| Bone cancer pain is the most serious and hardly be treated pain in patients with primary cancer or malignant tumors that have spread to bone. Breast carcinoma and prostate carcinoma are the most common cause of pain from mal ignant bone disease. In fact,70%of patients with advanced breast or prostate carcinoma have skeletal metastases, and metastases are presented in>90%of patients who die from breast or prostate cancer. The metastases have the most significant impact on quality of life for patients with malignant tumors or primary cancer, for example, breakthrough pain, pathological fracture, spinal cord compression and Hypocalcaemia. With the rapid development of medical diagnostic technology in the medical field, the survival time of patients with cancer was longer, it’s obviously important to raise the life quality of patients with cancer. Achievements have been made by WHO guided "analgesic ladder", but it still remains limitation in bone cancer pain treatment. Based on the animal models of bone cancer pain in recent years, the mechanisms of bone cancer pain have been studied, and laid the foundation of new drugs for bone cancer pain treatment.Mitogen-activated protein kinases (MAPKs) are important for intracellular signal transduction, are activated by phosphorylation and transduce a broad range of extracellular stimuli into diverse intracellular responses by both transcriptional and non-transcriptional regulation. The MAPK family consists of three major members: extracellular signal regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK), which represent three separate signaling pathways. Accumulating evidence shows that all three MAPK pathways contribute to pain sensitization after tissue and nerve injury via distinct molecular and cellular mechanisms. Activation (phosphorylation) of MAPKs under different persistent pain conditions results in the induction and maintenance of pain hypersensitivity and inhibition of MAPKs lead to decrease of hyperalgesia, the results suggested that MAPKs contributed central sensitization in inflammatory pain and neuropathic pain. So, the question of the role of MAPKs in bone cancer pain has been our first part of our research.Tradition Chinese medicine is an ancient Chinese therapy with an extensive history, now it has been spread worldwide. ShuiZhi (Hirudo) is a name of the Chinese materia medica, properties:Salty bitter, neutral and poisonous; related to the channels of the liver and bladder to remove stasis, simulate the circulation of blood, facilitate the flow of blood and remove blood stasis. Indications:Amenorrhea due to stagnation of blood, blood stasis in the channel, abdominal mass and stagnated pain due to traumatic injury. Hirudin is the main active ingredient of hirudo. An experiment showed that intrathecal hirudin prevented the development of neuropathic pain, and concluded that thrombin activated its’receptor in the spinal cord (lamina Ⅰ and Ⅱ), which may influence the release of PDGF, modulating neuropathic pain in the spinal cord. Cancer had been understood by Chinese doctors for a long history. Cancer had been divided to muscle cancer, bowel cancer and bone cancer based on the different parts which the cancer happened. The incoordination between Yin and Yang, lack of energy, dietary factors and depression were the main reasons of cancer. The treatment of cancer included promoting Blood circulation by removing Blood Stasis and restrain circulation. The pathological mechanism of cancer included general real cause pain and deficiency cause pain. So, the effect of hirudin on bone cancer pain and the mechanism of analgesic effect of hirudin are the other part of our research.According to the reported animal model of cancer pain, the presented study was aimed to:(1) Change of glial activity on day12after intra-tibia inoculation with carcinoma cells, and the effect of intrathecal injection of glia inhibitors on mechanical allodynia in bone cancer pain model.(2) The MAPKs activity after intra-tibia inoculation with carcinoma cells in different time points, and the effect of MAPKs inhibition on mechanical allodynia in bone cancer pain model.(3) The effect of hirudin on mechanical allodynia and the mechanisms of analgesic effect of hirudin in bone cancer pain model.The results are as follows:1. Intra-tibia inoculation of Walker256rat mammary gland carcinoma cells induced mechanical allodynia.4X105Walker256rat mammary gland carcinoma cells were injected into the right tibia of rats. The paw withdrawal thresholds were detected from day4and lasted until day27after intra-tibia inoculation of carcinoma cells. In the first three weeks, the paw withdrawal thresholds were detected the other day, in the last week, the paw withdrawal thresholds were detected every three days. The results suggested that compared to the normal rats, there was significant decrease of paw withdrawal thresholds in model rats.Summary:The model rats displayed significant mechanical allodynia on both side of hind limbs after intra-tibia inoculation with carcinoma cells.2. Change of glial cells activity and the effect of inhibitors of glial cells in bone cancer pain model2.1Change of astrocytes activity and the effect of fluorocirrate on bone cancer pain modelThe rats were randomized to normal or model group. The immunofluorescence results showed that GFAP-positive astrocytes were activated bilaterally in L4-L5spinal cord on day12after intra-tibia inoculation with carcinoma cells. The behavior results showed that a single intrathecal injection of fluorocitrate (an inhibitor of astrocytes, FC)1nmol attenuated bilaterally mechanical allodynia compared to control group on day12after intra-tibia inoculation with carcinoma cells. 2.2Change of microglia activity and the effect of minocycline on bone cancer pain modelThe rats were randomized to normal or model group. The immunofluorescence results showed that CDllb-positive microglia was activated bilaterally in L4-L5spinal cord on day12after intra-tibia inoculation with carcinoma cells. The first part of behavior results showed that a single intrathecal injection of minocycline (an inhibitor of microglia)100μg attenuated bilaterally mechanical allodynia compared to control group on day12after intra-tibia inoculation with carcinoma cells. The second part of behavior results showed that repetitious oral administration of three doses of minocycline (20mg,50mg and100mg) had no significant influence on mechanical allodynia in bone cancer pain model.Summary:Astrocytes and microglia were activated bilaterally in L4-L5spinal cord on day12after intra-tibia inoculation with carcinoma cells, and a single intrathecal injection of FC or minocycline increased the paw withdrawal thresholds in bone cancer pain model. Repetitious oral administration of minocycline had no significant effect on paw withdrawal thresholds in bone cancer pain model.3. The role of MAPKs in spinal cord of bone cancer pain model3.1The role of ERK in bone cancer pain modelBy using Western blot and immunofluorescence, we detect the change of phosphorylated ERK in L4-L5spinal cord of model day5, model day12and model day16. The results suggested that ERK was activated in bilaterally L4-L5spinal cord in different time points, and increased pERK was found in astrocytes and microglia on day12and day16. The first part of behavior results suggested that a single intrathecal injection of PD98059(an inhibitor of MEK, upstream of ERK)10μg attenuated bilaterally mechanical allodynia compared to control group (DMSO) on day12after intra-tibia inoculation with carcinoma cells. The second part of behavior results suggested that a single intrathecal injection of two doses of U0126(an inhibitor of MEK, upstream of ERK,1.25μg and3μg) attenuated mechanical allodynia compared to control group (DMSO) on day14after intra-tibia inoculation with carcinoma cells. DMSO as control had no effect on mechanical allodynia in bone cancer pain model.3.2The role of JNK in bone cancer pain modelBy using Western blot and immunofluorescence, we detect the change of phosphorylated JNK in L4-L5spinal cord of model day5, model day12and model day16. The results suggested that JNK was activated in bilaterally L4-L5spinal cord in different time points, and increased pJNK was found in astrocytes and microglia on day12and day16. The first part of behavior results suggested that a single intrathecal injection of SP600125(an inhibitor of JNK)10μg attenuated bilaterally mechanical allodynia compared to control group (DMSO) on day12after intra-tibia inoculation with carcinoma cells, the analgesic effect was disappeared on12hour after intrathecal injection. The second parts of behavior results suggested that repetitious intrathecal injection of SP600125(10μg) for3days from day10attenuated mechanical allodynia on12hour after intrathecal injection, and repetitious intrathecal injection of SP600125(10μg) for5days from day10attenuated mechanical allodynia on24hour after intrathecal injection. DMSO as control had no effect on mechanical allodynia in bone cancer pain model.3.3The effect of p38inhibition in bone cancer pain modelTo test the effect of the inhibitor of p38(SB203580), we intrathecal delivered three doses of SB203580(1μg,5μg and10μg) by using PE10tube daily for12days after intra-tibia inoculation with carcinoma cells. The behavior results suggested that SB20358010μg fully attenuated mechanical allodynia in bone cancer pain model from day1until day12(the end of administration), SB2035805μg partly attenuated mechanical allodynia, and SB2035801μg had no significant effect on mechanical allodynia in bone cancer pain model. DMSO as control had no effect on mechanical allodynia in bone cancer pain model. Summary:Intra-tibia inoculation of carcinoma cells induced ERK and JNK bilaterally activation in L4-L5spinal cord. The increased pERK was co-expressed with astrocytes and microglia, an intrathecal injection of MEK inhibitors (PD98059and U0126) increased the paw withdrawal thresholds in bone cancer pain model. The increased pJNK was co-expressed with astrocytes and neurons, a single intrathecal injection of JNK inhibitor (SP600125) increased the paw withdrawal thresholds in bone cancer pain model, and repetitious intrathecal injection of SP600125had additive analgesic effect in bone cancer pain model. Repeated intrathecal injection of SB203580(p38inhibitor) for12days attenuated the mechanical allodynia in bone cancer pain model.4. The effect of hirudin on bone cancer pain model and the mechanisms of analgesic effect of hirudin in bone cancer pain.4.1The effect of hirudin on paw withdrawal thresholds in bone cancer pain modelThe first part of behavior results suggested that an intravenous injection of shuxuetong (consists mainly of hirudin) attenuated mechanical allodynia on2hour and lasted until4hour on day12after intra-tibia inoculation of carcinoma cells, saline as control had no effect on mechanical allodynia. The second part of behavior results suggested that a single intrathecal injection of three doses of hirudin (50pmol,100pmol and200pmol) attenuated mechanical allodynia on day12, exhibited a dose dependent manner. The dose of200pmol hirudin had no effect on paw withdrawal thresholds in normal rats.4.2Expression of protease activate receptor1(PAR1) in spinal cordThe immunofluorescence results suggested that PAR1was expressed in L4-L5spinal cord and co-expressed with astrocytes. The RT-PCR results suggested that PAR1mRNA was decreased on day12and day16after intra-tibia inoculation of carcinoma cells compared to normal rats. The immunofluorescence results suggested that PAR1was co-expressed with pERK and pJNK. 4.3The effect of a single intrathecal injection of hirudin on MAPK activity in spinal cordThe Western blot results suggested that a single intrathecal injection of hirudin100pmol after2hours decreased the pJNK2level in spinal cord of bone cancer pain model on day12, but had no effect on pERK levels.Summary:PAR1was expressed in L4-L5spinal cord and co-expressed with pERK and pJNK. Both intravenous and intrathecal injection of hirudin increased paw withdrawal thresholds in bone cancer pain model. A single intrathecal injection of hirudin100pmol decreased pJNK2level in spinal cord on day12after intra-tibia inoculation of carcinoma cells.Conclusion:1. Astrocytes and microglia were activated in L4-L5spinal cord on day12after intra-tibia inoculation of carcinoma cells, the inhibitors of astrocytes and microglia attenuated mechanical allodynia suggested a role of glial cells activation in bone cancer pain.2. ERK and JNK were activated in glial cells of spinal cord in bone cancer pain model, intrathecal injection of three inhibitors of MAPK both attenuated mechanical allodynia suggested MAPK activation plays a role in bone cancer pain.3. Both intravenous and intrathecal injection of hirudin attenuated mechanical allodynia in bone cancer pain model. A single intrathecal injection of hirudin100pmol decreased pJNK2level and PAR1was co-expressed with pJNK in spinal cord suggested that hirudin inhibited thrombin then inhibited thrombin activate PAR1, which finally induced inhibition of JNK2phosphorylation. |
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