The Roles Of Chemokine CXCL13 In Morphine Analgesia Of Cancer-induced Bone Pain In Rats

BackgroundCancer is a group of diseases characterized by overgrowth and spread of abnormal cells.Each year worldwide there are approximately nine million cases of cancer reported.A significant portion of these patients will experience pain.Cancer pain of all types is reported to be suffered by 30%-50% of all cancer patients and 75%-90% of advanced,late-stage cancer patients.Of several categories of pain,metastatic CIBP is the most common type of pain reported.Cancer-nduced bone pain(CIBP)is a major clinical problem in patients with end-stage cancer,which affects the quality of life of cancer patients.It is still a difficult issue in clinical practice,because current treatments for this pain are limited due to inadequate knowledge of the mechanisms associated with CIBP.The World Health Organization’s(WHO)guidelines for cancer pain relief outline a treatment progression from non-opioid analgesics through strong opioids with adjuvant supplementation(eg,bisphosphonates,local radiotherapy)to treat progressively worsening pain.Based on this,it is necessary to do further research on the mechanism of bone cancer pain.To date,There are several changes in spinal cord associated with cancer-induced pain condition,including excitatory synaptogenesis,activation of glial cells,increasing of pro-hyperalgesic factors.The role of chemokines in the development of BCP has attracted much attention in addition to cytokines.Chemokines,which comprise a family of more than 50 family members,are chemotactic cytokines whose main function is to direct cell migration in the peripheral immune system1.A growing body of evidence shows that chemokines are also expressed in the central nervous system(CNS)to regulate the CNS function in both physiological and pathological conditions.The chemokine superfamily is divided into four classes based on the order of four conserved cysteine residues.Chemokine receptors are seven transmembrane G protein-coupled receptors(GPCRs)that can modulate intracellular signals after ligand binding.To date,it has been reported that CCL2/CCR2,CX3CL1/CX3CR1,CCL5/CCR5,CXCL12/CXCR4,CXCL10/ CXCR3,and CXCL1/CXCR2 may be correlated with CIBP.Morphine is a potent agonist of μ-opioid receptor and is widely used to relieve severe pain,including cancer pain.In our previous study,we found upregulation of(C-X-C motif)Ligand 13(CXCL13)in Rats with Cancer-Induced Bone Pain.Particularly,CXCL13 was recently found to be the most upregulated gene among detectable chemokines in the spinal cord afer spinal nerve ligation(SNL)-induced neuropathic pain.In addition,chemokines and opioids are important regulators of immune,inflammatory,and neuronal responses in peripheral and central pain pathways..Some chemokines,for example,CX3CL1,CXCL12,CXCL10,CCL2,CCL3 and CCL5,participate in the regulation of opioid santinociception.However,The exact relationship between the expression of CXCL13 and morphine analgesia in CIBP has not be clearly defined.Objectivewe investigated the role of CXCL13 in the regulation of morphine analgesia in CIBP rats and normal mice to provide a new target for cancer pain management.Method1.Preparation of CellsWalker 256 rat mammary gland carcinoma cells,kindly provided by the Laboratory of Huazhong University of Science and Technology,were injected into the abdominal cavities of young female Sprague-Dawley rats(weighing160-180 g).After it,we watch the rats every day,about 5-7 days,cancerous ascites developed,and the fluid was extracted for cancer cells.The ascites were washed with D-hanks fluid in 10 ml of medium for 5 min at 600 r per min for three times.After the last wash,The cell suspension was kept on ice until injection.2.AnimalsAdult female Sprague-Dawley rats,weighing 180–220 g,purchased from ExperimentalAnimal Center of Zhengzhou University,Zhengzhou,China,were housed in a temperature(24±0.5°C)-and light(12/12 h light-dark cycle)-controlled environment with free access to food and water.3.Animal experiment groupingModel of bone cancer pain : 24 Sprague-Dawley female rats,weighing about 180-220 g,were randomly divided into 3 groups,respectively,as the normal group,sham group,CIBP group eight rats of each group.We use the Von Frey flaments to detect the values of mechanical paw withdrawal threshold(MWT)at 1 day before operation and 0,1,3,5,7 and 14 day,respectively,then record them carefully.The expression of CXCL13 protein : 24 Sprague-Dawley female rats,weighs about 180-220 g,were randomly divided into 2 groups,respectively the sham and the CIBP group,Six rats in each group were sacrificed after measurement of MWT at 0,7,14,21 days for the detection of the expression of CXCL13 protion and its receptor CXCR5 at the spinal cord by western blot.the effect of recombinant rat CXCL13(rmCXCL13)on normal rats: 24 Sprague-Dawley female rats,weighs about 180-220 g,six rats of each group,randomly divided into 4 groups :saline group,morphine group,rmCXCL13 group and mixture group(rmCXCL13 + morphine)respectively,the same amount of drugs was given for the intrathecal injection(i.t.),the values of mechanical paw withdrawal threshold(MWT)were record before i.t.and 15,30,60,90 and 120 min after i.t.Then record them and compare the chang of the values.the effect of recombinant rat CXCL13(rmCXCL13)on morphine analgesia with bone cancer pain in rats: the same way with normal rats.4.Drugs and administrationMorphine,an agonist of u opioid receptor(MOR)was purchased from Northeast pharmaceutical group(NEPG China).Recombinant rat CXCL13 was purchased from PeproTech Inc.(PeproTech,USA).For intrathecal(i.t.)injection,intraspinal puncture was performed with a PE-10 catheter between the L5 and L6 interspace to deliver the drug.The volume for the intrathecal injection was 10 ul.5.Model of bone cancer painWalker 256 cell were injected as previously described.A rat model of bone cancer pain was established by the injections of Walker 256 mammary gland carcinoma cells(10 ul 4x106 cells/ml,purchased from Huazhong University of Science and Technology for Biological Sciences)into the intramedullary space of the right proximal tibia in rats.6.Intrathecal InjectionsBriefly,after the rats were anesthetized with the same way of model of bone cancer pain.Rats were intrathecally implanted with PE-10 catheter to the subarachnoid cavity at the level of lumbar enlargement,according to the method described previously.7.Behavioral ExperimentsEach rat was placed in a clear plastic box on an elevated metal mesh floor and acclimated for 30 min.Von Frey flaments were used to stimulate the plantar surface for a continuous period of 5 s.Each stimulus was repeated 5 times with 10-min intervals.8.Western blotWe detected the expression of CXCL13 protein and CXCR5 in the spinal cord on postoperative day of 0,7,14,21 in the Sham group and CIBP group.Results1.Compared with the normal group and sham group,the value of MWT of group CIBP was significantly decreased(P>0.05)at 1,3,5,7,10,14,21 days.The MWT value had no statistical significance(P > 0.05)at each point between the group normal and the group sham.we compared the value of MWT in the group of CIBP at each point,we found that it were statistically significant(P < 0.05)at the time of 3,5,7,10,14,21 days compared to the value before operation,especially at the day of 14 after CIBP,the value of MWT at the minimum,it suggested that the model of bone cancer pain model set up successfully.2.We detection the expression level of CXCL13 protein and its receptor CXCR5 at the spinal cord by western-blot,respectively,in group sham and group CIBP.the expression of CXCL13 protein and its receptor CXCR5 increase at 7,14,21 days after the establishment of CIBP modeling,the differences were statistically significant(P < 0.05)compared with the sham group,the level of CXCL13 protein and its receptor CXCR5 reached to the maximum at 14 days after modeling.we found that the expression of CXCL13 protein is consistent with the values of MWT with bone cancer pain in rats.we can guess that CXCL13 protein may increased the value of MWT in rats with bone cancer pain.3.In normal rats,compared with saline group,the MWT value increased obviously(P < 0.05)in the morphine group,and the value of MWT decreased obviously(P < 0.05)in the recombinant protein group.The differences was statistically significant(P < 0.05)between the morphine group and mixture group(rmCXCL13+morphine).We can guess that the rmCXCL13 protein can cause pain and reduce the analgesic action of morphine on normal rats.4.Bone cancer pain grouping in rats,Compared with saline group,the value of MWT was statistically significant(P < 0.05)at 60 min after intrathecal injection in the rmCXCL13 protein group,MWT value were statistically significant(P < 0.05)at all time points in morphine group and the MWT values are rising.Compared with the pure morphine injection,the differences were statistically significant(P < 0.05)in the mixed group(rmCXCL13 + morphine)at 30 min,60 min.Further illustrating that CXCL13 protein has a negative regulatory role on morphine analgesia in rats with bone cancer pain.ConclusionsChemokine CXCL13 is an algesic molecule and that up-regulation of CXCL13 plays a negative feedback role in the regulation of morphine analgesia in cancer bone pain in rats.