NLRP3信号通路相关因子参与夹脊电针改善脊髓损伤大鼠的作用机制研究

Action Mechanism of NLRP3 Signaling Pathway Related Factors Involving in Jiaji Electro-Acupuncture Improving SCI in Rats

目的:探讨夹脊电针对急性脊髓损伤大鼠NLRP3信号通路相关因子介导炎症反应的调控作用及其机制,为其临床应用提供理论依据。方法:选用120只大鼠,随机均分为假手术组(Sham组)、模型组(Model组)、夹脊电针组(EA组)、P2X7干扰组(P2X7R siRNA)和干扰对照组(Control siRNA),每组分1 d、3 d、7 d和21 d共4个时间点。BBB评分对大鼠肢体运动功能评定;免疫组化法检测大鼠脊髓组织中NLRP3、ASC和IL-18表达变化;免疫荧光双标法检测大鼠脊髓组织中NLRP3在小胶质细胞中的表达变化。结果:①BBB评分显示,与Sham组比较,各时间点Model组大鼠BBB评分显著降低,差异具有统计学意义(P<0.05);与Model组大鼠比较,术后3 d、7 d和21 d时间点EA组和P2X7R siRNA组大鼠BBB评分升高,差异有统计学意义(P<0.05)。②免疫组化显示:与Sham组比较,术后1 d、3 d、7 d和21 d时间点Model组大鼠脊髓组织NLRP3、ASC和IL-18增加,差异具有统计学意义(P<0.05);与P2X7R siRNA组比较,3 d、7 d和21 d时间点Control siRNA组NLRP3、ASC和IL-18表达仍较高,差异有统计学意义(P<0.05);与Model组比较,造模后3 d、7 d和21 d共3个时间点EA组大鼠脊髓组织NLRP3、ASC显著降低,差异具有统计学意义(P<0.05),造模后7 d、21 d两个时间点EA组大鼠脊髓组织IL-18显著降低,差异具有统计学意义(P<0.05);与Model组比较,造模后3 d、7 d和21 d共3个时间点P2X7R siRNA组大鼠脊髓组织NLRP3、ASC和IL-18显著降低,差异具有统计学意义(P<0.05)。③免疫荧光双标法结果显示:NLRP3与小胶质细胞标志物OX42存在共定位。结论:夹脊电针能够通过抑制脊髓损伤大鼠脊髓组织中NLRP3、ASC和IL-18表达,减轻炎性反应,促进运动功能恢复。

Objective:To investigate the regulation effect and mechanism of Jiaji electro-acupuncture on inflammatory response mediated by NLRP3 signaling pathway related factors in rats with acute spinal cord injury(SCI),so as to provide theoretical basis for its clinical application.Methods:120 rats were randomly divided into the sham operation group,the model group,the Jiaji electro-acupuncture(EA)group,the P2X7R siRNA group and the control siRNA group.Each group was subdivided into four time points of 1 d,3 d,7 d and 21 d after surgery.BBB score was used to evaluate the limb motor function of the rats.The expressions of NLRP3,ASC and IL-18 in rat spinal cord were detected by immunohistochemistry.The expression of NLRP3 in microglia cells in rat spinal cord was detected by immunofluorescence double-standard method.Results:In terms of the BBB scores,they were significantly lower in the model group than those in the sham group at each time point(P<0.05);the BBB scores were significantly higher in the EA group and the P2X7R siRNA group than those in the model group at the time points of 3 d,7 d and 21 d after surgery(P<0.05).Immunohistochemistry results showed that the expressions of NLRP3,ASC and IL-18 in spinal cord tissues were significantly increased in the model group than those in the sham group at the time points of 1 d,3 d,7 d and 21 d(P<0.05);compared to those in the P2X7R siRNA group,the expressions of NLRP3,ASC and IL-18 were still higher in the control siRNA group at the time points of 3 d,7 d and 21 d(P<0.05).Compared to those in the model group,the expressions of NLRP3 and ASC of spinal cord tissues were significantly decreased in the EA group at the time points of 3 d,7 d and 21 d(P<0.05);the expression of IL-18 was significantly reduced in the EA group at the time points of 7 d and 21 d(P<0.05).Compared to those in the model group,the expressions of NLRP3,ASC and IL-18 of spinal cord tissues were significantly reduced in the P2X7R siRNA group at the time points of 3 d,7 d and 21 d(P<0.05).The result of immunofluorescence double-standard method showed that NLRP3 and microglia marker OX42 shared the same location.Conclusion:By inhibiting the expressions of NLRP3,ASC and IL-18 in spinal cord tissues,Jiaji electro-acupuncture can reduce inflammatory response and promote the recovery of motor function of the rats with SCI.