| Bone cancer pain(BCP)is a complex refractory cancer pain caused by primary growth of bone tumor or secondary bone metastasis of tumor.At present,there is still no effective treatment in clinical practice,which seriously affects the quality of life of cancer patients.Therefore,in order to provide effective and feasible programs for the clinical treatment of BCP,it is of great clinical significance to explore the pathogenesis of bone cancer pain furtherly.Synaptic plasticity refers to the changes in the efficiency of transmission between synapses caused by the changes in the structure or function of synapses.AMPAR are the important glutamate receptors and mediate the most excitatory synaptic transmission in the nervous system.Glur1 is the most important subunit of AMPAR,of which the change in the phosphorylation can cause the movement and transfer of AMPAR on the synaptic membrane,affecting the transmission efficiency between synapses,and mediating synaptic plasticity.Studies have found that synaptic plasticity plays an important role in the conduction and regμlation of pain,and the central sensitization induced by synaptic plasticity at the spinal cord level is one of the important mechanisms of BCP.In recent years,studies have found that the extracellular matrix molecules(ECM)can participate in the process of chronic pain regμlation,and ECM take part in the synaptic plasticity by regulating the stability and plasticity of dendritic spines and synaptic structures.Decorin is one of the most abundant matrix proteins,and is widely involved in the processes of anti-inflammatory,antifibrosis,anti-tumor and other pathological and physiological processes.Meanwhile,in the nervous system,decorin has the ability to promote the differentiation and growth of neurons,as well as to promote the regeneration of axons in damaged nerve tissue.The results of the genome-wide microarray on the BCP of rats in our research group showed that the expression of decorin is significantly increased during the development of BCP,which suggests that decorin may be involved in the pathogenesis of BCP.The study was divided into two parts,in the first part,we first established the BCP model of rats to study the role of decorin in the formation of BCP and the possible mechanisms.At the second part,we studied the role of decorin in neuronal synaptic plasticity by culturing primary neurons in vitro to further explore the pathogenesis of BCP.Part1:Decorin induces the development of bone cancer pain by regulating the phosphorylation of Glur1Objective:BCP is clinically intractable pain and the pathogenesis of BCP is not completely clear.The aim of this study was to investigate the role and mechanism of decorin in BCP.Methods: Forty-eight female Sprague-Dawley(SD)rats were randomly divided into four groups: control group(sham),bone cancer pain group(BCP),bone cancer pain with negative control virus group(BCP + shctrl),bone cancer pain with virus group(BCP + shdecorin),twelve rats per group.The bone cancer pain model was established by inoculating the walker 256 breast cancer cells into the right tibia of the rats.The shctrl and shdecorin lentivirus were microinjected into the spinal dorsal horn of rats in the BCP + shctrl group and BCP + shdecorin group respectively.The paw withdrawal mechanical threshold(PWMT)of each group was tested on the day before modeling(0 d)and on days 3,5,7,10,14 and 21 after modeling to assess changes in the pain threshold in rats.At the 3rd week,the right tibia and the spinal cord of L 4-L 6 was removed for the following experiments.The HE staining were performed to detect the tumor cells in the tibia of rats.The immunofluorescence assays were performed to detect the distribution of decorin in the spinal cord of rats.And the changes of decorin,Glur1 and p-Glur1-ser 831 in the spinal cord were examined by RT-PCR and Western blot.Resμlts: In the sham group,the HE staining showed that no tumor cells were found in the bone tissue of rats,while in the BCP group,a large nμmber of the tumor cells were observed in the bone tissues,and the bone tissues were seriously damaged.Compared with the sham group,the PWMT of the BCP rats decreased significantly on the 7th day after modeling(P<0.05),and decreased progressively with time.Compared with the BCP group,the PWMT of rats in the BCP + shctrl group did not change significantly(P>0.05),while the PWMT of rats in the BCP + shdecorin group increased markedly(P<0.05).The immunofluorescence assay showed that decorin was abundantly expressed in the gray matter of the spinal cord,and the expression was up-regulated in the spinal dorsal horn of the BCP group.The quantitative results showed that compared with the sham group,the expression of decorin was significantly increased in the spinal cord of rats in BCP group(P<0.05);and compared with the BCP group,the expression of decorin were decreased significantly in the BCP + shdecorin group(P<0.05).Compared with the sham group,the expression of Glur1 in the BCP did not change markedly(P>0.05),while the expression of p-Glur1-ser 831 increased significantly(P<0.05);and compared with the BCP group,the level of Glur1 did not changed significantly(P>0.05),while p-Glur1-ser 831 expression were decreased significantly in the BCP + shdecorin group(P<0.05).Conclusion: Under the conditions of this study,it is found that decorin is participated in the development of the BCP,and the up-regulation of decorin in the spinal cord induces the development of the BCP by promoting the phosphorylation of Glur1.Part II: Decorin knockdown inhibits the neuronal excitatory synaptic plasticityObjective: To explore the role of decorin in synaptic plasticity,and clarify the mechanism of the BCP furtherly.Methods: Primary cultured cortical neurons of rats were randomly divided into three groups: normal group(normal),negative control virus group(shctrl)and virus group(shdecorin).The lentivirus was transfected on the 3rd day after the culture of the neurons.After 48 hours,the transfection effect was observed under the fluorescence microscope.The RT-PCR were performed to detect the interference effect of decorin on the 7th day,and on the 12 th day,the immunofluorescence assays were performed to examine the formation of spines and excitatory synapses,and to detect the expression of Glur1 and p-Glur1-ser 831.Resμlts: The immunofluorescence results showed that decorin was expressed in neuronal soma and dendrite.The quantitative analysis indicated that compared with the normal group,the mRNA of decorin did not change in the shctrl group(P>0.05),the mRNA of decorin was decreased significantly in the shdecorin group(P<0.05).Compared with the shctrl group,the density of dendritic spines(P<0.05)and the number of excitatory synapses in neurons were decreased in shdecorin group(P<0.01);the level of Glur1 remained unchanged(P>0.05),the expression of p-Glur1-ser 831 was decreased significantly(P<0.01).Conclusion: Under the conditions of this study,it is found that knockdown the expression of decorin in neurons significantly inhibits the excitatory synaptic plasticity. |
PDF Full Text Request