| Our previous studies have found that in the bone cancer pain(BCP)model of rats,sodium-potassium-chloride cotransporter 1(NKCC1)and potassium-chloride cotransporter 2(KCC2)affect the function of γ-aminobutyric acid(GABA)receptor by regulating intracellular Cl-and participate in the occurrence and development of bone cancer pain.The latest research shows that in pathological state,the expression of NKCC1/KCC2 protein is regulated by with-nolysine kinases 1(WNK1)through the pathway to Ste20/SPS1 related proline/alanine rich kinase(SPAK)and the intermediate oxidative stress-response kinase-1(OSR1),thus affects the intracellular concentration of Cl-.Therefore,we speculate that WNK1-SPAK/OSR1-NKCC1/KCC2 signal transduction pathway may be involved in the process of bone cancer pain,but there is no relevant report.In this study,we intend to establish a rat model of bone cancer pain.Based on this,we observed the expression of WNK1 and its downstream protein in the spinal dorsal horn(DH)and dorsal root ganglia(DRG)of BCP rats.Then,we used siRNA technology to inhibit the expression of WNK1,observed the expression of WNK1 and its downstream protein and the behavior of BCP rats.Then we intended to use neuropharmacological method to study whether WNK1-SPAK/OSR1-NKCC1/KCC2 signal transduction pathway is involved in the process of BCP.This study will enrich the mechanism of cancer pain and provide new ideas about the research and development of cancer pain drugs.Part Ⅰ Expression of WNK1 in spinal dorsal horn and dorsal root ganglion of rats with bone cancer painObjective To investigate the expression of WNK1 kinase in spinal cord DH and DRG of rats with bone cancer pain.Methods To establish the pain model of bone cancer in rats,Walker 256 breast cancer cells were inj ected into the metaphysis of left tibial.Female Sprague-Dawley(SD)rats who weight about 180-200g were randomly assigned to control group(n=5),BCP group(n=20)and sham group(n=5).The rats in control group administered nothing.In sham group,rats were injected 10 μl Hank’s solution into the metaphysis of left tibial.In BCP group,Walker256 breast cancer cells(1×105/10μl Hank’s solution)were injected by the same way.Rats in control and sham group were executed on the 12th day after observation.In BCP group,5 rats were executed separately on the 3th,6th,9th and 12th day after modeling.Spinal cord and dorsal root ganglion of L4-6 segments of rats were collected immediately after execution.The pain behavior of BCP rats was observed by ethological test.The expression of WNK1 was detected by real-time PCR and Western blot.The distribution and localization of WNK1 in spinal DH and DRG of BCP rats were detected by immunofluorescence.Results 1.Compared with sham or control group,the paw withdrawal mechanical threshold(PWMT)in BCP group decreased significantly from the 6th to 12th day after modeling(P<0.01).2.Compared with sham group,WNK1 mRNA and protein were up-regulated in spinal DH and DRG of rats with bone cancer(P<0.01 or 0.05).3,Immunofluorescence images showed the distribution of WNK1 located in spinal cord dorsal horn,especially in superficial layer of DH,and colocalization of WNK1 primarily with neurons,but not with astrocytes or microglial cells.In the DRG of BCP rats,WNK1 expression was also colocalized with neurons.Conclusion WNK1 kinase expression of spinal DH and DRG of BCP rats was significantly up-regulated.Part Ⅱ Effect of intrathecal injection of WNK1 siRNA on bone cancer pain in ratsObjective To observe the effect of intrathecal injection of WNK1 siRNA on bone cancer pain in ratsMethods Synthesis and screening of siRNA for WNK1(siWNK1)in vitro.Female SD rats that weight about 180-200g were randomly divided into sham+siWNK1 group(n=5),sham group(n=10),BCP group(n=10),BCP+siWNK1 group(n=10),BCP+vehicle group(n=10),BCP+scrambled group(n=10).On day 8,a PE-10 catheter was inserted into the L4 to L5 subarachnoid space of the rats under anesthesia with pentobarbital sodium(50mg/kg,intraperitoneal).Then in sham+siWNK1 and BCP+siWNK1 group,siWNK1 solution(3μg siRNA of 0.36μl jetPEI in 10μl 5%glucose solution)was injected intrathecally on days 9 through 11 into the rats.In BCP+scrabbled group,negative siRNA solution(3μg siRNA of 0.36μl jetPEI in 10μl 5%glucose solution)was injected intrathecally on days 9 through 11 too.In BCP+vehicle group,0.36μl jetPEI and 10μl 5%glucose solution were injected intrathecally from 9th to 11th day.The treatment of sham and BCP group was the same as the Part Ⅰ.In addition to sham+siWNK1 group,5 rats were executed on day 12 in other groups and spinal cord DH and DRG were collected immediately.The pain behavior of BCP rats was observed by ethological test.The mRNA level of WNK1,NKCC1/KCC2 and SPAK/OSR1 were detected by real-time PCR.The protein expression of WNK1,NKCC1/KCC2 and SPAK/OSR1 were detected separately in spinal DH and DRG by Western blot or immunofluorescence.Result 1.The chemical synthesis of siWNK1 can effectively inhibit the expression of WNK1 protein on PC 12 cells without obvious cytotoxicity.2.Compared with BCP group,PWMT value in rats of BCP+siWNK1 group increased(P<0.05),and limb use scores decreased(P<0.01).3.Compared with BCP group,WNK1 mRNA level of spinal DH and DRG in rats of BCP+siWNK1 group decreased significantly(P<0.01),accompanied by the decrease of WNK1 protein immunofluorescence intensity(P<0.01).4.Compared with BCP group,the expression of NKCC1 mRNA and protein in DRG of BCP+siWNK1 group decreased significantly(P<0.01),but there was no significant difference in spinal DH;5.Compared with BCP group,the expression of KCC2 mRNA and protein in spinal DH of BCP+siWNK1 group was significantly increased(P<0.01);6.Compared with BCP group,the expression of SPAK/OSR1 protein in DRG of BCP+siWNK1 group decreased(P<0.01),but there was no significant difference in spinal DH.Conclusion Intrathecal injection of siWNK1 can significantly alleviate the pain behavior of BCP rats,and partially inhibit the expression of SPAK/OSR1 and NKCC1/KCC2 protein,downstream of WNK1 kinase,in spinal DH or DRG.Part Ⅱ Effect of intrathecal injection of Closantel on bone cancer pain in ratsObjective To observe the effect of intrathecal injection of Closantel on bone cancer pain in ratsMethods Female SD rats who weight about 180-200g were randomly divided into sham+Closantel group(n=5),sham group(n=10),BCP group(n=10),BCP+vehicle group(n=10),BCP+Closantel group(n=10).In sham+Closantel and BCP+Closantel group,Closantel solution(60μg/10μl DMSO)was injected intrathecally on days 9 through 11 into the rats.In BCP+vehicle group,10μl DMSO was injected intrathecally from 9th to 11th day.The treatment of sham and BCP group was the same as the Part Ⅰ.In addition to sham+Closantel group,5 rats were executed on day 12 in other groups and spinal cord DH and DRG were collected immediately.The pain behavior of BCP rats were observed by ethological test.The level of NKCC1/KCC2 mRNA were detected by real-time PCR.The protein expression of NKCC1/KCC2 and SPAK/OSR1 were detected separately in spinal DH and DRG by Western blot or immunofluorescence.The colocalization of SPAK/OSR1 and NKCC1 were observed by immunofluorescence in DRG.Result 1.Compared with BCP group,PWMT value increased(P<0.05),and limb use scores decreased significantly(P<0.01)in rats of BCP+Closantel group.2.Compared with BCP group,after intrathecal injection of Closantel,the expressive level of NKCC1 mRNA and protein showed significant descending(P<0.01)in DRG of rats in BCP+Closantel group,but there was no significant difference in spinal DH.3.Compared with BCP group,the expressive level of KCC2 mRNA and protein enhanced obviously(P<0.01)in spinal DH of rats in BCP+Closantel group;4.Immunofluorescence image showed that NKCC1 and SPAK/OSR1 protein were co-expressed in DRG of rats.And compared with BCP group,the fluorescence intensity of SPAK/OSR1-NKCC1 protein decreased significantly(P<0.01)in DRG of rats in BCP+Closantel group after intrathecal injection of Closantel.Conclusion Intrathecal injection of Closantel can significantly alleviate the pain of BCP rats,and inhibit the expression of SPAK/OSR1-NKCC1 protein in DRG and KCC2 in spinal DH.Part Ⅳ Effect of NKCC1/KCC2 expression on bone cancer pain in ratsObjective To explore the effect of NKCC1/KCC2 protein expression on bone cancer pain in ratsMethods Female SD rats that weight about 180-200g were randomly assigned into BCP+vehicle group(n=10),sham group(n=10),BCP group(n=10),BCP+bumetanide group(n=5),BCP+CLP257 group(n=5).In sham group,rats were injected 10 μl Hank’s solution into the metaphysis of left tibial.In BCP group,Walker256 breast cancer cells(1×105/10μl Hank’s solution)was injected by the same way.In BCP+vehicle group,10μl DMSO were injected intrathecally from 9th to 11th day.In BCP+CLP257 and BCP+bumetanide group,CLP257 solution(100μg/10μl DMSO)and bumetanide(100μg/10μl DMSO)were injected intrathecally separately on days 9 through 11 into the rats.On days 12,5 rats were executed in each group and spinal cord DH and DRG were collected immediately.The pain behavior of BCP rats were observed by ethological test.The protein expression of NKCC1/KCC2 were detected separately in spinal DH and DRG by Western blot.Result 1.Compared with BCP group,after intrathecal injection of bumetanide,PWMT value increased from 1h to 4h(P<0.05 or 0.01),Limb use scores decreased(P<0.01)and the expression of NKCC1 protein in DRG of rats decreased(P<0.01)in BCP+bumetanide group.2.Compared with BCP group,the PWMT value of BCP+CLP257 group increased from 2h to 4h after intrathecal injection of CLP257(P<0.05),Limb use scores decreased(P<0.01)and the expression of KCC2 protein increased in spinal DH of rats in BCP+CLP257 Group(P<0.05).Conclusion NKCC1/KCC2 is involved in the process of bone cancer pain,and the expression of NKCC1/KCC2 affects the mechanical pain threshold of BCP rats. |
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