Study on Mechanism of"Yi Ceng"Application Regulating Macrophage M2 Polarization and Promoting DRG Neurons Autophagy to Relieve KOA Cold Pain Sensitivity
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.West-ern 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 significant-ly 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.
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