基于补体D因子介导的自噬探讨平喘颗粒抑制巨噬细胞M2型极化的机制  

Mechanism of Pingchuan Granules inhibiting M2 polarization of macrophages through complement D factor mediated autophagy

作　　者：王瑶[1,2] 田春燕[2] 孙丽丽[1] 刘琪 袁星星 李竹英[2] WANG Yao;TIAN Chun-yan;SUN Li-li;LIU Qi;YUAN Xing-xing;LI Zhu-ying(Heilongjiang University of Chinese Medicine,Harbin 150040,China;First Affiliated Hospital,Heilongjiang University of Chinese Medicine,Harbin 150040,China;Heilongjiang Academy of Traditional Chinese Medicine,Harbin 150006,China)

机构地区：[1]黑龙江中医药大学,哈尔滨150040 [2]黑龙江中医药大学附属第一医院,哈尔滨150040 [3]黑龙江省中医药科学院消化科,哈尔滨150006

出　　处：《中华中医药杂志》2022年第5期2488-2494,共7页China Journal of Traditional Chinese Medicine and Pharmacy

基　　金：国家自然科学基金面上项目(No.82074365);国家自然科学基金青年科学基金项目(No.82104761);黑龙江省自然科学基金面上项目(No.H2017068);哈尔滨市应用技术研究与开发项目(No.2017RAXXJ053);黑龙江中医药大学科研基金(No.201726);黑龙江省中医药科研项目(No.ZHY2020-041);黑龙江省博士后面上项目(No.LBH-Z21218)。

摘　　要：目的:观察平喘颗粒对巨噬细胞补体D因子(CF D)表达水平及其介导的自噬的影响,从而明确其抑制巨噬细胞M2型极化的作用机制。方法:采用白介素-4诱导U937细胞构建M2型巨噬细胞模型并给予平喘颗粒含药血清进行干预,分别以Western blot、RT-qPCR及免疫荧光法检测CFD的表达水平。以慢病毒转染CFD-shRNA及自噬诱导剂雷帕霉素作为对照,分别采用Western blot、RT-qPCR和流式细胞术巨噬细胞中M2型极化标记分子、自噬相关蛋白及CF D的表达水平,透射电镜观察巨噬细胞自噬小体的数量。结果:与模型组比较,平喘颗粒组显著降低U937细胞中CFD、Arg-1、FI ZZ1、YM1的表达水平及CD206阳性细胞百分比(P<0.01)。而在CFD-shRNA稳转U937细胞中加入CFD重组蛋白后,Arg-1、FIZZ1、YM1的表达水平及CD206阳性细胞数与模型组比较无显著性差异。与模型组比较,平喘颗粒组Beclin1与LC3Ⅱ/LC3Ⅰ显著降低(P<0.01),p62蛋白表达水平显著增加(P<0.01)。在CFD-shRNA稳转U937细胞中加入CF D重组蛋白后,自噬标记蛋白的表达水平与模型组比较无显著性差异。平喘颗粒+雷帕霉素组A rg-1、FIZZ1、YM1的表达水平及CD206阳性细胞数量显著高于平喘颗粒组(P<0.01)。结论:平喘颗粒可通过抑制CFD的表达水平进而抑制巨噬细胞的自噬水平,从而抑制巨噬细胞的M2型极化。Objective:To observe the effects of Pingchuan Granules on the expression level of complement D factor(CFD)and autophagy mediated by macrophages,to clarify its role in inhibiting M2 polarization of macrophages.Methods:Interleukin-4 was used to induce U937 cells to construct M2 macrophage model,and Pingchuan Granules containing serum was given to intervene.The expression levels of CFD were detected by Western blot,RT-qPCR and immunofluorescence.At the same time,using lentivirus transfected CFD-shRNA and autophagy inducer Rapamycin as control,the expression levels of M2 polarization marker molecules,autophagy related proteins and CFD in macrophages were measured by Western blot,RT-qPCR and flow cytometry,respectively.The number of autophagy bodies in macrophages was observed by transmission electron microscope.Results:Compared with the model group,Pingchuan Granules group significantly reduced the expression levels of CFD,Arg-1,FIZZ1 and YM1 and the percentage of CD206 positive cells in U937 cells(P<0.01).After adding CFD recombinant protein to CFD-shRNA stable U937 cells,the expression levels of Arg-1,FIZZ1 and YM1 and the number of CD206 positive cells had no significant change compared with the model group.Compared with the model group,the Beclin1 and LC3Ⅱ/LC3Ⅰin Pingchuan Granules group decreased significantly(P<0.01),and the expression level of p62 protein increased significantly(P<0.01).After adding CFD recombinant protein to CFD shRNA stable U937 cells,there was no significant change in the expression level of autophagy marker protein compared with the model group.The expression levels of Arg-1,FIZZ1 and YM1 and the number of CD206 positive cells in Pingchuan Granules+rapamycin group were significantly higher than those in Pingchuan Granules group(P<0.01).Conclusion:Pingchuan Granules can inhibit the autophagy level of macrophages by inhibiting the expression level of CFD,so as to inhibit the M2 polarization of macrophages.

关 键 词：平喘颗粒 巨噬细胞 细胞自噬 补体D因子 M2型极化 雷帕霉素 慢病毒 U937细胞 

分 类 号：R285[医药卫生—中药学]