The present study applied molecular docking to foretell the more promising chalcone-derived apocynin with greater stability, geometric, and energetic profiles for TRPV1-ligand complexes. In vitro studies using primary cell culture of trigeminal ganglion were carried out to analyze apocynin’s capacity to counteract TRPV1 activation by capsaicin. Furthermore, an in vivo model of neurogenic pain elicited by capsaicin was conducted to explore the therapeutic potential of apocynin in orofacial pain in rodents and the mechanism behind its effects. Lastly, in vivo toxicity of apocynin was performed in a survival assay on the Galleria mellonella larvae model. Herein, we demonstrated that apocynin has outstanding pharmacokinetics and binding properties against TRPV1 receptors and ultimately hindered trigeminal neuron activation upon the capsaicin challenge. In vivo, apocynin showed antinociceptive peripheral effects, decreasing the number of immunopositive TRPV1+ cells in the trigeminal ganglion, reducing extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and reducing substance P and CGRP release. In addition, the apocynin demonstrated a slightly toxic compound in the Galleria mellonela model. Overall, we provide proof of principles that apocynin can be elected as a potential pharmacological alternative to manage orofacial pain, particularly those of a neuropathic nature. However, clinical tests must be carried out to prove its effectiveness in orofacial pain.
The results of molecular docking for the apocynin ligand indicate its placement within the vanilloid pocket, a well-known binding site previously characterized for modulators like CAP and CZP. Given the vanilloid-like nature of the apocynin ligand, particularly when compared to the head region of CAP and CZP, it is reasonable to presume its localization within this binding site. Previous research has also indicated that compounds sharing this vanilloid anchoring region tend to occupy this specific site (Bustos et al., 2023). This higher RMSD profile observed for the apocynin ligand within the binding site may be attributed to a potential discrepancy in its initial spatial configuration originating from the molecular docking process. This effect was not observed when utilizing the CAP and CZP conformations obtained from structures derived from Cryo-Electron Microscopy methods. It is crucial to note that the TRPV1 channel complexes employed in this study, specifically TRPV1-CAP and TRPV1-Apo, originate from Ictidomys tridecemlineatus, a mammalian species. In contrast, the TRPV1-CZP complex originates from Rattus norvegicus. This distinction results in a shift in residue numbering within the vanilloid binding site. Regarding the intermolecular interactions between each ligand and the binding site in TRPV1, we observed that the Apo ligand maintains the same interactions previously reported for the vanilloid head of CAP and CZP. These interactions include hydrogen bonds (HBs) with TYR513 and GLU572 (TYR511 and GLU570 in TRPV1-CZP). Additionally, TYR513 forms π-π interactions with Apo, akin to what was found with CAP. Notably, the residue ILE571 forms HBs with Apo, in contrast to the interactions observed with the other two ligands. The remaining interactions that anchor Apo within the binding site are predominantly hydrophobic in nature.
The discovery that capsaicin could be used as a harmful agent in a pain model was a landmark in the pain and pharmacology field (Wood et al., 1988; Oh et al., 1996). It was also through this discovery that other studies were conducted to comprehend the involvement of TRPV1 receptors in this process (Sluka & Willis 1997). One of the first pieces of evidence on the mechanism of action of capsaicin revealed that it lowered the thermal threshold in rats and humans (Caterina et al., 1997; Caterina et al., 1999; Caterina et al., 2000). Furthermore, evidence was presented regarding the involvement of MAPKs and the neurotransmitter CGRP in this pain process (Sun et al., 2003; Sweitzer et al., 2004). In this sense, we opted for capsaicin as the noxious challenge to investigate the potential analgesic role of apocynin. Herein, we treated the rats with a dose titration of intra-articular injection of apocynin, and we found that the analgesic effect started at a dosage of 100 ng. However, only with 200 ng, the nociceptive score was equivalent to that of the negative control group (intra-articular saline injection). Therefore, we selected 200 ng of apocynin for the subsequent experiments. Similar findings were seen on the grimace scale, although the negative control demonstrated a statistical difference compared to apocynin. We further investigate whether this antinociceptive effect was exclusively local or if the dosage was high enough to reach the bloodstream. For that, we treated ipsilaterally of the side we challenged the animals with capsaicin (as illustrated in Fig. 3E). We demonstrated that apocynin's antinociceptive effect was exclusively local, which could prevent any side effect or first-pass metabolism that could shrink their therapeutic properties. By way of comparison, tramadol (an opioid used worldwide) induces antinociception ranging from 90 to 500 ug when injected into the temporomandibular joint (Abdalla et al., 2019; Lamana et al., 2017), which is 450–2500-fold more heightened, respectively.
We further examined, histologically, the impact of intra-articular injection of apocynin in the TRPV1 expression in the trigeminal ganglion. Our data clearly show that apocynin reduced the number of TRPV1+ immunopositive cells in the TG. TRPV1 is a receptor capable of transducing noxious (thermal) stimulus into nociceptor depolarization, mainly expressed in primary neurons, including TG (Caterina et al., 1997; Tominaga et al., 1998). For instance, mice with genetic deletion of TRPV1 show lower nociceptive reactions against heat and capsaicin challenges (Caterina et al., 2000). Furthermore, in thermal hyperalgesia induced by carrageenan, the disruption of TRPV1 counteracts the inflammatory pain mouse model of dry eye disease, where local inflammation triggers TRPV1, consequently causing corneal nerve damage (Pizzano et al., 2024). Therefore, inhibition of TRPV1 has been tested in distinct pain modalities with promising findings.
Transient receptor potential (TRP) ion channels, including members of the vanilloid TRP (TRPV), melastatin TRP (TRPM), canonical TRP (TRPC), and ankyrin TRP (TRPA) families, have drawn attention for their roles in myriad sensory functions (Fernandes et al., 2012). Both TRPV1 and TRPA1 play a critical role in inflammation and tissue damage and therefore have great potential as drug targets (Liang et al, 2023). We observed that intra-TMJ injection of apocynin significantly increased the expression of TRPA1, but not TRPV1. TRPA1 is widely expressed in neuronal and non-neuronal cells and has been proposed as a nociceptor mediating acute and inflammatory pain (Bautista et al., 2006; Kwan et al., 2006) through studies that demonstrated suppression of sensitivity to mechanical and cold stimuli and induced hyperalgesia in transgenic TRPA1-deficient mice (Obata et al., 2005; Kwan et al., 2006; Karashima et al., 2009). Interestingly, growing evidence has reported that activation of TRPV1 and TRPA1 causes increased intestinal motility and protective effects against gastrointestinal injury via neurotransmitter stimulation (Kojima et al., 2014; Liu et al., 2023). NADPH oxidase (NOX) inhibitors, as such the apocynin family, have been shown to activate TRPA1 in human embryonic kidney cells and human fibroblast-like synoviocytes (Suzuki et., 2014). Herein, we observed that the antinociceptive effects produced by apocynin were not changed, despite the boost in TRPA1 expression. We hypothesized that the increase in TRPA1 was a compensatory physiological response to TRV1 blockage. However, additional studies are needed to determine whether currently available TRPV1 or TRPA1 antagonists have any relative specificity for these TRP channels in different cell types (Fernandes et al., 2012).
We also found that apocynin-mediated inhibition of TRPV1 decreases the expression of ERKs and reduces the levels of substance P and CGRP. As mentioned previously, TRPV1 activation leads to the release of substance P and CGRP, which are neuropeptides widely distributed in areas of the central and peripheral nervous systems, playing an important role in pain neurotransmission (Fattori et al., 2016). TRPV1 has been shown to co-localize with substance P and CGRP in a dextran sulfate sodium-induced colitis model (Lapointe et al., 2015). Indeed, pharmacological blockage of TRPV1 inhibited capsaicin-induced substance P release in cultured dorsal root ganglion (DRG) neurons (Tang et al., 2008). Additionally, it was reported that Maresin-2 (MaR2), a specialized pro-resolution lipid mediator (SPM), inhibits the activation of TRPV1 and TRPA1, as well as reducing the release of CGRP and decreases pain and inflammation in a model of pain in lipopolysaccharide (LPS)-induced mice (Fattori et al., 2022).
In conclusion, we provide proof-of-principles that the newly synthesized apocynin compound effectively prevented nociception in a neurogenic model of orofacial pain. Mechanistically, the treatment with apocynin reduced the number of TRPV1+ immunopositive cells, decreased the release of substance P and CGRP, reduced the phosphorylation of ERK in the trigeminal ganglion, and demonstrated low toxicity.