蛇床子素对骨癌痛大鼠的镇痛作用及对TLR4/NF-κB信号通路的影响

Analgesic Effect of Osthole on Bone Cancer Pain Rats and Its Effect on TLR4/NF-κB Signaling Pathway

目的:探讨蛇床子素对骨癌痛大鼠的镇痛作用及其可能的作用机制。方法:将40只SPF级SD大鼠随机选取8只作为假手术组,其余32只大鼠通过左后肢胫骨注射Walker256乳腺癌细胞悬液的方法建立骨癌痛模型。将造模大鼠随机分为模型组、蛇床子素低剂量组、蛇床子素高剂量组及盐酸曲马多组,每组8只。术后第7天开始腹腔注射相应药物,1次/d,连续7 d。X摄片观测各组大鼠胫骨病理学改变;观察不同时间点各组大鼠机械痛阈值变化;免疫荧光染色观察各组大鼠脊髓背角处小胶质细胞标记物Iba-1的活化情况;酶联免疫吸附法(ELISA)检测各组大鼠脊髓中肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-6(IL-6)的含量;Western blotting检测各组大鼠脊髓中TLR4、NF-κB及p-NF-κB蛋白表达。结果:与假手术组比较,模型组大鼠骨质损伤严重,机械痛阈值明显降低(P<0.01),脊髓背角处小胶质细胞活化数量增加,炎症因子TNF-α、IL-1β及IL-6的释放增加(P<0.01),TLR4及p-NF-κB蛋白相对表达量升高(P<0.01);与模型组比较,蛇床子素可改善骨质损伤,升高骨癌痛大鼠机械痛阈值(P<0.05或P<0.01),抑制骨癌痛大鼠脊髓背角处小胶质细胞的活化(P<0.05或P<0.01),并抑制炎症因子TNF-α、IL-1β及IL-6的释放;蛇床子素还能抑制大鼠脊髓中TLR4及p-NF-κB蛋白表达(P<0.05或P<0.01)。结论:蛇床子素可能通过抑制炎症因子释放及脊髓小胶质细胞活化缓解乳腺癌转型诱导的骨癌痛,其作用机制可能与抑制TLR4/NF-κB信号通路有关。

Objective: To investigate the analgesic effect of osthole(Ost) on bone cancer pain rats and its possible mechanism. Methods: A total of 40 SPF SD rats were randomly selected from 8 rats as sham group.The remaining 32 rats were injected with Walker256 breast cancer cell suspension from the left hind leg to establish a bone cancer pain model. The model rats were randomly divided into model group, Ost low-dose group, Ost high-dose group and tramadol hydrochloride group, with 8 rats in each group. The corresponding drugs were injected intraperitoneally on the 7;day after operation, once a day for 7 days. X-ray was used to observe the pathological changes of tibia in each group, the changes of mechanical pain threshold were observed at different time points, the activation of Iba-1 was observed by immunofluorescence staining, the contents of TNF-α, IL-1β and IL-6 were detected by enzyme-linked immunosorbent assay(ELISA) and the protein expression of TLR4, NF-κB and p-NF-κB in the spinal cord of each group was detected by Western blotting.Results: Compared with the sham group, the bone injury in the model group was serious, the mechanical pain threshold decreased significantly(P<0.01), the activated number of microglia in the spinal dorsal horn increased,the release of inflammatory factors TNF-α, IL-1βand IL-6 increased(P <0.01), and the relative expression of TLR4 and p-NF-κB protein increased(P<0.01). Compared with the model group, Ost could improve bone injury,increase the mechanical pain threshold of rats with bone cancer pain(P<0.05 or P<0.01), inhibit the activation of microglia in the spinal dorsal horn of rats with bone cancer pain(P<0.05 or P<0.01), and inhibit the release of inflammatory factors TNF-α, IL-1β and IL-6. Ost could also inhibit the expression of TLR4 and p-NF-κB protein in rat spinal cord(P<0.05 or P<0.01). Conclusion: Ost may alleviate the bone cancer pain induced by breast cancer transformation by inhibiting the release of inflammatory factors and activation of spinal microglia.The mechanism may be related to inhibition of TLR4/NF-κB signaling pathway.