Study On Effects Of Tetramethylpyrasine On Pain Mediated By P2X₃ Receptor Of Primary Sensory Neuron And Its Mechanism

Background Pain is a common clinic symptom of many illnesses, and is a untoward sensation owned by both human being with individual difference. Pain can be divided into acute pain (physiology pain) and chronic pain (pathology pain). Acute pain is a kind of immediately pain caused by nociceptive stimulus, which mainly activating the nociceptors, so is called nociceptive pain. Chronic pain includes inflammation pain, neuropathic pain, and cancer pain, and is a common symptom in clinic. Burn is a severe injury, and burn pain is the very important acute pain. It is necessary to combine antinociceptive treatment with antineuropathic treatment for the method of burn pain management. At present, opioids are the primary method of pain management, but those drugs can produce the side effects such as depression of ventilation and consciousness, decreased gastrointestinal motility and constipation, nausea and vomiting, urinary retention, and physical dependence. In addition, opioid pharmacodynamics was altered in patients with pain, with requirements increasing over time so that even high doses of opioids may not totally relieve the pain in some patients. Neuropathic pain is one of the important kinds of chronic pain, and is the clinic tough problem. So it is a focal point to search a new mechanism of pain formation and analgesic drug. It was reported in British journal Nature that P2X3 receptors were localized predominantly on small nociceptive sensory neurons in dorsal root ganglion (DRG) and played a crucial role in facilitating pain transmission at peripheral and spinal sites. The purinoceptor belongs to two classes, the P1 and P2 receptor, ATP and its analogues can act on P2 receptor. It was proposed that ATP released from a variety of sources, such as damaged or stressed cells, could perhaps play a role in activating nerve P2X3 receptor, evoke action potential transmitting to central nervous system along neural axis. Tetramethylpyrazine(TMP)is one of the alkaloids contained in Ligustrazine which has been used in traditional Chinese medicine as an analgesic for injury. It was reported that TMP could inhibit inflammatory effect. Thus it was suggested TMP might bring out antinociceptive response. The action mechanisms of TMP may be associated with inhibiting the signals of P2X3 receptor activation. The present studies try to investigate the effects of TMP on the pain mediated by P2X3 receptor. The antinociceptive effect of TMP was investigated, on an acute nociception and a chronic constriction injury (CCI) model and a burn injury model, using behaviour methods, immunohistochemistry, in situ hybridization and whole cell patch clamp technique. This work hopes to look for a novel analgesic for pain study.PartⅠEffects of tetramethylpyrazine on acute nociception mediated by P2X receptorObjective: To observe the effects of tetramethylpyrazine (TMP) on acute nociception in rat hindpaw mediated by purine 2X (P2X) receptor.Methods: The effects of TMP administered subcutaneously and intrathecally on the acute nociception induced by P2X receptor agonists or co-applied with PGE2 in the rat hindpaw were investigated by the method of the behavioral study. Intracellular recording technique was used to study the effects of TMP on the membrane depolarization induced by ATP in freshly isolated rat dorsal root ganglion (DRG).Results TMP (10mmol/L ) significantly depressed the acute nociception induced by ATP (1μmol/L ) orα,β-meATP ( 0. 6μmol/L ) in the rat hindpaw. TMP (10mmol/L) could inhibit the acute nociception induced by PGE2 (5μmol/L ) orα,β-meATP (0.2μmol/L ) coinjected with PGE2 (5μmol/L ). TMP could not obviously affect the inflammatory edema in rat hindpaw induced by the local administration of ATP orα,β-meATP coinjected with PGE2 individually. By intracellular recording , TMP(300μmol/L) preapplied could inhibit the membrane depolarization induced by ATP in freshly isolated rat dorsal root ganglion (DRG).Conclusion: The antinociceptive effects of TMP may mainly be associated with inhibiting the transmission of nociceptive information mediated by P2X receptor activation. PartⅡEffect of TMP on neuropathic pain mediated by P2X3 receptorObjective: To investigate the effects of tetramethylpyrazine (TMP) on rat neuropathic pain induced by P2X3 receptor.Methods: Chronic constriction injury (CCI) rat model was adopted. Mechanical withdrawal threshold and thermal withdrawal latency were measured and P2X3 receptor expressions in L4/L5 spinal cord and DRG were detected by immunohistochemistry and in situ hybridization. Whole-cell patch-clamp technique was used to study the effects of TMP on P2X receptor agonists-activated currents in freshly isolated CCI rat dorsal root ganglion (DRG) neurons.Results: At day 14 after operation, the mechanical withdrawal threshold and thermal withdrawal latency in groupⅤ(CCI group) were lower than groupⅠ(NS group),Ⅱ(TMP group),Ⅲ(sham group) andⅣ(CCI+ TMP group) (p<0.05), while the expression of P2X3 receptor in L4/L5 spinal cord in groupⅤwas higher than those in groupⅠ,Ⅱ,ⅣandⅢ(p<0.01). The mechanical withdrawal threshold, thermal withdrawal latency and the expression of P2X3 receptor in L4/L5 spinal cord and DRG showed no significant difference among groupⅠ,Ⅱ,ⅢandⅣ(p>0.05). The expression of P2X3 receptor in L4/L5 spinal cord and DRG in groupⅣwas lower than that in groupⅤ(p<0.05). ATP-activated currents (IATP) on DRG in groupⅤwas higher markedly than those in groupⅠ,Ⅱ,ⅢandⅣ(p<0.05). There were no significant difference for IATP among groupⅠ,Ⅱ,ⅢandⅣ(p>0.05).Conclusion: TMP may alleviate neuropathic pain induced by P2X3 receptor. PartⅢEffect of TMP on burn injury pain mediated by P2X3 receptorObjective: To investigate the effects of tetramethylpyrazine (TMP) on rat burn injury pain induced by P2X3 receptor.Methods: First degree and superfacial second degree burn injury models were adopted. Mechanical withdrawal threshold and thermal withdrawal latency were measured and the P2X3 receptor expressions of never terminal in burn injury skin were detected by immunohistochemistry. Whole-cell patch-clamp technique was used to study the effects of TMP on P2X receptor agonists-activated currents in freshly isolated burn injury rat dorsal root ganglion (DRG) neurons.Results: At hour 1 after burn, the mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in groupⅢA (first degree foot burn +NS group) and groupⅢB (superfacial second foot burn +NS group) were lower than that in groupⅠ(sham foot burn), lasting for 24 or 96 hours respectively (p<0.01). After hour 24, there was no difference in MWT and TWL compared groupⅡA (first degree foot burn +TMP group) with groupⅠ, but there was difference between groupⅡB (superfacial second foot burn +TMP group) and group I and no difference between both after hour 72. At day 3 postburn, the P2X3 receptor expressions of never terminal of burn injury skin in groupⅥA (first degree back burn +NS group) and groupⅥB (superfacial second back burn +NS group) were significantly increased to compare with other groups (p<0.05). Post-treated with TMP, the P2X3 receptor expressions of never terminal in group groupⅤA (first degree back burn + TMP group) andⅤB (superfacial second back burn +TMP group) were markedly decreased. ATP-activated currents (IATP) of DRG neurons in groupⅥB was higher markedly than those in groupⅤB andⅥ(sham back burn)(p<0.05). There were no significant difference for IATP between groupⅤB andⅣ(p>0.05). P2X3 receptor agonistα,β-meATP activated current in groupⅥB was hiher than those in groupⅣand groupⅤB. Conclusion: TMP may alleviate burn injury pain induced by P2X3 receptor.