核心岩藻糖基化修饰调控NLRP3介导骨癌性疼痛的作用机制

Core Rockweed Glycosylation Modifications Regulate the Mechanism of Action of Inflammatory Vesicles 3 Mediating Bone Cancer Pain

目的研究核心岩藻糖基化(core fucosylation,CF)修饰对骨癌性疼痛的作用,并探究其作用机制。方法收集我院骨癌性疼痛患者30例,随机选取一半患者使用传统镇痛类药物吗啡治疗,设为吗啡组;另一半患者设为对照组。采用数字疼痛评分法(numerical rating scales,NRS)对所有患者进行疼痛评分;蛋白免疫印迹及ELISA实验观察患者血清及骨髓上清液中NLRP3、IL-1β、IL-18的表达情况;免疫沉淀观察NLRP3及CF修饰的变化。构建乳腺癌致骨癌性疼痛大鼠模型,将40只健康成年SD大鼠分为对照组、模型组、FUT8shRNA组、吗啡组。免疫荧光检测脊髓组织中CF修饰的变化特征;合成腺病毒包装的FUT8shRNA抑制大鼠体内CF修饰后检测脊髓组织中NLRP3、TNF-α、IL-1β、IL-18的表达变化;利用VonFrey纤维丝测痛仪测量后爪对VonFrey纤维丝刺激的触觉异常性疼痛敏感性,通过缩爪机械阈值(PWT)和自由行走疼痛评分观察骨癌性疼痛对大鼠疼痛行为的影响,比较两种干预方法对NLRP3、TNF-α、IL-1β、IL-18、p38MAPK、PI3K、Akt等蛋白表达变化的影响。结果与对照组相比,吗啡组疼痛强度明显降低、持续时间变短(P<0.05);吗啡组血清及骨髓上清液中NLRP3、IL-1β、IL-18表达明显下调(P<0.05);免疫沉淀实验结果显示,吗啡组NLRP3及CF修饰的水平明显下降(P<0.05)。在体内实验中,与对照组相比,模型组大鼠CF修饰表达明显升高(P<0.05);LCA、TNF-α、IL-1β、IL-18的水平显著上升(P<0.01);大鼠机械性触诱发痛和热痛觉过敏明显加重(P<0.05);NLRP3、TNF-α、IL-1β、IL-18、p38MAPK、PI3K、Akt表达显著上调(P<0.05);与模型组相比,FUT8shRNA组和吗啡组大鼠CF修饰表达明显降低(P<0.05);NLRP3、TNF-α、IL-1β、IL-18的水平显著下降(P<0.01);大鼠机械性触诱发痛和热痛觉过敏明显缓解(P<0.05);NLRP3、TNF-α、IL-1β、IL-18、p38MAPK、PI3K、Akt表达显著下调(P<0.05)。结论抑制CF修饰可对骨癌性疼痛发挥保护作用,其作用机制可能与调控p38MAPK/PI3K/Akt/NLRP3信号通路有关。

Objective To study the effect of core fucosylation(CF)modification on bone cancer pain and to investigate its mechanism of action.Methods Clinical samples of 30 patients with bone cancer pain in the oncology department of our hospital were collected.Half of the patients were randomly selected to be treated with the traditional analgesic drug morphine,and the other half were set as the morphine group and the other half as the control group.numerical rating scales(NRS)were used to score the pain of all patients.The expression of NLRP3,IL-1βand IL-18 in serum and bone marrow supernatant were observed by Western blot and ELISA.The changes of NLRP3 and CF modifications were observed by immunoprecipitation.The rat model of bone cancer induced by breast cancer was established,and 40 healthy adult SD rats were divided into control group,model group,FUT8shRNA group and morphine group.The changes of CF modification in spinal cord tissues were characterized by immunofluorescence;the expression changes of NLRP3,TNF-α,IL-1βand IL-18 in spinal cord tissues were detected after inhibition of CF modification in rats by synthetic adenovirus-packed FUT8shRNA;the sensitivity of hind paws to tactile abnormal pain stimulated by Von Frey fiber filaments was measured by using Von Frey fiber filaments meter.Pain behavior was expressed by the mechanical threshold of paw retraction(PWT)and the score of free walking pain to observe the effect of bone cancer pain on pain behavior in rats and to compare the effect of changes in the expression of NLRP3,TNF-α,IL-1β,IL-18,p38-MAPK,PI3K,Akt and other proteins in the two intervention methods.Results Compared with control group,pain intensity and duration in morphine group were significantly reduced(P<0.05).The expressions of NLRP3,IL-1βand IL-18 in serum and bone marrow supernatant of morphine group were significantly down-regulated(P<0.05).The results of immunoprecipitation experiment showed that NLRP3 and CF modification levels decreased significantly(P<0.05).In vivo experiment,compared with control group,CF modification expression was significantly increased in model group(P<0.05).The levels of LCA,TNF-α,IL-1βand IL-18 were significantly increased(P<0.01).Mechanical touch induced pain and thermal hyperalgesia were significantly aggravated in rats(P<0.05).The expressions of NLRP3,TNF-α,IL-1β,IL-18,p38-MAPK,PI3K and Akt were significantly up-regulated(P<0.05).Compared with model group,CF modification expression in FUT8shRNA group and morphine group was significantly decreased(P<0.05).The levels of NLRP3,TNF-α,IL-1βand IL-18 were significantly decreased(P<0.01).Mechanical touch induced pain and thermal hyperalgesia were significantly relieved in rats(P<0.05).The expressions of NLRP3,TNF-α,IL-1β,IL-18,p38-MAPK,PI3K and Akt were significantly down-regulated(P<0.05).Conclusion Inhibition of core rockweed glycosylation modification could exert protective effects on bone cancer pain,and its mechanism of action might be related to the regulation of p38-MAPK/PI3K/Akt/NLRP3 signaling pathway.