The Study Of Analgesia Effect And Mechanism Of CQ Compound On Tibia Metastatic Cancer Pain Rat Model

Cancer has now become a threat to human life. About3.5million cancer patients increase in our country every year. Among the cancer patients, pain is the most common symptom, about85%to90%of them suffer from cancer pain. Bone cancer pain is one of the most intractable pain in cancer pain and it has two main features-allodynia and hyperalgesia. The stimulation such as "touch" shouldn’t have caused the pain under normal physiological conditions caused the pain response, called allodynia; whereas the stimulation which can produce more intense pain in the normal state known as hyperalgesia. Now, we mainly still use the "three-step" therapy clinically, but have to face the problem of drug addiction. Therefore it is necessary for us to screen the drugs that have better analgesic effect and small side effect for bone cancer pain and to study the mechanism of analgesia occurred.CQ compound is a prescription developed by our research group. It has functions of promoting blood circulation and Qi, dredging collaterals and relieving pain, CQ compound showed well analgesic effect on multiple pain models such as sciatica, trigeminal neuralgia and incision pain. In previous studies, we found excessive excitatory amino acid, neuromodulator and inflammatory cytokines could produce central sensitization and outer periphery sensitization.This experiment uses Walker-256breast cancer cells to build a rat model with tibia metastatic cancer pain in order to observe the effect of CQ compound on pain behavior and the impact of drugs on amino acid neurotransmitters, SP and β-EP of central nervous system and cytokines of tumor tissue pain associated. Thus we can gain insight into the pathogenesis of bone cancer pain and the pharmacological mechanisms about how drugs exert analgesic effectiveness in peripheral and central nervous.These provide the basis for clinical use. Methods 1Build a rat model with tibia metastatic cancer pain1.1Preparation of ascites tumor cellsRecover the frozen Walker-256breast cancer cells, mixed it with saline to become tumor cell suspension. Use0.5ml suspension injected into the peritoneal cavity of normal female wistar rat. When the rat had ascites, extracted the ascites tumor cells and adjusted cell concentration to4.7*104/ul.1.2Rats grouped and modelingAll rats were vertical stimulated on the skin surface of the bottom right side of the rat paw by Von Frey hairs until they adjust to the stimulation before the experiment carried out. Rats whose mechanical pain thresholds above26g for3days were randomly divided into sham group and surgery group. Exposing the right hind tibia medial of the surgery group rats and injected4.4ul ascites tumor cells into the bone marrow cavity, meantime the sham group given4.4ul saline with the same method.2Measuring methods2.1Evaluation of mechanical pain thresholdRats were stimulated with Von Frey hairs from2g after they get used to the environment, once per second. When they had3positive reactions in5stimuli, the minimum number of gram was the mechanical pain threshold. The positive reaction is paw withdrawal. Rats whose pain threshold below or equal8g after modeling can be considered successful.2.2Evaluation of free walking painObserving the free walking state of rats who were placed in the box7days after modeling and scored:0:normal behavior;1:mild lameness, hind paws touch the ground, toes can’t outreach normally;2:protecting position,limping obviously;3: Unable to use part of the limb, limping obviously;4:Unable to use the whole limb. Rats whose score above0point after modeling can be considered successful.2.3Evaluation of radiographic studiesRats were subjected to X-ray13days after surgery to observe the changes of bone-breaking. Scored as follows:0:normal bone tissue;1point:1-3small punctate bone destruction;2points4-6small punctate bone destruction and medullary bone defects;3points: partial cortical bone destruction, medullary bone defects;4points: unilateral or full-thickness defects of cortical bone;5points:full thickness defects of bilateral cortical bone or fracture displacement. Rats that have bone destruction are deemed successful models.Rats who can meet the three evaluation methods which above-mentioned are seemed as successful models.3Rats grouped drug administration and effectThe surgery rats were randomly divided into model group、CQ-H group(i.p.200mg/kg)、 CQ-M group (i.p.150mg/kg), CQ-L (i.p.100mg/kg) and gabapentin group(i.p.100mg/kg).Sham group and model group were given normal saline i.p.. On the14th day after modeling, given drugs to rats for seven consecutive days and evaluate the mechanical pain threshold on the1th、3th、7th day. Measuring Omins (before drug administration),30mins,60mins,90mins,120mins,180mins,240mins, and300mins after i.p. drug administration.Drawn rats’cerebrospinal fluid、right tibia tumor tissue and L4-L6spinal cord tissue after the7th drug administration,4ISCUS examined the glutamate in cerebrospinal fluid of ratsAccording to reagents enzymatic reactions and colorimetric principle, using Sweden CMA’s ISCUS flex detector and special kits to detect cerebrospinal fluid in the sample pool automatically. Measuring wavelength:365and546nm; power:100VA. Using Laptop computers and LABplotTM software control instrumentation to analyze data.5Radioimmunoassay examine neuromodulator of spinalUse the balance and saturation of sample programs, y-radiation counting cpm value and draw the standard curve.6AimPlex Multiple Immunoassays for Flow detected cytokine of tumor local tissue. Put microspheres fluid, test samples, standards, biotin-labeled antibody and streptavidin-labeled PE into assay plate in order to prepare samples. Then examing the samples by fluorescence through flow cytometry with488nm excitation light of PE channel according to the size of microspheres and the strength of fluorescent labels and make a quantification of samples by standard curve.Results1Tibia metastatic cancer pain model was induced by Walker-256breast cancer cells.We successfully built a rat model with tibia metastatic cancer pain and this model has a success rate of55%. Rats’pain threshold declined10days after modeling. And12to22days, their behavioral indicators stabilized. The mechanical pain threshold of surgery group declined from70.00±17.77g to5.90±2.20g (P〈0.01)12days after modeling and decreased significantly compared to the sham group (P<0.01).The score of rats’free walk pain was higher obviously (P<0.05). The X-ray showed bone destruction and disappearance of trabecular bone.2Analgesic effect of CQ compound on tibia metastatic cancer pain rat modelThe results indicated CQ compound could significantly alleviate the mechanical pain sensitivity of tibia metastatic cancer pain. The onset time was30mins after administration, and60mins reached a peak efficacy. The threshold of mechanical pain of CQ high dose group was significantly higher (P<0.05) for seven time points compared with model group, and the high middle low-dose group showed a dose-dependent manner. Gabapentin group reached the peak in90mins, and could significantly elevate for six time points. Compared with administration for the first time, the analgesic effect of CQ compound was stronger when rats were administered for3days. Whereas when administered continuously for7days, the analgesic effect of CQ compound wasn’t enhanced.3The effect of CQ compound on glutamate in cerebrospinal fluid level of ratsThe glutamate level increased significantly after modeling. Sham group was4.78±4.00umol/l and model control group was43.78±20.00umol/l. It indicated that CQ compound showed inhibition on glutamate relieving and a dose-dependent manner among high middle low-dose group. CQ high dose group which was12.64±3.63umol/l showed the similar inhibition with Gabapentin group.4The effect of CQ compound on SP、 β-EP of spinalIt was found that SP and β-EP levels increased significantly after modeling between sham group and model control group. After administration, SP level of CQ high dose group declined (P<0.05), and the effect of CQ high-dose group was similar with gabapentin group. CQ compound group and gabapentin group both could reduce the level of β-EP.5The effect of CQ compound on cytokine expression of tumor local tissueIt was found that the level of IL-12P70decreased after modeling (P<0.05), then increased after administration. IFN-y expression showed the trend of declining after modeling and increasing after administration, but there was no significant difference between groups (P>0.05). It also could be seen β-NGF level increased obviously after modeling, and then reduced after treatment (P<0.05). It showed that CQ compound may have certain inhibiton to β-NGF.ConclusionWe successfully built a rat model with tibia metastatic cancer pain and observed the well analgesia effect of CQ compound for300min/d after administration, and its effect was similar with Gabapentin. The further study showed the mechanism of analgesia of CQ compound may be in connection with inhibiting the glutamate and SP level, adjusting cytokines(IL-12P70,IFN-γ,β-NGF)expressions of tumor-associated.