| 1. BackgroundPain is one of the most common symptoms of most clinical diseases, and is an unpleasant reaction in the body arising from damaged tissue or potential injury, including pain and pain response. Pain is a subjective feeling, and its classification is very complicated in clinically. According to the nature, causes and duration of pain, pain can be divided into acute pain and chronic pain. Acute pain is the acute symptoms caused by disease or tissue injury, it is caused by a variety of noxious stimulus, which mainly activating the nociceptors in the body, it is also known as nociceptive pain. Acute pain occurred mainly in surgical wounds, burns, acute inflammation, myocardial infarction, organ perforation, delivery in clinically. And chronic pain is a state that the pain still exists after damage caused by noxious stimulation, which is in itself a disease. In clinically, chronic pain occurred mainly in chronic low back pain, nerve pain (neuropathic pain), advanced cancer pain, and so on. In addition, based on pathological features, pain can be divided into nociceptive pain and neuropathic pain. Nociceptive pain is the reaction caused by harmful stimulus acting on the complete nociceptors, which is related to tissue injury or inflammation, also known as inflammatory pain, belonging to acute pain. Neuropathic pain is a chronic pain, which is mainly caused by peripheral or central nervous system dysfunction. Pain is a subjective feeling of human caused by noxious stimulation, which is often happened associated with other diseases, and it is known as neuropathic pain if accompanied by neurological abnormalities suggesting there may be neuropathic factors or nerve injury, which is the most common clinical refractory pain. Neuropathic pain included many types, mainly divided into peripheral neuropathic pain and central neuropathic pain. The formation mechanism of neuropathic pain is relatively complex, neuropathic pain arises from damage, or pathological change, in the peripheral or central nervous system. It is usually a chronic condition that can be difficult to treat because standard treatment with conventional analgesics does not typically provide effective relief of pain. Patients with neuropathic pain commonly present to primary care professionals, but making a diagnosis may be difficult. Neuropathic pain is usually associated with substantially greater impairment of quality of life compared with other types of chronic pain, and the disorder is a large cost burden on healthcare services. There is still no effective treatment measure for neuropathic pain in the present, and often facing the embarrassment of invalid or ineffective treatment, which make patients suffering great pain and seriously affect the patient's normal work and daily life. Burn is a severe, acute trauma, which is clinical common and often accompanied by severe pain. Burn pain is a special type of acute traumatic pain, which is generally occurred within the first 1 hour after burn and is appearing in the entire post-burn treatment. Severe burns will result in severe and persistent pain (basic pain), and also experiencing unbearable pain in wound management (operation pain) in a few weeks or even months. Burn pain is the most unbearable pain and the most common clinical symptoms experienced by burn patients, improper treatment of burn pain can lead to poor prognosis with the spirit in patients, and even suicidal behavior and violent tendencies, and severe pain will cause complications in patients with burns or even death. However, the treatment of burn pain was far from satisfactory at present, so it is an important part of burn treatment to find better, more practical and more economical method of treating burn pain. Internationally, the pain is considered the fifth vital signs following the respiration, pulse, blood pressure, body temperature, is an important indicator of vital signs. The treatment of pain has become an important component of modern medicine field, and has gradually developed into an acology of pain management. However, the chemicals analgesic treatment of pain is still the most important instruments in the present, mainly including opioid analgesics, non-opioid analgesics acting on the central, anti-inflammatory and analgesic drugs acting on the peripheral and anti-inflammatory analgesic compound. However, the incidence of side-effects of opioid analgesic drugs, such as the higher respiratory depression, nausea, vomiting, lethargy, urine retention, etc, and the long-term use will produce drug tolerance, dependence and withdrawal syndrome caused by a sudden withdrawal. Therefore, it is a problem that every clinician must be taken seriously how to regulate the treatment of pain, looking for a new molecular target-based analgesic drugs have become the research focus of medical workers and the primary task what they faced in the current. The studies found that the dorsal root primary sensory neurons play an important role in pain perception and transmission. Many molecules related to the process with the pain and feeling expressed in the nociceptor, especially the P2X3 purinergic receptor specific-expression in dorsal root sensory neurons, P2X3 receptor-binding ATP (P2X receptor agonist) released by sensory nerve endings involved in pain transmission of information. Purine and pyrimidine receptors containing P1 and P2 receptors, adenosine is a precursor of adenine nucleotides and metabolites, which mainly activate P1 receptors, while ATP and its analogues act on P2 receptors. It is generally believed that pain and nociceptive stimulation can induce damaged cells, stress cells and sensory nerve endings themselves to release large amounts of ATP, stimulating the P2X2/3 or P2X3 receptors, to lead to depolarization of sensory nerve endings and enhanced discharge of sensory neurons to cause pain. Thus show that ATP released from different types of cells plays a role in the pathogenesis of pain by activation of P2 receptors on (especially the P2X3 receptoror P2X3 receptor) in sensory nerve terminals. Therefore, P2 purinergic receptor agonists and antagonists have broad prospects for clinical application, and the specific P2X3 receptor antagonist may be new treatments for pain and other pathological states. Radix puerariae is the dried root of Pueraria lobata (Willd.) Ohwi and Pueraria thomsonii berth [Fabaceae]. In China, R. puerariae is known as'Ge Gen', and has been used as a traditional Chinese medicine for the management of various diseases including cardiovascular disorders. R. puerariae is also known as Kadzu root in the West and contains significant amount of the isoflavonoid puerarin. The uses of Ge-gen described in pharmacopoeias and in traditional Chinese medicine are for the treatment of fever, pain, diabetes, measles, acute dysentery or diarrhea, etc. Puerarin [4H-1-benzopyran-4-one,8-β-D-glucopyranosyl-7-hydroxy-3-(4-hydroxyphenyl), C21H20C9] is a major active ingredient extracted from the traditional Chinese medicine Ge-gen (Radix Puerariae, RP). Puerarin is mainly used for myocardial infarction, coronary heart disease, angina pectoris, retinal artery and vein obstruction, sudden deafness, and diabetes treatment in clinical practice. Reported in the literature, Pueraria decoction, Kudzu as the main ingredient, has anti-inflammatory effect; Joint injection of steroids and puerarin in the joint cavity can significantly improve the long-term efficacy of the treatment of osteoarthritis. On the other hand, pain is the performance of inflammation, and inflammation is closely related to injury response, thus shows that puerarin extracted from Pueraria may have anti-nociceptive response and analgesic effect. In this study, the sciatic nerve chronic constriction injury (CCl) and the superficialⅡdegree burn rat model rat model will be used to observe the effects of puerarin on mechanical allodynia and thermal hyperalgesia behaviors of rats and effects of puerarin on P2X3 receptor-mediated burn pain and neuropathic pain by using immunohistochemistry, in situ hybridization, RT-PCR and western blot technique, and combining nociceptive behavior studies to observe the effects of puerarin on P2X3 receptor-mediated acute nociception, so that we understand the role of puerarin in the pathogenesis of the P2X3 receptors involving in neuropathic pain, burn pain and acute nociceptive behavior, which will enlarge the scope of application of traditional Chinese medicine puerarin and provide a new experiment foundation for exploring new analgesic drugs for the treatment of neuropathic pain, burn pain and acute injury pain.2. Effects of puerarin on acute nociception mediated by P2X3 receptorObjective:To observe the effects of puerarin (PUE) on acute nociception mediated by purine 2X3 (P2X3) receptor.Methods:The effects of PUE administered intrathecally on the acute nociception induced by P2X3 receptor agonists in the rat hindpaw were investigated by the method of the behavioral study. And the effects of puerarin on the expressions of P2X3 protein and mRNA induced by intraplantar injection formalin in L4-L6 dorsal root ganglion were detected by immunohistochemistry and RT-PCR.Results:Intraplantar injection of ATP (10μmol/L),α,β-meATP (1μmol/L) or 2.5% formalin could induce obvious acute nociception in the rat hindpaw. Intrathecal injection of ATP (10μmol/L) orα,β-meATP (1μmol/L) could enhance the acute nociception induced by plantar subcutaneous injection of ATP (10μmol/L) or α,β-meATP (1μmol/L). Puerarin (10mmol/L) could obviously inhibit the acute nociception induced by intraplantar injection of formalin in rat. Co-administration of intrathecal puerarin with ATP into the rat, produced a significant reduction in nociceptive paw flinching, licking and guarding behavior increased by intrathecal injection of ATP in rat (p<0.05). Co-administration of intrathecal puerarin (2,10,50mmol/L) withα,β-meATP into the rat, produced a significant(p<0.01) and dose-dependent reduction in nociceptive paw flinching, licking and guarding behavior increased by intrathecal injection ofα,β-meATP in rat. Intraplantar injection of puerarin could decrease the upregulation of P2X3 receptor expression induced by intraplantar injection of formalin in DRG via immunohistochemistry and RT-PCR.Conclusion:The anti-nociceptive effects of puerarin may mainly be associated with inhibiting the transmission of nociceptive signaling mediated by P2X3 receptor activation in primary sensory neurons.3. Effects of Puerarin on neuropathic pain mediated by P2X3 receptorObjective:To investigate the effects of puerarin on rat neuropathic pain mediated by P2X3 receptor.Methods:Chronic constriction injury (CCI) rat model was adopted. Sprague-Dawley rats were randomly divided into blank control group (Ctrl), sham group (Sham), puerarin-treated control group (Ctrl+PUE), chronic constriction injury (CCI) group and puerarin-treated CCI group (CCI+PUE). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and the expressions of P2X3 protein and mRNA in L4/L5 dorsal root ganglion (DRG) were detected by immunohistochemistry, RT-PCR, western blot and in situ hybridization.Results:At day 4-7 after operation, CCI model has been established; MWT and TWL in group CCI and CCI+PUE were lower than those in group Ctrl, Sham and Ctrl+PUE, and there was significant difference (p<0.01), while there was no difference among group Ctrl, Sham and Ctrl+PUE (p>0.05); At day 7-10 after operation, MWT and TWL in group CCI+PUE was higher than those in group CCI and there was significant difference (p<0.01), but there was no significant difference compared with those in group Ctrl (p>0.05); At day 14 after operation, the expressions of P2X3 proteins and mRNAs in L4/L5 DRG of group CCI were higher than those in group Ctrl, Sham and Ctrl+PUE (p<0.05 or 0.01), while the expressions of P2X3 proteins and mRNAs in group CCI+PUE was significantly decreased compared with those in group CCI(P<0.05 or 0.01).Conclusion:Puerarin may alleviate neuropathic pain mediated by P2X3 receptor in dorsal root ganglion neurons.4. Effects of puerarin on burn injury pain mediated by P2X3 receptorObjective:To investigate the effects of puerarin on rat burn injury pain mediated by P2X3 receptor.Methods:Superficial second degree foot/back burn injury models were adopted. Sprague-Dawley rat were randomly divided into sham paw burn group (SPB group), puerarin-treated paw unburned control group (PPC group), untreated superficial second degree paw burn group (UPB group), puerarin-treated superficial second degree paw burn group (PPB group), blank paw control group (BPC group), sham back burn group (SBB group), puerarin-treated back unburned control group (PBC group), untreated superficial second degree back burn group (UBB group), puerarin-treated superficial second degree back burn group (PBB group) and blank back control group (BBC group). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were measured and the expressions of P2X3 receptor in dorsal root ganglion from test rats were detected by immunohisto-chemistry, RT-PCR, western blot and in situ hybridization.Results:The mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) in group UPB and PPB were lower than those in group SPB, PPC and BPC (p<0.01) after burn, and the MWT and TWL in group PPB were increased compared with those in group UPB after burn 48 hours and 1 hour respectively (p<0.05). After 96 hours, there was no difference in MWT and TWL between group PPB and group SPB (p>0.05), and there also was no difference in MWT and TWL between PPC and BPC (p>0.05). The MWT and TWL in group UPB were still decreased, there was difference between group UPB and group SPB (p<0.01), and there also was difference between PPC and BPC (p<0.01). At day 3 post burn, the expressions of P2X3 protein and mRNA in dorsal root ganglion from burn injury model rat in group UPB (untreated superficial second degree back burn group) were increased significantly compared with those in other groups (p<0.01), while in group PPB (puerarin-treated superficial second degree back burn group), post-treated with Puerarin, the expressions of P2X3 receptor were decreased markedly compared with those in group UBB(p<0.05 or 0.01), and there was no difference compared with those in group SBB, PBC and BBC (p>0.05).Conclusion:Puerarin may alleviate burn injury pain induced by P2X3 receptor.
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