Effect Of Lappaconitine On P2X₃ Receptors-mediated Pain Transmission In Rat Dorsal Root Ganglion Neurons And Relative Mechanism

The pain does serious harms to the human health, reduces the quality of life and adds great burden to the families and the society. The neuropathic pain caused by the peripheral nerve injury and lesion has a complex pathogenesis and is a kind of chronic pain clinically difficult to cure, to which much efforts has been committed in terms of research and development of the analgesic drugs. In recent years, the role of the purinergic receptors in the pain has gradually aroused the attention of researchers and the analgesic mechanism exploration and the drug development with the purinergic receptors as targets have become a hotspot in the field of pain research. The dorsal root ganglion (DRG) neurons are primary afferent neurons, where distributed a variety of P2X receptors (P2X_1-6) especially the P2X₃ receptor that was highly selectively expressed in the nociception-related small- and medium-size DRG cells. The nociceptive stimulus to the body may induce the activation of the purinoceptor released from the cells by adenosine 5'-triphosphate (ATP), as results in excessive excitability of the primary afferent neurons. In the meantime, ATP could activate the pain pathways through increasing the calcium influx and regulating the effects of the pain-relative substances including glutamate (Glu), the substance P (SP), GABA, PGE2 and GNF. Therefore, reduction of the excitability of the primary afferent neurons has become one of the treatment measures for the neuropathic pain.Lappaconitine (LA), also called as thorn aconitine, is the alkaloid extracted from the aconitum roots of a ranunculaceae plant. For a very long time, the people in China and Japan used aconite root decoction for Qufeng (expelling the wind), dehumidification, dissipation of the wounds and alleviation of the pain, etc. As the non-narcotic analgesics, LA was first discovered in China and has been accepted as the three-step analgesic medication for the cancer patients, with advantages of nil-clinical addiction, nil-teratogenicity, nil-mutation, nil-toxic accumulation and low adverse reaction. The previous studies on LA have focused on the clinical application of LA. In recent years, there reported certain researches on the mechanism of LA, which was scattered, superficial and nonsystematic in nature however. In fact, the exact mechanism of LA still remained unclear at present. Most previous studies argued that the analgesic action site of LA was not in the spinal cord but in the nerve center above the spinal cord, including the hippocampus, periaqueductal gray (PAG), nucleus raphes magnus (NRM), etc. Some researches have proved that LA was the Na⁺ channel blocker and central calcium channel blocker and could affect the potassium current, when the central norepinephrine system, the opioid system and the downlink modulation system were involved. However, LA has been clinically and widely used for epidural anesthesia, patient-controlled epidural analgesia (PCEA), subarachnoid block and regional anesthesia and obtained sound outcome. Some studies suggested that the intraperitoneal injection of LA could inhibit the effect of SP and somatostatin (SOM) on the spinal cord, reduce the expression of c-fos of the spinal cord nerve cells in rats with formalin-induced pain, significantly alleviate the thermal hyperalgesia of the rats with chronic constriction injury (CCI) of the sciatic nerve and decrease the expression of NF-?B in the spinal cord, all of which could not be explained by the supraspinal analgesic mechanism. Accordingly, we speculated that there may exist some analgesia mechanism at the spinal cord level or at subspinal cord level. In order to better guide clinical medication, it is urgent to carry out study on the mechanism of LA, especially its action site and mechanism in the spinal cord, low-level center, peripheral nervous system and non-nervous system.P2X₃ receptors are the non-selective cation channel and the previous studies suggested that LA may be the cation channel blocker (Na⁺, Ca2⁺). However, whether there existed correlation between LA and the P2X₃ receptors has not reported so far. Our previous experiments found that the intraperitoneal injection of LA could alleviate the pain caused by the plantar injection of ATP, suggesting that the analgesic effect of LA may be associated with the P2X₃ receptors. In order to further clarify the correlation between LA and the P2X₃ receptors of the DRG neurons and the possible action mechanism, we, based on the study on the purinergic receptors of our laboratory, have integrated the methods including morphology, molecular biology, oligonucleotides and electrophysiology under in vivo and in vitro conditions to observe the following phenomena: (1) the effect of LA on the behavior (flinching reaction) of rats with ATP-induced pain and on the expression of the P2X₃ receptor in the DRG neurons; (2) the effect of LA on the behavior (thermal hyperalgesia and mechanical hyperalgesia) of CCI rats and on the expression of the P2X₃ receptor in the DRG neurons; (3) the effect of LA on the ATP and?,?-meATP activated current (ATP- and?,?-meATP-induced current, IATP and I?,?-meATP) of CCI rats in the DRG neurons; (4) the effect of LA on the evoked and spontaneous discharges of ATP in the DRG neurons of CCI rats; (5) the effect of LA on the behavior (thermal hyperalgesia and mechanical hyperalgesia) of CCI rats after the antisense oligonucleotide of the P2X₃ receptor was used to lower the expression of the P2X₃ receptor in the DRG neurons; (6) the effect of LA on the P2X₃ receptor-mediated neuropathic pain from different perspectives and the preliminary study on the mechanism of this regulation.The main results were as follows:1. LA inhibited the flinching response induced by the plantar injection of ATP and the increase of the expression of P2X₃ receptors in the DRG neurons.The flinching response was significantly increased in rats after the plantar injection of ATP. The intraperitoneal injection of LA could significantly alleviate the hyperalgesia induced by the plantar injection of ATP. The immunohistochemistry experiments showed that the expression of the P2X₃ receptors was increased in the small- and medium-sized neurons of the ipsilateral L4-6 DRG segment. While the expression of the P2X₃ receptor in the small- and medium-sized neurons of the ipsilateral L4-6 DRG segment was significantly decreased posterior to LA treatment, when the expression of the P2X₃ was consistent with change of the hyperalgesia.2. LA inhibited the CCI-induced mechanical allodynia and thermal hyperalgesia, the increase of the CCI-induced expression of the P2X₃ receptor of the DRG neurons and the inward current mediated by the P2X₃ receptor of the DRG neurons. With the intrathecal injection of the P2X₃ receptor antisense oligonucleotide, there showed a weakened effect of LA on inhibiting the CCI-induced hyperalgesia.?LA inhibited the CCI-induced mechanical allodynia and thermal hyperalgesia and the CCI-induced expression increase of the P2X₃ receptor in the DRG neurons.CCI reduced the mechanical pain threshold and the heat pain threshold in SD rats but increased the expression of the P2X₃ receptor in the DRG neurons. The intraperitoneal injection of LA could increase the mechanical pain threshold and the heat pain threshold but decrease the expression of the P2X₃ receptors in the DRG neurons in the CCI rat model.?The intraperitoneal injection of LA could inhibit the CCI-induced increase of the P2X₃ receptor-mediated inward current amplitude in the rat DRG neurons.Both ATP and?,?-meATP in the rat DRG neurons had three types of the activated currents, ie, rapid desensitization response current, slow desensitization response current and mixed current from fast to slow. CCI neuropathic pain model increased the number of rat DRG neurons that generated the IATP and I?,?-meATP and powered up the current amplitude of the fast and mixed IATP and I?,?-meATP. The pretreatment with the intraperitoneal injection of LA could inhibit the increase of the current amplitude of the CCI-induced fast and mixed IATP and I?,?-meATP but had no inhibitory effect on the slow current.?Posterior to the intrathecal injection of the P2X₃ receptor antisense oligonucleotide, the inhibitive effect of LA on the CCI-induced hyperalgesia was weakened. The intrathecal microinjection of the P2X₃ receptor antisense oligodeoxynucleotide (A-ODN) for 5 consecutive days decreased the expression of the P2X₃ receptor in the DRG neurons and significantly weakened the inhibitive effect of LA on the CCI-induced hyperalgesia.3. Extracellular application of LA inhibited the ATP-activated currents mediated by the P2X₃ receptors in the rat DRG neurons as well as the CCI- and ATP-induced discharge. Extracellular application of LA inhibited the ATP-activated currents of the DRG neurons.Extracellular application of LA exerted inhibitive effect on the rapid desensitization components of the fast IATP and the mixed IATP of the rat DRG neurons in a dose-dependent manner, while no inhibitive effect was found on the slow IATP. No inhibitive effect of the Na⁺ channel blocker TTX was found in the role of LA in inhibiting the fast IATP of the DRG neurons. The PLC inhibitor neomycin and the PKC inhibitors Che could block the inhibitive effect of LA on the fast IATP of the DRG neurons, which was free from the PKA inhibitor H89 and the GPCR inhibitor GDP-?-s.LA inhibited the spontaneous discharge of the CCI rat DRG neurons and suppressed the ATP-evoked discharge of the DRG neurons in the normal and CCI rats. In the CCI rat DRG neurons, there recorded the spontaneous discharge and LA inhibited the CCI-induced spontaneous discharge frequency of the DRG neurons. Extracellular application of ATP may induce the evoked discharge of the DRG neurons in the normal rats and increased the discharge frequency of the DRG neurons in CCI rats. LA exerted inhibitive effect on the discharge frequency of the normal and CCI rats. The PKC inhibitor Che and the PLC inhibitor neomycin could significantly weaken the inhibitive effect of LA on the ATP-evoked discharge in the rat DRG neurons in both groups, for which the PKA inhibitor H89 and G protein activation inhibitor GDP-?-s exerted no effect.The results indicated that the increased excitability and sensitivity of the DRG neurons due to the increased expression and sensitivity of the P2X₃ receptors in the rat DRG neurons may play an important role in the development of the neuropathic pain caused by sciatic nerve injury and induced by the plantar injection of ATP. LA exerted the inhibitive effect on the injury-induced expression increase of the P2X₃ receptor in the DRG neurons, on the ATP current mediated by the P2X₃ receptors in the DRG neurons as well as on the discharge increase of the DRG neurons induced by CCI and ATP. The mechanism of LA relieving the pain is through changing the expression and function of the P2X₃ receptors, weakening the sensitivity and excitement of the primary neurons and reducing the generation and transmission of the pain information, which may involve the intracellular PKC and PLC signaling pathways. This study can help deepen the understanding of the analgesic mechanism of LA and provide new ideas for clinical application of LA and treatment of pain.