| BackgroundAs advances in detection and treatment of cancer,patients with cancer have a longer survival time.However,pain that accompanies the cancer has become a main public health problem world widely,significantly compromising quality of life of many patients.Bone represents the preferential site of metastasis for breast and prostate cancers,with an incidence up to 70%?90%.Bone metastasis may cause bone fractures,hypercalcemia,spinal cord compression and severe bone pain,the consequences of which are devastating.Up to 75%of patients with advanced cancer endure bone cancer pain(BCP),which is a common kind of moderate and severe pain.BCP is a complex pain state involving a combination of background,spontaneous and incident(movement-evoked)pain.Spontaneous and incident pain often referred to as breakthrough pain.Their intermittent nature makes these types of pain hard to treat,because the episodes tend to be rapid in onset and of short duration.Moreover,high-dose opioids analgesics may cause severe adverse effects.The poor understanding of BCP mechanisms makes it difficult to manage.Therefore,the identification of novel therapies and the elucidation of the mechanism of BCP are important for pain relief.The development of chronic pain causes the change of related genes in dorsal root ganglion,dorsal horn and pain-related brain regions,the products of which can produce long-term and stable neural plasticity regulation.Epigenetic mechanisms enhance or suppress gene expression without alterations of the primary DNA sequence.Based on recent evidence,epigenetic modifications,including DNA methylation,histone modification and RNA interference,contribute to the development and maintenance of BCP.SIRT1,a member of Sirtuin family,is a type of nicotinamide adenine dinucleotide(NAD+)-dependent deacetylase.SIRT1 plays an important role in regulation of pathogenesis of metabolic disease,aging,and tumorigenesis.Recently,growing evidence suggests that SIRT1 may be a novel therapeutic target for the prevention of chronic pain.However,the expression and activity of SIRT1 during the development and maintenance of BCP remain unknown.The actions of glutamate,the main excitatory neurotransmitter in the mammalian central nervous system,are mediated by ligand-gated ionotropic glutamate receptors(iGluRs)and metabotropic glutamate receptors(mGluRs).mGluRs,which are expressed abundantly in the spinal cord,have been reported to play critical roles in pain transmission and central sensitization.Eight mGluRs,mGluR1 to mGluR8,have been identified to date.These receptors are subdivided into three groups based on sequence identity,pharmacology,and signal transduction.Group ? mGluRs(mGluR1 and mGluR5)mainly lead to phospholipase C(PLC)activation,while group ?(mGluR2 and mGluR3)and group ? receptors(mGluR 4,6,7,and 8)predominantly inhibit adenylate cyclase(AC).Based on accumulating evidence,the inhibition of Group ? mGluRs(mGluRl and mGluR5)may exert analgesic effects.A recent study demonstrated that spinal SIRT1 activation can functionally reverse pain behavior and epigenetically downregulate the expressions of mGluR1/5 in type 2 diabetic rats.Accordingly,we hypothesized that SIRT1 attenuates BCP by inhibiting mGluR 1/5 expression.This study investigated the underlying mechanisms of spinal SIRT1 in BCP mouse model to provide new insights into the clinical treatment of BCP.This study was divided into two parts.Part I:Changes of spinal SIRT1 in the BCP mice;Part ?:Spinal SIRT1 activation attenuated BCP in mice by inhibiting mGluR 1/5 expression.ObjectivesPart ?:The time course of SIRT1 expression and activity in the spinal cord of mice with BCP was detected to identify that SIRT1 is involved in the development and maintenance of BCP.Part ?:The effect of spinal SIRT1 activation on pain behavior and mGluRl/5 expression in BCP mice was explored to clarify the underlying mechanisms of SIRT1 in BCP.This part may provide new insights into the clinical treatment of BCP.MethodsPart I:Osteosarcoma NCTC 2472 cells were incubated in NCTC 135 medium containing 10%horse serum according to the American Type Culture Collection(ATCC)recommendations.Experiments were performed on male C3H/HeN mice(age,4 to 6 weeks;weight,20 to 25 g).The method for inducing BCP was established by implanting osteosarcoma NCTC 2472 cells into the right femur of mice.Mice were randomly divided into sham group and BCP group(n=20 each).Behavioral tests,including paw withdrawal mechanical threshold(PWMT)and the number of spontaneous finches(NSF),were performed on days 0,4,7,10,14 and 21 after implantation(n=8 each).The L3-L5 spinal cords of mice in each group(n=6 each)were harvested quickly on days 7,14 and 21 after implantation.The levels of SIRT1 protein,mRNA,and activity in the spinal cord were measured by Western blot,Real-time PCR,and Fluorometric Assay,respectively.The right femur of each mouse was removed to reveal the extent of tumor infiltration and bone destruction by a histological method on day 21 after implantation.Part ?:There are two experiments in this part.Experiment ?:The effect of intrathecal administration of SRT1720,a selective SIRT1 agonist,on pain behavior and spinal mGluRl/5 expression in BCP mice.Experiment ?:The effect of intrathecal administration of AAV-SIRT1-shRNA on pain behavior and spinal mGluR 1/5 expression in normal mice.Experiment I:Mice were divided into four groups(n=12 mice each),namely,the sham group,BCP group,DMSO group,and SRT1720 group.Drugs were intrathecally administered from days 14 to 16 after inoculation for 3 consecutive days.20%DMSO,and 5 ?g SRT1720(dissolved in 20%DMSO)were administered intrathecally at a dose of 5 ?l in DMSO group,and SRT1720 group,respectively.The L3-L5 spinal cords of mice in each group(n=6 each)were harvested on day 1 after intrathecal administration.The levels of SIRT1 and mGluRl/5 protein,SIRT1 and mGluR 1/5 mRNA,and SIRT1 activity in the spinal cord were measured by Western blot,Real-time PCR,and Fluorometric Assay,respectively.Behavioral tests were performed on days 1,2,3,and 4 after intrathecal administration in each group(n=6 each).Experiment ?:Mice were divided into three groups(n=6 mice each),namely,the Naive group,AAV-GFP control,and SIRT1 shRNA group.AAV-GFP and AAV-SIRT1-shRNA were administered intrathecally at a dose of 5 ?l in AAV-GFP control group,and SIRT1 shRNA group,respectively.Behavioral tests were performed on day 21 after intrathecal administration in each group(n=6 each).Then,The L3-L5 spinal cords of mice in each group were harvested.The levels of SIRT1 and mGluR1/5 protein,SIRT1 and mGluR1/5 mRNA,and SIRT1 activity in the spinal cord were measured by Western blot,Real-time PCR,and Fluorometric Assay,respectively.ResultsPart ?:Compared with the levels on the day before implantation,the PWMT decreased and NSF increased on day 4 after implantation in sham mice;the PWMT decreased and NSF increased on days 4,7,10,14,and 21 after implantation in BCP mice(P<0.05).Compared with the sham mice,BCP mice exhibited a significant increase in PWMT and a decrease in NSF on days 7,10,14,and 21 after implantation(P<0.01).Bone histology showed that the tumor significantly infiltrated and eroded the cortical bone in mice with BCP,while no obvious bone destruction was observed in sham mice on day 21 after implantation.The levels of the SIRT1 protein,mRNA,and activity were decreased in the spinal cord of mice with BCP on days 7,14,and 21 after implantation compared with the levels in sham mice(P<0.01).Part II:Experiment I:Compared with the levels in sham mice,the levels of the SIRT1 protein,mRNA,and activity were decreased in the spinal cord of BCP mice and DMSO mice(P<0.01).Compared with the levels in BCP mice,the levels of the SIRT1 protein,mRNA,and activity were increased in the spinal cord of SRT1720 mice(P<0.01).Compared with the sham mice,BCP and DMSO mice exhibited a significant decrease in PWMT and an increase in NSF on days 1-4 after intrathecal administration(P<0.01).Compared with the BCP mice,SRT1720 mice exhibited a significant increase in PWMT and a decrease in NSF on days 1-3 after intrathecal administration(P<0.01).Compared with the levels in sham mice,the levels of the mGluR1/5 protein and mRNA were increased in the spinal cord of BCP mice and DMSO mice(P<0.01).Compared with the levels in BCP mice,the levels of the mGluRl/5 protein and mRNA were decreased in the spinal cord of SRT1720 mice(P<0.01).Experiment ?:The levels of spinal SIRT1 protein,mRNA,and activity did not show significant differences between naive mice and AAV-GFP control mice(P>0.05).The levels of spinal SIRT1 protein,mRNA,and activity were decreased in SIRT1 shRNA mice compared with the levels in naive mice(P<0.01).The PWMT and NSF did not show significant differences between naive mice and AAV-GFP control mice(P>0.05).Compared with the naive mice,SIRT1 shRNA mice exhibited a significant decrease in PWMT and an increase in NSF(P<0.01).The levels of spinal mGluR1/5 protein and mRNA did not show significant differences between naive mice and AAV-GFP control mice(P>0.05).The levels of spinal mGluR1/5 protein and mRNA were increased in SIRT1 shRNA mice compared with the levels in naive mice(P<0.01).ConclusionPart ?:Spinal SIRT1 is involved in the development and and maintenance of BCP.Part ?:The activation of spinal SIRT1 functionally attenuates BCP in mice by inhibiting mGluR1/5 expression. |
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