Objective To investigate the relationship between C-C chemokine receptor type 2 (CCR2) and P38 mitogen-activated protein kinase (P38MAPK) signaling pathway in the spinal cord of rats and further clarify the mechanism of bone cancer pain (BCP).Methods A total of 92 healthy female SD rats, of which 60 were subjected to behavioral tests using a ciliary mechanical stimulation needle .SD rats were randomly divided into six groups:sham operation group (group S), bone cancer pain group (group B), sham operation +DMSO solvent group ( group SD ) , bone cancer pain +DMSO solvent group ( group BD) , sham operation +RS102895 CCR2 inhibitor group ( group SR ) , bone cancer pain +RS102895 CCR2 inhibitor group ( group BR ) , and Von Frey was used in the behavioral test .Another 32 SD rats were randomly divided into the following 8 groups (n=4):sham operation group (group S), bone cancer pain 5 d group (group B5), bone cancer pain 9 d group (group B9), bone cancer pain 14 d group (group B14), bone cancer pain +DMSO solvent group ( group BD ) , bone cancer pain +RS102895 CCR2 inhibitor 0.5 h group ( group BR0.5 h) , bone cancer pain +RS102895 CCR2 inhibitor 4 h group ( group BR4 h) , bone cancer pain +RS102895 CCR2 inhibitor 12 h group ( group BR12 h).Western blot was used to detect the expression of P38, p-P38 and CCR2 in spinal cord of rats.Results At day 5,7,9,14,21 post-injection,mechanical withdrawal thresholds of group S were (30.9 ±1.5),(31.9 ±1.2),(32.0 ±1.1), (31.6 ±1.5),(32.2 ±1.4)g respectively, the mechanical withdrawal thresholds of group B were ( 26.4 ± 0.7),(24.4 ±0.8),(21.4 ±0.8),(13.5 ±0.4),(9.9 ±0.2)g respectively,the mechanical withdrawal thresholds in group B decreased obviously versus group S , and the differences were statistically significant (t=-13.177,-16.660,-23.778, -35.574, -48.401,all P<0.01).At day 9 post-injection,the mechanical withdrawal thresholds in SD,BD,SR and BR groups were (32.4 ±1.7),(19.4 ±1.1), (32.1 ±1.3), (26.3 ±1.0) g respectively,the difference was statistically significant (F=224.681,P<0.01 ) ,and the mechanical withdrawal thresholds in group BD decreased obviously versus group SD ,while the mechanical withdrawal thresholds in group BR increased obviously versus group BD .The expression levels of p-P38 in spinal cord of group S, group B5, group B9 and group B14 were(0.08 ±0.03),(0.20 ±0.05), (0.40 ±0.17),(0.65 ±0.14)respectively,the expression levels of CCR2 were(0.08 ±0.04),(0.18 ± 0.05),(0.30 ±0.09),(0.58 ±0.07)respectively,the difference was statistically significant (F=19.123, 40.746,all P<0.01),and the expression of p-P38 and CCR2 in group B9 were showed a significant up-regulation versus group S .The expression levels of p-P38 in spinal cord of group BD , group BR0.5 h, group BR4 h and group BR12 h were ( 0.57 ±0.06 ) , ( 0.17 ±0.11 ) , ( 0.03 ±0.01 ) , ( 0.25 ±0.11 ) respectively,and the difference was statistically significant (F=29.582,P<0.01).The expression of p-P38 in group BR0.5 h, BR4 h, BR12 h showed a significant down-regulation versus group BD.Conclusion CCR2 in the spinal cord may be involved in the development of bone cancer pain by activating P 38MAPK signaling pathway in rats .%目的 探讨骨癌痛大鼠脊髓趋化因子受体2(CCR2)与P38丝裂原活化蛋白激酶(P38MAPK)信号通路的关系,进一步明确骨癌痛的发生机制.方法 健康雌性SD大鼠共92只,其中60只利用纤毛机械刺激针方法用于行为学试验,采用随机数字表法分为以下6组(n=10):假手术组(S组)、骨癌痛组(B组)、假手术+二甲亚砜(DMSO)溶剂组(SD组)、骨癌痛+DMSO组(BD组)、假手术+RS102895 CCR2抑制剂组(SR组)、骨癌痛+RS102895 CCR2抑制剂组(BR组).另外32只SD大鼠采用随机数字表法分为以下8组(n=4):假手术组(S组)、骨癌痛5 d组(B5组)、骨癌痛9 d组(B9组)、骨癌痛14 d组(B14组)、骨癌痛+DMSO溶剂组(BD组)、骨癌痛+RS102895 CCR2抑制剂后0.5 h组(BR0.5 h组)、骨癌痛+RS102895 CCR2抑制剂后4 h组(BR4h组)、骨癌痛+RS102895 CCR2抑制剂后12 h组(BR12 h组),通过蛋白质印迹法(Western Blot)检测大鼠脊髓P38蛋白、磷酸化的P38(p-P38)和CCR2的表达水平.结果 S组造模后5、7、9、14、21 d的机械缩足反应阈值分别为(30.9±1.5)、(31.9±1.2)、(32.0±1.1)、(31.6±1.5)、(32.2±1.4)g,B组分别为(26.4±0.7)、(24.4±0.8)、(21.4±0.8)、(13.5±0.4)、(9.9±0.2)g,与S组比较,B组造模后5、7、9、14、21 d时机械痛阈值降低,差异均有统计学意义(t=-13.177、-16.660、-23.778、-35.574、-48.401,均P<0.01).造模后第9天,SD组、BD组、SR组和BR组给药后4 h机械缩足反应阈值分别为(32.4±1.7)、(19.4±1.1)、(32.1±1.3)、(26.3±1.0)g,差异有统计学意义(F=224.681,P<0.01);与SD组比较,BD组机械痛阈值降低;与BD组比较,BR组机械痛阈值升高.S组、B5组、B9组、B14组大鼠脊髓p-P38表达水平分别为(0.08±0.03)、(0.20±0.05)、(0.40±0.17)、(0.65±0.14),CCR2表达水平分别为(0.08±0.04)、(0.18±0.05)、(0.30±0.09)、(0.58±0.07),差异均有统计学意义(F=19.123、40.746,均P<0.01);与S组比较,B9组大鼠脊髓p-P38和CCR2表达上调.造模后第9天,BD组、BR0.5 h组、BR4 h组和BR12 h组大鼠脊髓p-P38表达水平分别为(0.57±0.06)、(0.17±0.11)、(0.03±0.01)、(0.25±0.11),差异有统计学意义(F=29.582,P<0.01);与BD组比较,BR0.5 h组、BR4 h组、BR12 h组大鼠脊髓p-P38表达明显下调.结论 脊髓CCR2可能通过激活P38MAPK信号通路参与了大鼠骨癌痛痛觉过敏的发生.
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