“易层”贴敷调控巨噬细胞M2型极化促进DRG神经元自噬缓解KOA冷痛敏的机制研究

Study on Mechanism of “Yi Ceng” Application Regulating Macrophage M2 Polarization and Promoting DRG Neurons Autophagy to Relieve KOA Cold Pain Sensitivity

目的研究“易层”贴敷(YC)改善膝骨关节炎(KOA)冷痛敏的作用及机制。方法用IL-4处理骨髓来源的巨噬细胞成功构建M2型巨噬细胞模型。将60只雄性C57BL/6J小鼠随机分为假手术(Sham)组、KOA组、M2型巨噬细胞鞘内注射(M2-II)组、M2型巨噬细胞鞘内注射+“易层”贴敷(M2-II+YC)组,每组15只。KOA模型通过内侧半月板失稳术(DMM)构建,造模成功后,M2-II组和M2-II+YC组予以鞘内注射M2型巨噬细胞,M2-II+YC组使用“易层”贴敷28天,给药完成后提取各组背根神经节(DRG)组织。采用冷板实验和丙酮低温实验进行冷痛行为学检测;免疫荧光法检测小鼠DRG组织中CGRP、PGP9.5、c-Fos和M2型巨噬细胞标志物荧光强度;Western blot和qPCR检测巨噬细胞CD206、Arg-1和DRG组织TRPA1、TRPM8、LC3B、Beclin1、P62蛋白和基因表达量。结果与KOA组比较,M2-II组和M2-II+YC组小鼠冷板实验和丙酮低温实验所测定的冷痛阈值均明显提高(P<0.05,P<0.01);DRG组织中疼痛标志物CGRP、PGP9.5和c-Fos平均荧光强度明显降低(P<0.001);冷痛伤害感受器TRPA1、TRPM8蛋白和基因表达明显降低(P<0.01,P<0.001);M2表型CD206和Arg-1蛋白荧光强度明显升高(P<0.01,P<0.001);DRG神经元自噬LC3B、Beclin1和P62蛋白和基因表达明显升高(P<0.05,P<0.01,P<0.001);与M2-II组比较,M2-II+YC组小鼠上述各指标均明显改善(P<0.05,P<0.01,P<0.001)。结论“易层”贴敷可能通过调控巨噬细胞M2型极化促进DRG神经元自噬,发挥对KOA小鼠冷痛敏的抑制作用。

Objective To study the effect and mechanism of “Yi Ceng” application on cold pain sensitivity of knee osteoarthritis(KOA).Methods The M2-type macrophage model was successfully established by treating bone marrow derived macrophages with IL-4. 60 male C57BL/6J mice were randomly divided into sham operation group, KOA group, intrathecal injection of M2-type macrophages(M2-II) group, intrathecal injection of M2-type macrophages +“Yi Ceng”(M2-II+YC) group, 15 mice in each group. KOA model was established by medial meniscal instability(DMM). After successful establishment of model, M2-II group and M2-II+YC group were injected intrathecally with M2-type macrophages. M2-II+YC group was applied with “Yi Ceng” application for 28 days. Dorsal root ganglia(DRG) tissues were extracted from each group after administration. Cold plate experiment and acetone hypothermia experiment were used to detect cold pain behavior. The fluorescence intensity of CGRP, PGP9.5, c-Fos and M2-type macrophage markers in DRG tissues of mice was detected by immunofluorescence method. Western blot and qPCR were used to detect protein and gene levels of CD206, Arg^(-1) in macrophages and TRPA1, TRPM8, LC3B, Beclin1, P62 in DRG tissues. Results Compared with the KOA group, the cold pain thresholds measured by cold plate test and acetone hypothermia test were significantly higher in both the M2-II group and the M2-II+YC group of mice(P<0.05, P<0.01);the average fluorescence intensities of the pain markers CGRP, PGP9.5, and c-Fos in the DRG tissues were significantly lower(P<0.001);and the cold pain injury receptor TRPA1, TRPM8 protein and gene expression was significantly lower(P<0.01, P<0.001);M2 phenotype CD206 and Arg^(-1) protein fluorescence intensity was significantly higher(P<0.01, P<0.001);DRG neuronal autophagy LC3B, Beclin1 and P62 protein and gene expression was significantly higher(P<0.05, P<0.01, P< 0.001);compared with the M2-II group, all of the above indexes were significantly improved in the M2-II+YC group of mice(P<0.05, P<0.01, P<0.001). Conclusion “Yi Ceng” application may exert an inhibitory effect on cold pain sensitivity in KOA mice by regulating macrophage M2-type polarisation to promote autophagy in DRG neurons.