of 54 ± 7% and 90 ± 7% at 1 h, respectively) after its administration. As expected, sub-nociceptive doses of the Bk, DABk, or AITC agonists did not induce overt nociceptive behavior, mechanical allodynia, or muscle strength loss in animals previously treated with vehicle ( Figure 3).

Interaction of Kinin B 2 and B 1 Receptors and the TRPA1 Channel Sustain the Anastrozole-Induced Pain
Intraplantar Bk (1 nmol/paw, i.pl.) and DABk (3 nmol/paw, i.pl.), at sub-nociceptive doses, induced significant overt nociceptive behavior ( Figure 4B,E) and enhanced the mechanical allodynia (Figure 4C,F) and the muscle strength loss (Figure 4D,G) in anastrozoletreated animals compared to the vehicle plus vehicle group. A967079 (100 mg/kg, p.o., 0.5 h prior) markedly prevented the sensitizing effect induced by Bk ( Figure 4B) and DABk ( Figure 4E) in anastrozole-treated animals on the overt nociceptive behavior with inhibitions of 79 ± 7% and 66 ± 26%, respectively, compared to the anastrozole plus Bk or DABk groups. A967079 also prevented Bk-and DABk-induced responses on mechanical allodynia from 0.5 h up to 1 h for both agonists (inhibition of 77 ± 20% and 30 ± 5% at 1 h, respectively) ( Figure 4C,F) and muscle strength loss from 0.5 h up to 2 h for both agonists (inhibition of 100% and 65 ± 11% at 0.5 h, respectively) ( Figure 4D,G).  The sensitizing effect induced by TRPA1 agonist AITC (0.3 nmol/paw, i.pl.) on overt nociceptive behavior, mechanical allodynia, and muscle strength loss was prevented by pre-treatment with B2R and B1R antagonists. Icatibant (100 nmol/kg, i.p., 0.5 h prior) markedly inhibited the responses induced by AITC (0.3 nmol/paw, i.pl.) on the overt nociceptive behavior ( Figure 5B; inhibition of 80 ± 16%), mechanical allodynia ( Figure 5C), and muscle strength loss ( Figure 5D) from 0.5 h up to 3 h (inhibition of 49 ± 17% at 2 h) and from 0.5 h up to 2 h (inhibition of 90 ± 7% at 1 h) after its administration, respectively. The sensitizing effect induced by AITC in anastrozole-treated animals also was prevented by the pre-treatment with DALBk (150 nmol/kg, i.p., 0.5 h prior) on the overt nociceptive The sensitizing effect induced by TRPA1 agonist AITC (0.3 nmol/paw, i.pl.) on overt nociceptive behavior, mechanical allodynia, and muscle strength loss was prevented by pre-treatment with B 2 R and B 1 R antagonists. Icatibant (100 nmol/kg, i.p., 0.5 h prior) markedly inhibited the responses induced by AITC (0.3 nmol/paw, i.pl.) on the overt nociceptive behavior ( Figure 5B; inhibition of 80 ± 16%), mechanical allodynia ( Figure 5C), and muscle strength loss ( Figure 5D) from 0.5 h up to 3 h (inhibition of 49 ± 17% at 2 h) and from 0.5 h up to 2 h (inhibition of 90 ± 7% at 1 h) after its administration, respectively. The sensitizing effect induced by AITC in anastrozole-treated animals also was prevented by the pre-treatment with DALBk (150 nmol/kg, i.p., 0.5 h prior) on the overt nociceptive behavior ( Figure 5E; inhibition of 81 ± 10%), mechanical allodynia ( Figure 5F), and muscle strength loss ( Figure 5G) from 0.5 h up to 2 h (inhibition of 62 ± 18% at 1 h and inhibition of 99 ± 1% at 1 h, respectively). These results suggest the cooperation between kinin B 2 R and B 1 R and TRPA1 channel to mediate nociceptive parameters induced by anastrozole.
Pharmaceutics 2023, 15, x FOR PEER REVIEW 12 of 23 behavior ( Figure 5E; inhibition of 81 ± 10%), mechanical allodynia ( Figure 5F), and muscle strength loss ( Figure 5G) from 0.5 h up to 2 h (inhibition of 62 ± 18% at 1 h and inhibition of 99 ± 1% at 1 h, respectively). These results suggest the cooperation between kinin B2R and B1R and TRPA1 channel to mediate nociceptive parameters induced by anastrozole. The desensitization protocol by intraplantar injection of Bk ( Figure 6B) or DABk (Figure 6E) completely abrogated the AITC-induced overt nociceptive behaviors in anastrozole-treated mice (inhibitions of 93 ± 3% and 91 ± 7%, respectively) compared to The desensitization protocol by intraplantar injection of Bk ( Figure 6B) or DABk ( Figure 6E) completely abrogated the AITC-induced overt nociceptive behaviors in anastrozole-treated mice (inhibitions of 93 ± 3% and 91 ± 7%, respectively) compared to anastrozole plus AITC-treated non-desensitized mice. In these same animals, the local desensitization with Bk and DABk also abolished the mechanical allodynia from 0.5 h up to 3 h (inhibition of 47 ± 10% at 0.5 h and 27 ± 5% at 1 h, respectively) ( Figure 6C,F) and muscle strength loss from 0.5 h up to 1 h (inhibition of 33 ± 2% and 32 ± 2% at 0.5 h, respectively) ( Figure 6D,G) after its injections, when compared to the non-desensitized group anastrozole plus AITC. anastrozole plus AITC-treated non-desensitized mice. In these same animals, the local desensitization with Bk and DABk also abolished the mechanical allodynia from 0.5 h up to 3 h (inhibition of 47 ± 10% at 0.5 h and 27 ± 5% at 1 h, respectively) ( Figure 6C,F) and muscle strength loss from 0.5 h up to 1 h (inhibition of 33 ± 2% and 32 ± 2% at 0.5 h, respectively) ( Figure 6D,G) after its injections, when compared to the non-desensitized group anastrozole plus AITC.
After that, we assessed the contribution of signaling pathways downstream from the B 1 R. The overt nociceptive behavior induced by a sub-nociceptive dose of DABk in animals treated with anastrozole was attenuated when DABK was co-injected with inhibitors of PLC (U73122) or PKA (H89), with inhibitions of 60 ± 23% and 67 ± 23%, respectively ( Figure 7E). Furthermore, both inhibitors also reduced the responses induced by DABk on mechanical allodynia (inhibition of 35 ± 6% and 33 ± 5% at 1 h after its injections, respectively) ( Figure 7F) and on muscle strength loss (inhibition of 89 ± 3% and 66 ± 6% at 1 h after its injections, respectively) ( Figure 7G) in anastrozole-treated mice. The PKC inhibitor (GF109203X) at a dose of 3 but not 1 nmol/paw attenuated the overt nociceptive behavior (inhibition of 89 ± 4%) ( Figure 7E), mechanical allodynia (inhibition of 38 ± 10% at 1 h after its injection) ( Figure 7F), and muscle strength loss (inhibition of 76 ± 4% at 1 h after its injection) ( Figure 7G) induced by a sub-nociceptive dose of DABk in anastrozoletreated animals.
Next, we evaluated whether signaling pathways downstream from B 2 R and B 1 R activation could be contributing to TRPA1 activation in this model. The inhibition of PLC and PKC by U73122 and GF109203X, respectively, reduced the overt nociceptive behavior induced by a sub-nociceptive dose of AITC in animals treated with anastrozole compared to the anastrozole plus AITC group ( Figure 8B), with inhibitions of 94 ± 3% and 97 ± 2%, respectively. Furthermore, both inhibitors, U73122 and GF109203X, reduced AITC-induced responses on mechanical allodynia (inhibition of 39 ± 9% and 27 ± 2% at 0.5 h after its injections, respectively) ( Figure 8C) and muscle strength loss (inhibition of 75 ± 2% at 1 h and 69 ± 3% at 0.5 h after its injections, respectively) in previously anastrozole-treated mice ( Figure 8D). Moreover, the inhibition of PKA by H89 attenuated the AITC-induced responses on overt nociceptive behavior (inhibition of 49 ± 1%) ( Figure 8B), mechanical allodynia (inhibition of 28 ± 7% at 1 h after its injection) ( Figure 8C), and muscle strength loss (inhibition of 52 ± 7 % at 1 h after its injection) in previously anastrozole-treated mice ( Figure 8D). were assessed until treatments with the inhibitors showed an effect. Baseline (BL) values were measured before the anastrozole or vehicle administration. ### p < 0.001 vs. Veh plus Veh group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. ANS plus Bk group. Data were expressed as the mean + SEM (n = 6/group) and analyzed by one-or two-way ANOVA followed by the Bonferroni post hoc test, except for the U73122 effect on overt nociceptive behavior induced by ANS plus Bk and the H89 effect on overt nociceptive behavior induced by ANS plus DABk, which were analyzed by Student's t-test. Veh: intraplantar or oral vehicle injections; ANS: oral administration of anastrozole to induce the pain model; Bk: bradykinin intraplantar treatment; U731: U73122, a PLC inhibitor; GF109: GF109203X, a PKC inhibitor; PWT: paw withdrawal threshold.
Next, we evaluated whether signaling pathways downstream from B2R and B1R activation could be contributing to TRPA1 activation in this model. The inhibition of PLC and PKC by U73122 and GF109203X, respectively, reduced the overt nociceptive behavior induced by a sub-nociceptive dose of AITC in animals treated with anastrozole compared to the anastrozole plus AITC group ( Figure 8B), with inhibitions of 94 ± 3% and 97 ± 2%, respectively. Furthermore, both inhibitors, U73122 and GF109203X, reduced AITC-induced responses on mechanical allodynia (inhibition of 39 ± 9% and 27 ± 2% at 0.5 h after its injections, respectively) ( Figure 8C) and muscle strength loss (inhibition of 75 ± 2% at 1 Figure 7. The signaling pathways downstream from the kinin B 2 and B 1 receptors are activated and contribute to the nociceptive behaviors induced by kinin agonists in anastrozole-treated mice. (A) Male C57BL/6 mice were treated by the oral route (p.o.) with anastrozole (0.2 mg/kg) or its vehicle (0.5% CMC) to induce painful behaviors. At 3 h after anastrozole administration (Time 0), the animals received the intraplantar (i.pl.) co-injection of U73122 (30 pmol/paw, i.pl., PLC inhibitor), GF109203X (1 or 3 nmol/paw, i.pl., PKC inhibitor), or H89 (1 nmol/paw, i.pl., PKA inhibitor), and a sub-nociceptive dose of Bk (1 nmol/paw, i.pl.) or DABk (3 nmol/paw, i.pl.). The overt nociceptive behavior was immediately evaluated for 10 min (B,E); the PWT (C,F) and strength muscle (D,G) were assessed until treatments with the inhibitors showed an effect. Baseline (BL) values were measured before the anastrozole or vehicle administration. ### p < 0.001 vs. Veh plus Veh group. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. ANS plus Bk group. Data were expressed as the mean + SEM (n = 6/group) and analyzed by one-or two-way ANOVA followed by the Bonferroni post hoc test, except for the U73122 effect on overt nociceptive behavior induced by ANS plus Bk and the H89 effect on overt nociceptive behavior induced by ANS plus DABk, which were analyzed by Student's t-test. Veh: intraplantar or oral vehicle injections; ANS: oral administration of anastrozole to induce the pain model; Bk: bradykinin intraplantar treatment; U731: U73122, a PLC inhibitor; GF109: GF109203X, a PKC inhibitor; PWT: paw withdrawal threshold.

Discussion
AIs are the mainstay of endocrine treatment for postmenopausal women diagnosed with hormone receptor-positive breast cancer [3,7]. However, they cause musculoskeletal pain, leading to non-adherence or discontinuation of therapy [8]. Although the participation of the TRPA1 channel and kinins B1R and B2R in AI-induced pain is well characterized [13,44], the interaction between these receptors in the development and maintenance of pain remains unknown. In this study, single or repeated (for 15 days) anastrozole administration caused pain symptoms (mechanical allodynia and muscle strength loss) in mice. The local administration of B2R, B1R, or TRPA1 agonists induced overt nociceptive behav- Veh plus Veh group. ** p < 0.01, and *** p < 0.001 vs. ANS plus AITC group. Data were expressed as the mean + SEM (n = 6/group) and analyzed by one-or two-way ANOVA followed by the Bonferroni post hoc test. Veh: intraplantar or oral vehicle injections; ANS: oral administration of anastrozole; U731: U73122, a PLC inhibitor; GF109: GF109203X, a PKC inhibitor; PWT: paw withdrawal threshold.

Discussion
AIs are the mainstay of endocrine treatment for postmenopausal women diagnosed with hormone receptor-positive breast cancer [3,7]. However, they cause musculoskeletal pain, leading to non-adherence or discontinuation of therapy [8]. Although the participation of the TRPA1 channel and kinins B 1 R and B 2 R in AI-induced pain is well characterized [13,44], the interaction between these receptors in the development and maintenance of pain remains unknown. In this study, single or repeated (for 15 days) anastrozole administration caused pain symptoms (mechanical allodynia and muscle strength loss) in mice. The local administration of B 2 R, B 1 R, or TRPA1 agonists induced overt nociceptive behavior and enhanced and prolonged the pain parameters in anastrozole-treated mice. All painful behaviors mentioned above were attenuated by B 2 R, B 1 R, or TRPA1 antagonists. The interaction between B 2 R and B 1 R and the TRPA1 channel was confirmed using agonists and antagonists of these receptors and an in vivo desensitization protocol. Furthermore, we showed that signaling pathways downstream from B 2 R and B 1 R activation involving PLC, PKC, and PKA are crucial factors in sensitizing TRPA1 and contributing to sustaining anastrozole-induced pain symptoms.
In clinical practice, patients receive anastrozole for long periods, and many develop musculoskeletal pain that persists throughout the treatment [3,8]. In this study, we showed for the first time that anastrozole treatment for 15 days induced pain behavior in mice without causing desensitization, similar to other AIs (exemestane and letrozole) [13]. The maximum pain behavior occurred 3 h after anastrozole treatment, which is consistent with the peak plasma concentration of anastrozole in humans (2-4 h after administration) [55].
Local exposure to agonists of kinin receptors (B 2 R or B 1 R) and TRPA1 (at doses that generally do not cause nociception) is associated with immediate nociceptive responses lasting for a few minutes and more prolonged nociceptive behaviors in animals previously sensitized [13,38,40,56]. Although we have utilized sub-nociceptive doses of B 2 R and B 1 R and TRPA1 agonists, it is worth mentioning that bradykinin (a B 2 R agonist) and AITC (a TRPA1 channel agonist) can evoke pain behaviors in a dose-dependent manner in naive animals [35,57,58]. However, even with increasing DABk doses (a B 1 R agonist), it does not induce painful behaviors in naive animals [23,35,59], only in animals previously sensitized by different pain models [23,57,59], such as after anastrozole treatment. Corroborating with these results, we showed for the first time that the intraplantar administration of B 2 R, B 1 R, or TRPA1 agonists caused overt nociceptive behavior and enhanced the mechanical allodynia and loss of muscle strength of anastrozole-treated but not vehicle-treated mice, which were reduced by pre-treatment with their respective antagonists. In fact, systemic B 2 R, B 1 R, or TRPA1 antagonists also reduced the mechanical allodynia and loss of muscle strength of AIs-treated mice [13,44].
Although AIs activate the TRPA1 channel, the AI concentration needed for its activation in vitro is higher than that in patient plasma [13], suggesting that additional factors interact to increase the sensitivity of TRPA1 to AIs. Indeed, previous studies have reported that TRPA1 endogenous agonists are pro-algesic mediators that contribute to sensitizing TRPA1 and generating AI-induced painful conditions [13,40]. Furthermore, PAR-2 activation also sensitizes TRPA1, causing AIs-induced pain [13]. Therefore, the current findings indicate that other pro-algesic factors or signaling pathways that might activate or sensitize TRPA1 may act along with AIs to trigger pain symptoms. In this sense, B 1 R and B 2 R activation could sensitize the TRPA1 channel once they are co-expressed in sensory neurons and participate in the painful stimulus transmission [25,[27][28][29]60]. Once studies have shown that B 2 R and B 1 R interact with TRP channels, including TRPA1 [30,31,35,37,61], we hypothesized that B 2 R and B 1 R activation is a prerequisite for enhancing the sensitivity of TRPA1 to AIs.
Confirming this hypothesis, B 2 R and B 1 R antagonists prevented the sensitizing effect of the TRPA1 agonist on pain parameters in anastrozole-treated animals. Furthermore, the TRPA1 antagonist also prevented pain behaviors induced by B 1 R and B 2 R agonists, similar to other studies [33,62]. Our results are also consistent with previous studies showing that the pharmacological inhibition or genetic deletion of TRPA1 reduces pain responses to intraplantar injections of B 2 R and B 1 R agonists [30,33].
The repeated or continuous stimulation of G protein-coupled receptors (GPCRs) results in a desensitization process, i.e., a loss or reduction of their response [63]. Utilizing an in vivo desensitization protocol, we reinforced the interaction between B 2 R and B 1 R and the TRPA1 channel in the pain symptoms induced by anastrozole. The intraplantar TRPA1 agonist injection in previously anastrozole-treated mice failed to cause pain-related behaviors in animals submitted to desensitization protocol by B 2 R and B 1 R agonists. To date, there have been no studies performing the B 1 R desensitization protocol. However, both B 2 R and B 1 R receptors can be expressed in sensory neurons [29]. Once Ferreira et al. [35] demonstrated that repeated administration of Bk resulted in B 2 R desensitization, a GPCR, we adapted the experimental protocol for B 1 R desensitization. Together, these results suggest that the TRPA1 channel might be sensitized downstream from B 2 R and B 1 R activation, contributing to pain symptoms induced by anastrozole. However, the underlying pathways activated by kinin receptors and the putative sensitizers of TRPA1 involved in this process have not yet been elucidated. In this sense, we investigated the involvement of PLC/PKC-and PKA-dependent signaling pathways on TRPA1 sensitization from B 2 R and B 1 R activation. B 2 R and B 1 R stimulation leads to the activation of the PLC/PKC-dependent signaling pathway [64], resulting in increased cytosolic calcium (Ca 2+ ) levels and diacylglycerol release. Diacylglycerol can activate PKC or be hydrolyzed by diacylglycerol lipase to generate 2-arachidonolyglycerol, followed by arachidonic acid formation dependent on monoacylglycerol lipase [64,65]. The PLC/PKC pathway activation contributes to sensitizing sensory neurons, and therefore, it is critical for developing acute and chronic pain conditions [37,[66][67][68]. Once PLC/PKC pathway inhibition reduces the painful behaviors [37,[67][68][69], we evaluated the involvement of this signaling pathway dependent on B 2 R and B 1 R activation in anastrozole-treated mice. Our findings indicate that PLC and PKC activation sustains the sensitizing effect of B 2 R and B 1 R agonists in anastrozole-treated animals once inhibition of their activity significantly attenuates the pain symptoms in animals. Consistent with these findings, previously published data has confirmed that activating the PLC/PKC signaling pathways dependent on kinin receptors is essential to assist hypersensitivity in several pain conditions in rodents [31,33,35,37,59,70].
Although less evident, some studies suggest that kinin receptors might sensitize nociceptors dependent on PKA activation [31,35,71]. Indeed, PKA signaling mediates different types of pathological pain [72][73][74][75], while its inhibition reduces the neural response [31,32] and pain behaviors [74,76,77]. Here, we identified that PKA activation seems to be critical in mediating the anastrozole-induced pain because its blockade reversed B 2 R and B 1 R agonist-induced sensitizing effects. It is plausible that kinin receptor activation leads to the activation of PKA by indirect mechanisms dependent on prostaglandin production. Arachidonic acid, formed by PLC/PKC signaling, is rapidly converted by cyclooxygenases to prostaglandins, which prostanoid receptors activate, stimulating cAMP production by adenylyl cyclase. cAMP activates PKA, which phosphorylates signaling proteins, including the TRPA1 channel, contributing to its activation [31,34,78]. Nonetheless, more studies are necessary to elucidate PKA activation followed by B 2 R and B 1 R activation.
The TRPA1 channel might be sensitized by intracellular mechanisms dependent on B 2 R and B 1 R activation [30,31,33,71]. The signaling molecules that activate TRPA1 downstream of PLC/PKC, or PKA are widely debated in the literature [30,32,34]. The substances metabolized during arachidonic acid hydrolysis, the release of Ca 2+ from intracellular stores, and TRPA1 phosphorylation by PKC and PKA are well-elucidated mechanisms [30][31][32]34]. These mechanisms are potentially linked and could enhance TRPA1 activity and/or trafficking to the membrane, allowing the amplification of pain symptoms [32]. We found that inhibition of the PLC/PKC and PKA pathways attenuated the overt nociceptive behavior and the sensitizing effect on pain symptoms induced by a TRPA1 agonist in anastrozoletreated animals. Our findings suggest that PLC/PKC and PKA may act downstream from B 2 R and B 1 R activation to sensitize the TRPA1 channel, leading to painful anastrozoleinduced symptoms. Since anastrozole action is peripherally restricted [79] and the agonists of the B 2 R, B 1 R, and TRPA1 channels and PLC/PKC and PKA signaling pathways inhibitors were locally administered, it is plausible that all the painful symptoms observed in this study are due to peripheral effects.
We reported that the B 2 R and B 1 R activation-dependent PLC/PKC and PKA intracellular signaling pathways seem to interact to sensitize the TRPA1 channel in anastrozoleinduced pain. Thus, direct TRPA1 activation by anastrozole and indirect TRPA1 stimulation from B 2 R and B 1 R activation appear to converge in a common nociceptive pathway. The interaction between kinin receptors and the TRPA1 channel suggests a novel paradigm to explain the development of the pain symptoms induced by anastrozole. Therefore, regulating the activation of signaling pathways downstream of B 2 R and B 1 R would be a promising alternative for developing drugs to treat AIs-related pain, such as anastrozole. The prompt and targeted treatment of pain symptoms might contribute to (i) re-establishing the quality of life of breast cancer survivors, (ii) improving adherence to AI therapy, and (iii) consequently contributing to disease control.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/pharmaceutics15041136/s1. Figure S1: The B 2 R, B 1 R, and TRPA1 channels contribute to the development of painful behaviors induced by anastrozole in mice.

Institutional Review Board Statement:
The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Federal University of Santa Maria (CEUA, process numbers 2304280220/2020 and 3026220520/2020).
Informed Consent Statement: Not applicable.