Study On Mechanisms Of Lappaconitine Anti-hyperalgesia And Preliminary Study On The Compatibility Between Oxycodone And Lappaconitine

Hypetalgesia is a variety of incerased sensitvity to pain at the injuried,surrounding or distent tissues under the noxious stimuli,otherwise the intense pain at the noxious stimuli site. The inflammation of peripheral tissue,and nerve injury ofen leads to hyperalgesia,which is produced by periphetal and central sensitization.And spinal sensitization plays a very important role of the hypetalgesia-production. The recent sudies have shown that there is a large number of important factors controling the information-tansmission about pain in the spinal cord dorsal. Astrocyltes and microglial cells are very important to the generation and maintenance of neuropathic pain.The activated glial cells can release not only substances exciting pain-neurons,but also the proinflammatory cytokines inducing hyperalgesia. The actived c-fos as the marker of the excite pain-nerve can change the level of the peptide at the spinal cord such as dynorphin,so that the long phase of cell functions has been changed. Meanwhile,it means c-fos plays a imprtant role in the central mechanisms of hyperalgesia formed that the expression of c-fos and the occation of hyperalgesia are at the same time. NO with the dual role of both the messenger and effector molecule was found to play a key role in thermal hyperalgesia. The increasing of nertonal citric oxide synthase(nNOS) at the spinal cord neuronal leads to the incereased expression of NO causing hyperalgesia. Therefore,nNOS is considered the marker of expressed NO in spinal cord. P2X3receptors belonging to P2X receptors highly-selecticedly distributs the middle and small diameter neurons with the relevant of nociception in the dorsal root ganglion(DRG). The inerasing adenosine5’-triphosphate(ATP) under noxious stimuli can induce the expression of P2X3receptors,resulting in hyperexcitability of primary afferent neurons.Lappaconitine (LA) is the alkaloid extracted from the aconitum roots of a ranunculaceae plant. 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. 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, subarachnoid block and obtained sound outcome. Accordingly, we speculated that there may exist some analgesia mechanism at the spinal cord level or at subspinal cord level.The study observe the impact of LA to chronic inflammatory rats mechanical nociceptive threshold as well as the expression of GFAP,OX-42(CDllb/c),c-fos and NOS and explore the anti-hyperalgesia effect and mechanism of LA. According to previous studies, oxycodone is compound with LA and the efects of compound drugs is being expired by examining the effects of mechanical stimulation and thermal stimulation. Part I Rat model of chronic inflammatory pain1.1Effecs of the morphological changes and mechanical nociceptive threshold of rats dealed with CFA plantar subcutaneous injectionObjective:To observe the rat plantar subcutaneous injection of complete Freund’s adjuvant,through the changes of morphological and maximal anti-allodynic,whether the judgment the CFA rats of chronic inflammatory pain model was established. MethodsrUse the model rats with left-rear plantar of subcutaneous injection CFA100ul,to measure maximalanti-allodynic of rats on before moldeing day,the1st day,the3rd day,the6th day,the9th day,the1lth day and the13th day by Homemade measuring instruments.and to oberve the appearance and gait of model rats. Results:Modeling rats is in good condition,but the locomotor activities decrease such as exploration and upright action. And the injection feetsof rats has been sustained swelling and showed physical protection status under the resting condition. While measuring the maximal anti-allodynic,the injected feet of model rats have strong muscle contraction,and there is no autophagy limb phenomenon either. The maximal anti-allodynic of model rats has been decreased significantly,and the pain peak phenomenon occerde on the6th day. Conclusion:Plantar subcutaneous injection of CFA can cause chronic inflammatory pain model.1.2Experssion of spinal cord glial cells, c-fos and NOS of rats dealed with CFA plantar subcutaneous injectionObjective:To observe the expression of glial cells, c-fos and NOS in the injected side spinal cord of rats dealed with the plantar subcutaneous injection of CFA,classify the inflammatory pain allergy of model rats. Methods:Remove the spinal cord L4-6segments after perfusion,and detective the AOD of GFAP, OX-42(CDllb/c), c-fos and NOS in spinal cord dorsal horn by using immunohistochemical methods,comparing injected side and contralateral spinal cord dorsal horn. Results:Glial cells, c-fos and NOS in the injected side of the spinal cord dorsal horn have highly expression.The AOD of each index of model rats has significant statistical difference compared with the control group, positive model animals injected contralateral spinal cord dorsal horn.ConcIusion:There is hyperalgesia exited in model rats with CFA plantar injection. Part Ⅱ Study on the anti-hyperalgesia mechanism of LA2.1Effects of LA on rat-chronic-inflammatory painObjective:To the inhibition of LA to the maximalanti-allodynic of rats induced inflammation by CFA.Methods:Use the CFA rat-model,measure the maximalanti-allodynic of rats on before molding day,the6th day,the9th day and he13rd day.On the7th day,LA was given to model rats by gavage,once a day for6days.Results:LA of16mg/kg can improve the maximalanti-allodynic,but LA of8mg/kg does not. Conclusion:LA has inhibition in rats with chronic inflammatory pain but its analgesic strength less than oxycodone’.LA may have a inhibition of hyperalgesia generate.2.2Effects of LA on GFAP, OX-42(CD1lb/c), c-fos, NOS and P2X3of CFA rats’spinal cordObjective:To study the inhibition of LA on the expression of glial cells, c-fos, NOS and P2X3reception in spinal glial of CFA rats. Method:After the last mesuring the maximalanti-allodynic,remove the spinal cord L4-6the section segments and L4-6DRG on the injection side.To detective the AOD of GFAP, OX-42(CDllb/c), c-fos, NOS in injected side of the spinal cord dorsal and P2XS in DRG by the Immunohistochemical chemical method. Results:LA of16mg/kg can reduce the AOD of GFAP, OX-42(CDllb/c), c-fos, NOS and P2X3.Conclusion:LA has the inhibited of nflammatory pain and hyperalgesia. PartⅢ Preliminary Study on the pharmacodynamics of the compatibility between oxycodone and LA3.1Study on ompatibility between oxycodone and LAObjective:To determine the best ratio of the application compatibility between oxycodone and LA by mouse hot plate test and acetic acid writhing test, under the orthogonal test design.Methods:According to the clinical amount of oxycodone and LA,the selected experimental dose is that oxycodone4mg/kg,5mg/kg,6mg/kg and LA4m/kg,8mg/kg and12mg/kg with the L9(34) of orthogonal experimental designs.To determine licking latent of mice and calculate the pain threshold percentage of mice with the mouse hot plate test method at30min,60min,90min and120min time points afer administration.To count the mice writhing in mouse acetic acid writhing test in20minutes afer lacial acetic acid intraperitoneal injected.Results:The orthogonal analysis showed that in the mouse acetic acid writhing testthere was no difference of different doses of oxycodone and LA;in the hot plate test in mice, the compatibility between oxycodone of5mg/kg and LA of8mg/kg had the best effect. Conclusion:the compatibility between oxycodone and LA in accordance with the5:8ratio,has the best effects.3.2Effects of the compatibility between oxycodone and LA on chronic inflammatory painObjective:To study the compatibility between oxycodone and LA on the improvement of maximalanti-allodynic of CFA rats. Methods:to measure maximalanti-allodynic on the before modeling day,the6th day,the9th day, the12th day and the13rd day.Results:there is no effects on maximalanti-allodynic of rats with the administration of the comatiblity,but on the12th day,the compatibility with oxycodone of1.4ing/kg and LA of2.24mg/kg,incrased the maximalanti-allodynic,the results under a significant difference compared with he model group.. Conclusion:there is almost no effects in the compatibility on the pain response of Mechanical stimulation.3.3Effects of the compatibility between oxycodone and LA on the pain response of thermal stimulationObjective:To inspect the effent of the compatibility on thermal stimulation pain by the mouse hot plate test.Method:to measured licking latent and calculate the pain threshold increasing the reaction rate at the time points afer administration30min,60min,90min,120min.Results:the compatibility with oxycodone of4mg/kg and LA of6.4mg/kg had the best effects for extending mouse hot plate the licking latency and increasing the pain threshold percentage,but the compatibility with oxycodone of2mg/kg and LA of3.2mg/kg. Conclusion:the compatibility has better analgesia on thermal stimulation pain.In summary.the rat plantar subcutaneous injection of CFA is concered a uccessfully established chronic inflammatory pain model;LA can effectively improve the maximalanti-allodynic of CFA rats;LA has the inhibition of hyperalgesia;there is the mechanism of analgesic anti-inflammatory action at the spinal cord for LA.The compatibility between oxycodone and LA has no advantages of the combination drugs.