| Objective and BackgroudCancer has become the first major ill and chronic disease which is a serious hazard to human health and leads to modern human death. As one of the most common symptoms of cancer patients, cancer pain can occur at any stage of cancer. Cancer invasion pain (CIP) induced by tumor infiltration is prone to cancer patients and refractory to treat. Our study found that mirror image pain (MIP) did exist in CIP animal model, which refers to the phenomenon unilateral injury results in pain hypersensitivity in the ipsilateral and contralateral sides respective to the injury site. Studies have shown that a variety of animal models such as neuropathic pain, inflammatory pain, skin and tissue injury induced pain have observed the phenomenon of bilateral hyperalgesia. So did exist in rat model of tibia metastatic cancer pain. MIP is clinically common in patients with some severe chronic pain, complex regional pain syndrome, amputation and stroke sequelae so on. Among them the MIP of Cancer pain patients was a thorny issue facing clinicians, because it not only seriously affected the quality of life of patients but also interfered with the treatment of cancer.CQ prescription is formulated by our research group according to the theory of TCM, which can promote blood and qi circulation, expel wind and remove dampness and activate meridians to relieve pain. The major components of CQ prescription are ligustrazine and sinomenine. Our previous studies have shown that CQ prescription can relieve pain of neuropathic and cancer pain animal models, and the analgesic effect and potency corresponds is similar with that of gabapentin which was commonly used to analgesic as non-morphine medicine in clinical. The analgesic mechanism may be mediated by inhibiting the level of neurotransmitters in striatal extracellular such as NE and Glu. But its analgesic effect of CIP and MIP is still not clear.We built a mouse model of cancer invasion induced mirror image pain (CIIMIP) by injecting S180 sarcoma cell and observed the analgesic effect of CQ prescription on bilateral hyperalgesia. Then, we detected the level of excitatory and inhibitory amino acids in the spinal cord which was closely related to pain, the content of endogenous-induced pain and analgesic substance in the brain cortex and the level of cytokines in the serum and spinal cord. Thus we could get insight into the pathogenesis mechanisms of CIIMIP and analgesic mechanism of CQ prescription involved in these substances. Further we observed the influence of NMD A, GABAa receptor antagonists on analgesic effect of the CQ prescription to discussion analgesic mechanism of CQ compound involved in EAA/IAA. So we can provide a reference for the study of mechanisms and treatment of CIIMIP.Method1 To establish the CIIMIP model miceMale BALB/c mice whose mechanical withdraw threshold were above 0.4g on the 4th day after continuous 3 day stimulation were randomly divided into native group, sham group and surgery group. The mice in surgery group was injected 0.2mL,1×107/mL of S180 cell sap on the right leg near the greater trochanter of femur. S180 cell sap was replaced by the inactivated S180 cell sap in sham group, and no operation in native group. Then we evaluated the mechanical withdraw threshold of bilateral hind paw and spontaneous pain behavior of contralateral hind at pre-surgery and 2-7d post-surgery.2 Grouping, administration and pharmacodynamics evaluation of miceThe mice whose mechanical withdraw threshold were below 0.07g during 5-7 day after surgery were then randomly divided into CQ high dose group (H-CQ,200mg/kg), CQ middle dose group (M-CQ, group 150mg/kg), CQ low dose group (L-CQ, 100mg/kg) and positive drug group (GBP,120mg/kg). The mice in Native, Sham and Model group were intraperitoneally administrated normal saline instead of drug. The drug was intraperitoneally administrated once daily in the 8th,9th,10th day. Mechanical withdraw threshold of the bilateral hind paw was evaluated pre-administration and 30,60,90,120, 180 and 240 min post administration.3 To detect the levels of neurotransmitters and proinflammatory cytokinesThe mice were sacrificed to drawn blood, cerebral cortex and L3-L5 spinal cord tissue at 45 min after administration in the day after the end of behavioral assessment. The level of excitatory amino acids (Asp, Glu) and inhibitory amino acids (Gly, GABA, Tau) in the L3-L5 spinal cord was measured by the high performance liquid chromatography-fluorescence detector (HPLC-FLD). Radioimmunoassay was used to detect the concentration of SP, DynA1-13 and P-EP in the cerebral cortex. Detection technology with AimPlex multiple immunoassays for flow was used to analysis the content of IL-6, MCP-1 in serum and RANTES, MCP-3 in the spinal cord.4 Administration of NMDA and GABAA receptor antagonistThe model mice were randomly divided into bicuculline+CQ prescription group (Bic+CQ) and amantadine+CQ prescription group (Ama+CQ). The mice were administered bicuculline as a GABAA receptor competitive antagonist (2mg/kg) via tail vein 15 min before injecting CQ prescription in Bic+CQ group, and administered amantadine as the noncompetitive NMDA receptor antagonist (200mg/kg) by gavage 30min before injecting CQ prescription in Ama+CQ group in the 8th day. Then mechanical withdraw threshold of the bilateral hind paw was evaluated at-15 min (pre-administration of Bic) or-30 min (pre-administration of Ama),0 min (pre-administration of CQ prescription) and 30,60,90,120,180 and 240 min post administration of CQ prescription. Then we observed the influence of NMDA and GABAA receptor antagonist on analgesic effect of the CQ prescription.Results1 To establish the CIIMIP model miceMechanical withdraw threshold of ipsilateral hind paw of mice in surgery group began to decline in 2th day, then stably keep at (0.031+0.006) g to (0.057+0.029) g and decreased significantly compared to Sham group and Native group during 4-7d (P＜0.05 or P＜0.01). Mechanical withdraw threshold of contralateral hind paw of mice in surgery group also began to decline in 2th day, then stably keep at (0.044+0.012) g to (0.101+ 0.038) g and decreased significantly compared to Sham group and Native group during 4-7d(P<0.05 or P<0.01).2 Analgesic effect of CQ prescription on CIIMIP model miceMechanical withdraw threshold of bilateral hind paw of model mice began to increase at 30 min and the efficacy peaked in the 30 and 60 min after administrating CQ prescription firstly. For CIP, mechanical withdraw threshold of CQ high dose group was significantly higher for five time points within 180 min than model group (P＜0.01 or P＜0.05), and M-CQ group significantly increased the mechanical withdraw threshold at 60,90 min compared with Model group (P<0.05). It has similar effect for MIP, mechanical withdraw threshold of CQ high dose group was significantly higher for five time points within 180 min than model group (P<0.01 or P＜0.05), and M-CQ group significantly increased the mechanical withdraw threshold at 30,60,90 min compared with Model group (P＜0.05). There was a slight improvement for analgesic effect of CQ compound prescription on bilateral hind paw of mice model after administered for three consecutive days, but no statistical difference was found.However, the analgesic efficacy peaked at 60 and 90 min after administrating GBP firstly. Mechanical withdraw threshold of bilateral hind paw of mice in GBP group significantly improved for all time points within 240 min than model group (P<0.01 or P<0.05). There was a decreasing trend of analgesic effect of gabapentin on bilateral hind paw of mice model after administered for three consecutive days, but no statistical difference was found.3 Levels of EAA and IAA of the spinal cord in CIIMIP mice and effect of CQ prescriptionCompared with native group, the levels of Glu in the L3-L5 spinal cord in model group significantly increased (P＜0.05), and there was no significant change in Asp, While the levels of Gly, Tau and GABA in the spinal cord significantly decreased (P＜0.05, P＜0.01). CQ prescription could inhibit the levels of Glu (P＜0.05 or P＜0.01) and elevate the levels of Gly, GABA and Tau in spinal cord of model mice (P＜0.01). Moreover, the effects of CQ prescription on the contents of Glu, GAB A and Tau had a dose-dependent relationship.4 Influence of NMD A and GABAA receptor antagonists on analgesic effect of the CQ prescriptionBicuculline pretreatment could significantly prevent the analgesic effects of CQ high dose for CIP at 180 min after administration (P＜0.05) and for MIP at 180,240 min after administration (P＜0.05). Amantadine pretreatment could significantly enhance the analgesic effects of CQ middle dose for CIP for all time points within 180 min after administration (P＜0.01 or P＜0.05) and for MIP for five time points within 180 min after administration (P＜0.01 or P＜0.05).5 Contents of SP, DynAl-13 and β-EP of the cerebral cortex in CIIMIP and effect of CQ prescriptionCompared with Native groups, the contents of endogenous-induced pain substance SP in the cerebral cortex in model group significantly raised (P＜0.01), endogenous analgesic substance DynA1-13 significantly reduced (P＜0.05), while β-EP had no significant change but had the decreased trend. CQ prescription (low and middle dose) could significantly reduce the content of SP (P＜0.05, P＜0.01), but DynAl-13and β-EP no significant increased.6 Contents of RANTES and MCP-3 of the spinal cord in CIIMIP and effect of CQ prescriptionThe contents of RANTES and MCP-3 in spinal cord in model group were significantly higher than those of Sham group (P＜0.01, P＜0.05), and CQ prescription (low and high dose) could significantly reverse the changes of above chemokines (P＜0.05).7 Levels of IL-6 and MCP-1 of the serum in CIIMIP and effect of CQ prescriptionThe levels of IL-6 and MCP-1 in serum in Model group significantly increased compared with Native group (P＜0.05), and CQ prescription could significantly reduce the content of IL-6 and MCP-1 (P＜0.01).Conclusion1 The mechanical withdraw threshold of contralateral mirror sites in CIIMIP model mice inoculated S180 sarcoma appeared same trend and approximate degree of decline, following the generation of cancer invasion pain of ipsilateral hind paw.2 CQ prescription had significant dose-dependent analgesic effect on CIP and MIP in CIIMIP model mice. The analgesic effect of CQ prescription at this test dose for CIP and MIP was weaker than gabapentin, but the time to reach peak effect slightly was earlier than gabapentin.3 The level of Glu in the spinal cord of CIIMIP model mice significantly increased, and the level of GABA significantly decreased. CQ compound could relieve pain by adjusting the levels of Glu and GABA and balancing EAA/IAA. Then we study the influence of NMDA and GABAA receptor antagonists on analgesic effect of the CQ prescription. The results suggested CQ compound analgesia partly through NMDA, GABAA receptors.4 In mice model of CIIMIP, the content of SP in the cerebral cortex significantly increased, the content of DynA1-13 significantly decreased, and the levels of proinflammatory cytokines such as IL-6, RANTES, MCP-1 and MCP-3 were significantly higher. CQ prescription might inhibit SP and proinflammatory cytokines to relieve pain furtherly. |
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