PAR2 has been highlighted as a target for multiple proteases and it can mediate pain in different conditions [7]. PAR2 in the peripheral nerve endings are implications of the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states [32]. The focus on the pathophysiology and therapeutic potential of purinergic signaling has been raised more recently [73]. The close relationship between P2 × 3 and the pain-sensing system and the specific actions of P2 × 3 on pain sensation attract us to explore P2 × 3 as a novel potential therapeutic target on relieving pain. Sensitization of TRPV1 and TRPA1 via PLC and PKC kinases is correlated with the role of PAR2-mediated nociceptive signaling and pain has been recognized recently [74]. In the present study, both in vitro model of DRG with PAR2 agonist SL-NH2 challenge and SL-NH2-induced pain rat model were used to investigate the relationship between PAR2 activation and nonselective cation channels in mediating pain. The results showed that PAR2 agonist SL-NH2 induced upregulation of P2 × 3, TRPV1, and TRPA1 and phosphorylation of PLCβ3 and PKCε in DRG neurons in vitro and in vivo. SL-NH2 also elevated the proportion of P2 × 3-, TRPV1-, and TRPA1-expressing neurons. The upregulation of P2 × 3, TRPV1, and TRPA1 and phosphorylation of PLCβ3 and PKCε in DRG neurons was paralleled with mechanical allodynia and thermal hyperalgesia behaviors in rats. These findings represent a novel peripheral mechanism underlying PAR2 signaling in mechanical allodynia and thermal hyperalgesia by regulating the expression of different nonselective cation channels.
The relationship between nonselective cation channels P2 × 3, TRPV1, and TRPA1 and PAR2 activation in mediating mechanical allodynia and thermal hyperalgesia deepened our knowledge about PAR2-mediated pain-sensing system. P2 × 3-expressing non-peptidergic small-diameter unmyelinated C-fibers represent a large proportion of somatic nociceptors in pain-sensing processing system [65]. From the nociceptor's receptive field in peripheral tissue, excitatory P2 × 3 drives the initial nerve impulse along with the sensitizing C-fibers to facilitate pain signaling transmission through their central terminals to the spinal dorsal horn (SDH). Co-activation of PAR2 and TRPV1 or TRPA1 resulting in the release of proinflammatory neuropeptide transmitters is a new mechanisms in mediating pain responses [66, 67]. The PLC and PKC signaling in neurons is commonly considered as the pathway of pain and is usually the target for treatment. However, the extent of nonselective cation channel P2 × 3, TRPV1, and TRPA1 as the common effector of PAR2 remains uncertain. In this way, the involvement of PLCβ3 and PKCε signaling pathway as well as P2 × 3, TRPV1, and TRPA1 in the context of PAR2 activation were investigated. In this study, we used pain behavior animal model mediated by PAR2 activation, as well as cultured DRG neurons with PAR2 activation to examine the possible signaling and cation channel mechanisms involved. The intraplantar injection of the PAR2 agonist SL-NH2 caused the pain behavior associated with c-Fos elevation in SDH. The signaling molecules in pain behavior animal model DRG and SL-NH2 treated DRG culture were upregulated. PAR2 agonist also upregulates the expression of TRPV1, TRPA1 and P2 × 3 channels suggested that the direct activation of PAR2 can induce TRPV1, TRPA1, and P2 × 3 channel expression through PLCβ3 and PKCε signaling pathway. Furthermore, pain behavior observed in animal model in the presence of PAR2 activation suggested P2 × 3, TRPV1, and TRPA1 is related to animal pain behavior.
These results provided additional support for the development of new therapeutic alternatives that can focus on pain induced by activation of PAR2. The expression of c-Fos is inducted after DRG inflammation, DRG neuron hyperexcitability, and neuropathic pain hypersensitivity [75, 76], including PAR2 involved nociception [77]. c-Fos increases the expression of proinflammatory cytokines and causes neurochemical alterations in the DRG neurons [18, 78]. In the present study, the pain induced by SL-NH2 injection indicated that PAR2 is activated. The c-Fos protein was expressed in the SDH of SL-NH2-induced pain model. PAR2 contributes to the onset and maintenance of SL-NH2-induced pain hypersensitivity in a c-Fos dependent manner. PAR2 is involved in this characteristic behavior because c-Fos induces the synthesis of proinflammatory cytokines or neurotransmitters causing peripheral inflammation.
Interestingly, TRPA1-mediated heat pain sensitivity may indirectly modulate TRPV1 channels which were co-expressed with TRPA1 in nociceptors. These two receptor channels are in a synergistic and/or conditional relationship with noxious stimulation [79]. In this present study, the expression of TRPV1 and TRPA1 were significantly increased by SL-NH2-induced PAR2 activation in cultured and pain behavior animal model DRG neurons. P2 × 3 channel expression was affected similarly by PAR2 activation in this experiment.
In the peripheral terminals of sensory neurons, PLC-PKC cascade can directly activate or sensitize neighboring TRPV1 and TRPA1 channels in DRG [16, 28, 80, 81]. According to the subdivision of DRG neurons, TRPV1- and TRPA1-positive neurons are related to both peptidergic and non-peptidergic nerve fibers, whereas P2 × 3-expressing neurons represent non-peptidergic DRG neurons [82–87]. It has been shown that the co-expression of other receptors with P2 × 3 in C-fibers can alter neuronal sensitization induced P2 × 3 activation. The key role of co-expression of TRPV1 and P2 × 3 in nociceptive neuronal subpopulations in mediating severe pain complicated the concept and mechanism of nonselective cation channels on pain signaling processing [65]. The close relationship between PAR2 and TRPV1 is reflected by the co-expression of PAR2 and TRPV1 in nociceptive DRG neurons. Blocking PAR2 decreases expression of TRPV1, and the blockade of TRPV1 prevents the activation of PAR2-induced persistent thermal hyperalgesia [32, 66]. PAR2 is also co-expressed with TRPA1 in small DRG neurons [74]. The attenuation of TRPA1 signaling by blocking PAR2 activation and inhibition of the TRPA1 inducing decreases of PAR2-related hyperactivity and pain suggests the close relationship and interaction between PAR2 with TRPA1 in mediating hyperactivity and pain [44, 67]. The difference in pain processing mechanisms between PAR2-positive neurons that co-expresses with P2 × 3-, TRPV1-, or TRPA1-expressing neurons and PAR2-positive neurons that do not co-expresses with other neurons should be further investigated in future studies. The previous studies have reported the modulation of TRPV1, TRPA1, or P2 × 3 by activation of PAR2 in a various neuropathic pain and inflammatory pain [88–92]. We draw on the previous research to get inspiration: Since TRPV1, TRPA1 or P2 × 3 are involved in the regulation of PAR2 during the process of mediating pain, is there a common signaling pathway which simultaneously mediates TRPV1, TRPA1 and P2 × 3 via PAR2 activation. We made the hypothesis and conducted the experiment and finally got a positive conclusion. This discovery provides a considerable clinical prospect. Additionally, PAR2 agonist also induces itch by activating superficial SDH neurons [93–96]. Although the effect of itching induced by SL-NH2 injection is difficult to rule out in our present study, we only focused on pain behaviors and their relationship with the modulation of TRPV1, TRPA1, and P2 × 3 by activation of PAR2. It is worth noting that SL-NH2 is not only active PAR2 but also Mrgpr GPCRs, particularly MrgprC11. But MrgprC11 activated by SL-NH2 will mediated histamine-independent itch rather than pain [97]. So this does not affect our conclusions. The transmission of pain information is closely related to the ion channel that precisely controls pain information on nociceptors. Non-selective cation channels P2 × 3, TRPV1, and TRPA1 have all been shown to play important roles in different types of initiation or transmission processes of pain. Previous studies demonstrated that inhibition of PAR2 can enhance the analgesic effect of opioids, thereby reducing the addictiveness of such drugs, while reducing the side effects such as itching after the application of opioids. This effect is based on the integration of PAR2-mediated TRPV1 and µ-opioid receptor [17, 98]. According to our results, PAR2 can mediate the progression of pain by non-selective cation channels P2 × 3, TRPV1, and TRPA1 via PLCβ3/PKCε signaling pathway. Applying this discovery to the development of clinical analgesic drugs by inhibiting PAR2, which can simultaneously relieve pain related with P2 × 3, TRPV1, or TRPA1, not only enhance the efficacy and application range of analgesic drugs, but also reduce side effects. As a result, TRP channels mediated enhancement of neuronal excitation and a variety of neuronal signals is closely related to the pain sensations [99].