骨癌痛大鼠脊髓趋化因子受体2与P38丝裂原活化蛋白激酶信号通路的关系

Relationship between C-C chemokine receptor type 2 and P38 mitogen-activated protein kinase signaling pathway in the spinal cord of rats with bone cancer pain

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5d组（B5组）、骨癌痛9d组（B9组）、骨癌痛14d组（B14组）、骨癌痛＋DMSO溶剂组（BD组）、骨癌痛＋RS102895 CCR2抑制剂后0．5h组（BR0．5h组）、骨癌痛＋RS102895 CCR2抑制剂后4h组（BR4h组）、骨癌痛＋RS102895 CCR2抑制剂后12h组（BR12h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、21d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、21d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4h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、BR0．5h组、BR4h组和BRl2h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，BR0．5h组、BR4h组、BR12h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。

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 Van 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 v0. 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 upregulation versus group S. The expression levels of p-P38 in spinal cord of group BD, group BRO. 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-regnlation versus group BD. Conclusion CCR2 in the spinal cord may be involved in the development of bone cancer pain by activating P38MAPK signaling pathway in rats.