Traditional Chinese Medicine (tcm) Pain Analgesia Mechanism Research

Purpose：Cancer pain is a special pain that differ from the inflammatory andneuropathic pain, it is the most accompanied by pain in advanced cancer patientsclinically, common in advanced breast cancer, lung cancer, prostate cancer, andseriously affecting the quality of life of cancer patients, to some extent inpatients with shortened life span, so it has been paid attention to.The limitations and side effects exist in Western medicine treatment cannot effectively controlled about45%of the patients with cancer pain. Chinesemedicine analgesic plaster in the treatment of pain has exact curative effect,the selection of Corydalis, safflower, nux vomica, Caulis, walnuts and otherdrugs, role in Xin bulk temperature, Tongluo Sanjie, promoting blood circulationand removing blood stasis, detumescence acetanilide wait. But its mechanism ofaction is unclear, this research established a model of bone cancer pain, andreseach the effects and possible mechanisms of of Chinese medicine analgesicplaster on cancer pain by behavioral science, imaging, pathology andimmunohistochemistry method.Material and method：1.Establishment of bone cancer pain model in ratMaterial: Wistar rats(female),Walker256,Morphine hydrochloride injection(Northeast Pharmaceutical Group, Shenyang Pharmaceutical Co., Ltd), Voltarencream (Beijing Novartis Pharmaceutical Co., Ltd.), Chinese medicine pain paste(homemade), Bone wax (Nantong Hua Likang Medical Supplies Co., Ltd.)Methods:178female Wistar rats were randomly divided into6groups,respectively, into normal control group, sham operation group, model group,Chinese medicine analgesic plaster group, morphine group, Votalin group, with 30rats in each group. The method of making model: model group, Chinese medicineanalgesic plaster group, morphine group, Votalin group120in total, Models oftibial cancer pain were made according to the method described by Medhurst etal.Rats were incised the skin from the left side of the upper tibia, separatedthe subcutaneous tissue until exposed tibia, then use the dental hand turn topunch in the upper tibia, using the10μl micro syringe into the marrow cavityand slowly infusing Walker256tumor cell liquid5μl (about5×105tumor cells),then injecting air1μl, and sealing pinholes with aseptic bone wax, flushingthe wound with gentamicin, coated with erythromycin ointment. Sham operationgroup, the same operateing with the same amount of PBS fluid as model group.Not disposed of the Normal control group.Detection of indexes: the general state observation, pain behavior detection(including preoperative and postoperative spontaneous pain behaviorobservation, mechanical allodynia and thermal hyperalgesia in the paw withdrawalthreshold detection), imaging (partial X ray), histopathological examination(based on local bone tissue slices, for hematoxylin-eosin staining). Themechanical allodynia and thermal hyperalgesia in the paw withdrawal thresholdin the preoperative and postoperative are determined weekly. Imagingexamination and histopathological detection can be observed in bone destruction.The above indicators of outcome can determined the mold whether success, as wellas analgesic drug treatment is effective.2. Study on the analgesic mechanism of Chinese medicine analgesic plasterMaterial: Anti-of NMDAR1(Wuhan Boster Biological Engineering Co.,Ltd.),Anti-of COX2(Wuhan Boster Biological Engineering Co., Ltd), Anti-PKA(Wuhan Boster Biological Engineering Co., Ltd.), Anti-GFAP (Wuhan BosterBiological Engineering Co., Ltd.), Anti-NK2R,(Beijing Boaosen biologicalTechnology Co., Ltd), Anti-ADCY8,(Beijing Boaosen biological Technology Co.,Ltd), Anti-phosphors-CAMKK2,(Beijing Boaosen biological technology Co., Ltd), The Anti-phospho-of ERK1/2(the Beijing Boaosen bio-technology Co., Ltd),.Anti-phospho-MAPK14(Thr180/Tyr182)(the Beijing Boaosen bio-technology Co.,Ltd.), Anti-phospho-CREB-1(Ser133)(the Beijing Boaosen bio-technology Co.,Ltd.), Anti-c-fos in (the Beijing Boaosen bio-technology Co., Ltd.), DABconcentrate (the Beijing Boaosen Biotechnology Co., Ltd), Type SABCimmunohistochemical staining kit (Wuhan Boster Biological Engineering Co.,Ltd)，Universal DAB staining kit (the Beijing Boaosen Biotechnology Co., Ltd),Methods:178female Wistar rats were randomly divided into6groups,respectively, into normal control group, sham operation group, model group,Chinese medicine analgesic plaster group, morphine group, Votalin group, with24rats in each group. Infusing Walker256tumor cell liquid5μl (about5×105tumor cells). Sham operation group injected the same amount PBS liquid intothe left tibia bone marrow cavity, no intervention in normal control group. Fromthe seventh postoperative day in each group according to the experimental schemewere given Chinese medicine analgesic plaster, morphine, Votalin, applying fora therapeutic intervention. Preoperative determination of six groups of ratswith lateral MWT and PWT, Postoperative1～21d determinated weekly (after7,14,21days). first of all take6～8rats,4%paraformaldehyde was used toperfuse the rat, takeing the rat spinal cord L4-6segment, then usingimmunohistochemistry technology to observe the positive product distributionand change of the following indicator in the rat lumbar spinal cord of bone cancerpain:(1) receptor and effector enzymes: NMDAR2B (N-methyl-D-aspartic acidreceptor2B), NK1(neurokinin1receptor), AC VIII (adenylate cyclase VIII),COX-2(COX2).(2) the intracellular signal transduction pathways: PKA (protein kinase A),pCaMK II (the phosphorylation of calmodulin kinase II), pERK (phosphorylationof extracellular signal regulated kinase, p-p38(phosphorylation of p38). (3) nuclear transcription and Translation: pCREB (phosphorylation of cAMPresponse element binding protein), and Fos protein.(4) bone cancer pain feature indicator: GFAP (astrocytic glial fibrillaryacidic protein).3．statistical methodsGroup method of data using mean±s, n represents the number of samples.Groups were compared using analysis of variance (Two-way Repeated MeasuresANOVA), the same group at different time data were compared using analysis ofcovariance (One-way ANOVA), when P<0.05think difference of statisticalsignificance. Each rate compared with X2testing, comparison between two groups,the X2segmentation computing, P<0.005, think difference of statisticalsignificance. Application of statistical analysis software is SPSS11.0.Results：1．Establishment of the rat model of bone cancer painResults:(1) pain behavior observation:①spontaneous pain behaviorobservation: the model of rats after the corresponding-side limb weight-bearingability with bone cancer progressed gradually reduced, to bone cancer laterperiod, the corresponding-side limb is in persistent crouch state, disease ratcan be manifested as dragging feet. On the side of the body of sham operationgroup, normal control group showed no this phenomenon.Normal control group during the period of whole experimental observation,the hind limbs were not observed flinching behavior. In Sham operation group,the side of postoperation started flinching behavior only after postoperation3d, comparing with baseline differences (P<0.05), but after1weeks, thedifference disappeared.Model group lateral postoperative3d started flinching behavior, comparedwith sham operation group had no difference (P>0.05),7d flinch flinchingfrequency increased significantly, and total time in the observation period gradually extend, after3,7,10,14d observed in the paw withdrawal timerespectively45.94±20.78s,95.32±24.92s,156.39±23.52s,237.72±27.51s,compared with the normal control group and sham operation groups had significantdifference (P<0.05).②Mechanical allodynia (MWT) and thermal hyperalgesia (PWT) of the pawwithdrawal threshold: after1～6d, the rats of model group in side clawmechanical allodynia and thermal hyperalgesia paw withdrawal threshold comparedwith the normal control group showed no significant difference (P>0.05); after14～21d the rats in the model group, operation and claw mechanical allodyniaand thermal hyperalgesia paw withdrawal threshold was lower than that in normalgroup (P<0.05).(2)X-ray shows, model group rats with lateral tibial marked bone destruction,the damage degree and moulding process is related to time. The rats in the modelgroup in the postoperative7d only injection site intra bone marrow density isslightly low, no clear area of bone destruction; after14d observed local bonedensity is uneven, and the emergence of a small number of cortical bone loss;after the21d visible bone destruction degree aggravate, cortical bone loss morevisible, even fracture. Sham operation group, normal control group and modelgroup in the contralateral hind at postoperative7,14,21d were not observed inbone destruction phenomenon.(3)Histopathological examination after7,14,21d respectively, material,operation hind tibiae vaccination side partial paraffin sections, stained withHE,7d after operation, the tibial bone marrow cavity tumor cell growth and active,but the cortical bone, intact trabecular bone, no obvious damage of bone marrowcavity;14d large number of tumor cells infiltration, trabecular bone destruction,bone cortex is missing, thinning;21d cortical bone defect, medullary cavityfilled with tumor cells, and perforation of cortical bone outward growth,invasion of the surrounding muscle tissue. While the Sham operation group, normal control group with no intact tissue damage.By the multifaceted, multi-angle evaluation, we can confirm the bone cancerpain model is success.2．The evaluation of drug analgesia effectivenessResults: mechanical allodynia(MWT) and thermal hyperalgesia (PWT) pawwithdrawal threshold: after1～6d, model rats with lateral claw MWT and PWT innormal control group, sham operation group and basic value comparison showedno significant difference (P>0.05); from postoperative7d, model group, Chinesemedicine analgesic plaster group, morphine group, Votalin group began todecrease, as compared with the normal control group differences werestatistically significant (P<0.05), model group21d after operation (P<0.05)continued to decline. The morphine group and Votalin group after14d visibleincreased (P>0.05),21d after surgery and decreased (P<0.05), and the normalcontrol group and sham operation group compared with baseline differences werenot statistically significant (P>0.05). Chinese medicine analgesic plastergroup after14～21d became MWT and PWT increased (P <0.05). Normal control group,sham operation group and the basic values of no significant difference (P>0.05)..3. Study on the analgesic mechanism of Chinese medicine analgesic plasterResults: immunohistochemistry showed,(1) NMDAR2B, NK1, AC Ⅷ, COX-2,PKA,pCaMK Ⅱ, pERK, p-p38, pCREB, Fos, GFAP detection, model group most after14～21d various indicators of immune reaction positive neurons or positive cell ratiogradually increased, and the normal control group sham operation group, therewas significant difference (P<0.005).(2) the morphine group in earlyintervention (7d), NK1, COX-2, NMDAR2B PKA, pCaMK Ⅱ, pCREB, Fos positiveneurons were decreased, and the normal control group, sham operation groupcompare difference not to have statistical significance (P>0.005), but withthe long time use, the intervention of14d again escalated, and normal controlgroup and sham operation group had statistical significance (P<0.005). AC VIII in morphine intervention early to see increased, and continues through the endof the intervention, and the normal control group and sham operation group hadstatistical significance (P<0.005). PERK, p-p38in morphine intervention earlysign of decline, and continues through the intervention ended, and the normalcontrol group and sham operation group, no significant difference (P>0.005).GFAP in morphine intervention occurs early intervention increased, until late,compared with the control group the differences were statistically significant(P<0.005).(3) Votalin group drug intervention in7d, NMDAR2B, PKA, visible pCaMKⅡ, pERK, p-p38, pCREB, Fos positive neurons were decreased, and the normalcontrol group and sham operation group compare difference not to have statisticalsignificance (P>0.005), but after a slow increase, and the normal control group,sham operation group compared with the presence of statistical significance(P<0.005). NK1in applying intervention in the early to see its positive neuronsincreased ratio, and continues through the Votalin intervention ended, andnormal control fake operation group had statistical significance (P<0.005). ACⅧand COX-2in applying intervention early to see its positive neurons wereincreased, and continues through the Votalin intervention ended, and normalcontrol combination of sham operation group compare difference not to havestatistical significance (P>0.005). GFAP in applying intervention occurs earlyintervention increased, until late, compared with the control group thedifferences were statistically significant (P<0.005).(4) Chinese medicineanalgesic plaster group (CM) on drug intervention is visible on NMDAR2B,7d,COX-2, PKA, AC ⅧpCREB, Fos, GFAP index decreased until the end of14d,intervention, and normal control group and sham operation group comparedifference not to have statistical significance (P>0.005). PCaMK Ⅱ, pERK,p-p38in pain medicine paster on7d, its positive neurons ratio remains high,and the normal control group and sham operation group had statisticalsignificance (P<0.005), the intervention of14d began to decline, and the normal control group and sham operation group difference was not statisticallysignificant (P>0.005). NK1manifestations of early decline, later increased(P<0.005).Conclusion：1．On one hand, traditional Chinese analgesic plaster can influence the effectof NMDA and NK1receptor activity, thereby blocking intracellular signaltransduction pathways, reduced transcription and translation, on the other handby inhibiting the activation of astrocytes, a decrease in excitatoryneuromodulator and pro-inflammatory mediators are released to inhibition ofcentral sensitization formation, thus its analgesic effect.2．During early intervention of morphine, it can suppress NMDA and NK1receptoractivity, blocking intracellular signal transduction pathways, reducedtranscription and translation, than follow its inhibiting role is activation.In addition, the early intervention can activated astrocytes, which leads toits analgesic effect gradually weakened, and the emergence of the phenomenonof reduced pain threshold, manifested as allodynia and hyperalgesia.3．Nonsteroidal anti-inflammatory and analgesic drug showed the inhibitory roleon NMDA receptors and COX-2activity, blocking intracellular signal transductionpathways, reduced transcription and translation, moreover, its intervention inearly activated astrocytes, and as the disease progresses, the effect decreasedgradually, and the analgesic effect is limited.