华蟾素对骨癌痛大鼠的镇痛效应及对脊髓胶质细胞活化的影响

Treatment of Cionbufagin on analgesic effect and activation of glial cells in the spinal cord of cancer-induced bone pain rats

目的:探讨华蟾素对骨癌痛大鼠的镇痛效应及对脊髓胶质细胞活化的影响。方法:选用雌性SD大鼠48只,随机分为正常对照组、假手术组(胫骨内注射20μL PBS)、模型组和华蟾素干预组,每组12只。模型组与华蟾素干预组大鼠胫骨内注射20μL Walker 256乳腺癌细胞构建骨癌痛模型。华蟾素干预组于造模第7天开始腹腔注射华蟾素注射液,其余各组每天予以等量生理盐水,各组于造模前以及造模后第2、5、7、9、13、15天分别测定热缩足阈值和机械缩足阈值,造模第7天行胫骨X线摄片观察骨质破坏程度,末次行为学检测后处死大鼠,取外周血、L4~L6节段脊髓,利用免疫组化检测星形胶质细胞标记物(GFAP)和小胶质细胞标记物(Iba-1)的表达情况,ELISA检测外周血及脊髓中相关炎性因子(TNF-α和IL-1β)的表达。结果:X线显示模型组大鼠胫骨骨皮质破坏明显、骨质连续性缺乏、骨组织肿胀明显,正常对照组、假手术组未见明显骨组织肿胀、骨连续性较好,提示造模成功。行为学检测结果表明与模型组比较,华蟾素干预可明显缓解骨癌所诱发的机械痛敏、热痛敏(P<0.01)。免疫组化结果提示与假手术组相比,模型组大鼠脊髓胶质细胞活化明显增强(P<0.05);与模型组相比,华蟾素能显著抑制脊髓胶质细胞活化(P<0.05)。ELISA检测结果显示与假手术组相比,模型组大鼠脊髓及血清中TNF-α和IL-1β表达增强(P<0.05);与模型组相比,华蟾素能降低TNF-α和IL-1β表达(P<0.05)。结论:华蟾素对骨癌痛大鼠有较好的镇痛效应,其镇痛作用可能是通过抑制脊髓星形胶质细胞及小胶质细胞活化发挥作用的。

OBJECTIVE:To explore whether the intraperitoneal administration of Cionbufagin has an analgesic effect on cancer-induced bone pain(BICP)rats,and to assess whether the inhibition of astrocytic and microglia activation is involved in the analgesic effects of Cionbufagin.METHODS:A total of 48 adult female Sprague-Dawley(SD)rats were divided into the following groups with 12 rats per group:Control,Sham,CIBP,and Cionbufagin.The groups of CIBP and Cionbufagin rats were prepared by injection of 20μL Walker 256 breast cancer cell suspension into the tibial bone marrow cavity.For the sham-operated group,20μL PBS was used to replace the tumor cells injected into the tibia.Cionbufagin was injected intraperitoneally on the seventh day post-operation and once daily for 7 days whereas normal saline was used for the other groups.Mechanical withdrawal threshold and radiant heat threshold of hind paws of rats were measured to evaluate analgesic effects of Cionbufagin.Moreover,tibia was detected by X-ray assay on the seventh day after operation.Immunofluorescence staining was used to determine the expression of spinal astrocytes and microglial activation.Enzyme-linked immunosorbent assay(ELISA)was used to detect expression of pro-inflammatory cytokines[interleukin(IL)-1βand tumor necrosis factor(TNF)-α].RESULTS:After operation,mechanical pain and thermal pain threshold decreased in the BICP group,and the differences were statistically significant compared with the control and sham groups on the 7th day(P<0.01).After injected with Cinobufagin,the pain threshold of the Cinobufagin group was increased and the difference was statistically significant compared with the CIBP group(P<0.01).X-ray assay showed tibia were obviously broken,suggesting that model was successful.The activation of microglia and astrocytes in the spinal cord in BICP group were higher than that of the sham group(P<0.05),the activation of microglia and astrocytes of the Cinobufagin group was significantly lower than that of the CIBP group(P<0.05).The concentration of TNF-αand IL-1βin peripheral blood and spinal cord in BICP group were increased when compared with the sham rats,but there were low on the Cionbufagin group when compared with the BICP group(P<0.05).CONCLUSION:The study showed that Cionbufagin has an analgesic effect on CIBP rats,the analgesic effects were mainly mediated through inhibition of activation of microglia and astrocytes in the spinal cord.