CAJ | 학술논문

目的研究脊髓P2Y12受体在大鼠骨癌痛中的作用及其对p38丝裂原活化蛋白激酶(p38MAPK)表达的影响.方法雌性SD大鼠40只,用随机数字表法分为5组(n=8):正常组(N)、假手术组(S)、DMSO溶剂骨癌痛组(DA)、骨癌痛组(A)、骨癌痛镇痛组(MA).大鼠左胫骨上段注入经腹水培养的Walker256癌细胞建立骨癌痛模型.建模后第9～12天分别行鞘内注药,S组、A组注射0.9％生理盐水,DA组注射5％二甲基亚砜(DMSO)溶剂,MA组注射MRS2395(400 pmol/μl,溶于5％ DMSO溶液),容量各15μl.注药前后测试大鼠左后足机械痛阈值,术后第12天测完痛阈后取L4～6脊髓,采用免疫组化及免疫荧光法测定脊髓背角磷酸化p38MAPK(p-p38MAPK)表达水平.结果建模后第9天,与N组(36.1 ±4.0)g、S组(38.9±5.2)g比较,MA组机械性痛阈值下降为(19.8±5.0) g(P＜0.01),第12天p-p38MAPK表达增加(P＜0.01).与DA组(17.7±3.0)g、A组(19.1 ±2.5)g比较,MA组鞘内给药后大鼠机械痛阈值增高,峰值为(26.5±4.7)g(P＜0.05);与DA组(细胞数:43.4±3.8、IOD值:569 ±27)、A组(细胞数:45.0±2.6、IOD值:594±22)比较,MA组(细胞数:20.9±2.2、IOD值:246±25)第12天脊髓p-p38MAPK表达减少(P＜0.01)；但与N组、S组比较,MA组机械痛阈值仍低于正常(P＜0.01),脊髓p-p38MAPK蛋白表达高于N组(细胞数:9.9±2.4、IOD值:82±28)、S组(细胞数:10.9±2.2、IOD值:109 ±25)(P＜0.01).免疫荧光双标显示脊髓背角p-p38 MAPK与小胶质细胞共表达.结论鞘内注射P2Y12受体抑制剂MRS2395可能通过抑制脊髓背角的p38MAPK磷酸化程度部分减轻大鼠骨癌疼痛.
목적 연구척수P2Y12수체재대서골암통중적작용급기대p38사렬원활화단백격매(p38MAPK)표체적영향.방법 자성SD대서40지,용수궤수자표법분위5조(n=8):정상조(N)、가수술조(S)、DMSO용제골암통조(DA)、골암통조(A)、골암통진통조(MA).대서좌경골상단주입경복수배양적Walker256암세포건립골암통모형.건모후제9～12천분별행초내주약,S조、A조주사0.9％생리염수,DA조주사5％이갑기아풍(DMSO)용제,MA조주사MRS2395(400 pmol/μl,용우5％ DMSO용액),용량각15μl.주약전후측시대서좌후족궤계통역치,술후제12천측완통역후취L4～6척수,채용면역조화급면역형광법측정척수배각린산화p38MAPK(p-p38MAPK)표체수평.결과 건모후제9천,여N조(36.1 ±4.0)g、S조(38.9±5.2)g비교,MA조궤계성통역치하강위(19.8±5.0) g(P＜0.01),제12천p-p38MAPK표체증가(P＜0.01).여DA조(17.7±3.0)g、A조(19.1 ±2.5)g비교,MA조초내급약후대서궤계통역치증고,봉치위(26.5±4.7)g(P＜0.05);여DA조(세포수:43.4±3.8、IOD치:569 ±27)、A조(세포수:45.0±2.6、IOD치:594±22)비교,MA조(세포수:20.9±2.2、IOD치:246±25)제12천척수p-p38MAPK표체감소(P＜0.01)；단여N조、S조비교,MA조궤계통역치잉저우정상(P＜0.01),척수p-p38MAPK단백표체고우N조(세포수:9.9±2.4、IOD치:82±28)、S조(세포수:10.9±2.2、IOD치:109 ±25)(P＜0.01).면역형광쌍표현시척수배각p-p38 MAPK여소효질세포공표체.결론 초내주사P2Y12수체억제제MRS2395가능통과억제척수배각적p38MAPK린산화정도부분감경대서골암동통.
Objective To investigate the role of P2Y12 receptor in rat bone cancer pain model and its influence on p38MAPK (Mitogen-activated protein kinase).Method A total of forty female SD rats,weighting 200-250 g,were randomly divided into 5 groups (n =8):normal group (group N),sham group (group S),vehicle group (group DA),cancer group (group A),and analgesia group (group MA).Rats in group N were untreated,rats in group S were injected with Hank's solution 10 μl into the left tibal metaphysis; rats in group DA,group A and group MA were injected with Walker256 cancer cells (10 μl,2 × 107 cells/ml) into the left tibal metaphysic to establish the model of bone cancer pain.Catheterization was simultaneously made in four groups between L3 and L4 vertebra except group N.Saline (0.9％,15 μl)was injected in group S and group A,DMSO (5％,15 μl) was injected in group DA,and MRS2395 (400 pmol/μl,15 μl) was injected in group MA on day 9 to 12 post-inoculation.Mechanical withdrawal thresholds were measured on left hind paw before and every 10 min after intrathecal injection.Rats were euthanized after measuring mechanical withdrawal threshold at day 12 post-inoculation.L4-6 sections of spinal cord were collected to determine the expression of p-p38MAPK by immunohistochemistry and immunofluorescent.Result Compared to that in group N (36.1 g ± 4.0 g) and group S (38.9 g± 5.2 g),mechanical withdrawalthresholds in group MA (19.8 g±5.0 g) were decreased ou day 9 post-inoculation (P ＜ 0.01),and the expression of p-p38MAPK in spinal cord was increased on day 12 (P ＜ 0.01).Compared to that in group DA (17.7 g ± 3.0 g) and group A (19.1 g ± 2.5 g),mechanical withdrawal threshold in group MA was obviously increased after intrathecal injection,peaked at (26.5 g ± 4.7 g) (P ＜ 0.05) ; compared with group DA (number 43.4 ± 3.8,IOD 569 ± 27) and group A (number 45.0 ± 2.6,IOD 594 ± 22),the expression level of p-p38MAPK in spinal cord in group MA at day 12 was significantly decreased (number 20.9 ± 2.2,IOD 246 ± 25) (P ＜ 0.01) ; Mechanical withdrawal threshold in group MA was still lower than group N and group S,while the expression of p-p38MAPK was higher than group N (number 9.9 ± 2.4,IOD 82 ± 28) and group S (number 10.9 ± 2.2,IOD 109 ± 25) (P ＜ 0.01).Immunofluorescent showed that p-p38MAPK was colocalized with microglia in spinal dorsal horn,but not with neurons and astrocytes.Conclusions These results demonstrate rat bone cancer pain could partially relieved after intrathecal injection of P2Y12 receptor inhibitor MRS2395 through inhibiting the phosphorylation of p38MAPK in spinal dorsal horn.