N-Ac-PGP通过激活TLR4诱导巨噬细胞的M1极化并提高慢性阻塞性肺疾病炎症水平  

Activation of TLR4 by N-Ac-PGP induces M1 macrophage polarization to promote inflammation in chronic obstructive pulmonary disease

作　　者：刘倩[1] 杨姣[1] 石西南 徐悦[1] 杜晓华[1] LIU Qian;YANG Jiao;SHI Xinan;XU Yue;Du Xiaohua(Department of Respiratory and Critical Care Medicine,The First Affiliated Hospital of Kunming Medical University,Kunming 650032,China;Department of Pathology,Yunnan University of Chinese Medicine,Kunming 650500,China)

机构地区：[1]昆明医科大学第一附属医院,云南昆明650032 [2]云南中医药大学,云南昆明650500

出　　处：《中国病理生理杂志》2023年第11期2060-2066,共7页Chinese Journal of Pathophysiology

基　　金：云南省教育厅科学研究基金项目(No.2019J1233);云南省科技厅科技计划项目(No.202101AY070001-089);云南省卫生高层次人才培养计划(No.H-2018095)。

摘　　要：目的:探究N-乙酰化脯氨酸-甘氨酸-脯氨酸(N-acetylated proline-glycine-proline,N-Ac-PGP)通过Toll样受体4(Toll-like receptor 4,TLR4)诱导巨噬细胞M1极化对慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)炎症反应的影响。方法:检测COPD患者痰液中N-Ac-PGP和M1型巨噬细胞炎性细胞因子的表达。体外培养人单核细胞白血病细胞THP-1,使用佛波脂(phorbol 12-myristate 13-acetate,PMA)诱导使之分化为M0型巨噬细胞,然后使用脂多糖(lipopolysaccharide,LPS)和N-Ac-PGP诱导M0型巨噬细胞极化为M1型。采用RTqPCR和Western blot法分别检测巨噬细胞表面标志物CD11b、CD45、CD86和诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的mRNA和蛋白表达水平。使用ELISA检测M1型巨噬细胞炎性细胞因子肿瘤坏死因子α(tu-mor necrosis factor-α,TNF-α)、白细胞介素1β(interleukin-1β,IL-1β)、IL-6和IL-23的水平。最后抑制TLR4通路,再次检测巨噬细胞的极化情况。结果:N-Ac-PGP与M1型巨噬细胞炎性细胞因子表达量呈显著正相关。PMA处理THP-1细胞成功诱导分化为M0型巨噬细胞。LPS和N-Ac-PGP处理后M1型巨噬细胞的标志蛋白表达显著增加(P<0.05),证明经过LPS和N-Ac-PGP处理的M0型巨噬细胞向M1型极化。同时,炎性细胞因子TNF-α、IL-1β、IL-6和IL-23的表达上调(P<0.05)。进一步的实验中,TLR4抑制剂与N-Ac-PGP联用逆转了N-Ac-PGP对M0型巨噬细胞向M1型极化的促进作用,也显著降低了炎性细胞因子的表达水平(P<0.05)。结论:N-Ac-PGP通过激活TLR4诱导的巨噬细胞M1极化促进COPD炎症反应。AIM:To investigate the effect of N-acetylated proline-glycine-proline(N-Ac-PGP)-induced M1 macrophage polarization by Toll-like receptor 4(TLR4)on the inflammatory response in chronic obstructive pulmonary disease(COPD).METHODS:The expression levels of N-Ac-PGP and M1 macrophage inflammatory cytokines in the sputum of COPD patients were clinically tested.Human monocytic leukemia THP-1 cells were cultured in vitro,and were induced to differentiate into M0 macrophages using phorbol 12-myristate 13-acetate(PMA).Subsequently,the M0 macrophages were induced to polarize to M1-type by lipopolysaccharide(LPS)and N-Ac-PGP.RT-qPCR and Western blot were performed to detect the mRNA and protein expression levels of the macrophage surface markers CD11b,CD45,CD86,and inducible nitric oxide synthase(iNOS),respectively.The levels of M1 macrophage inflammatory cytokines tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-6 and IL-23 were detected using ELISA.Finally,the TLR4 path‑way was inhibited and the macrophage polarization was detected.RESULTS:The level of N-Ac-PGP was positively corre-lated with the expression of inflammatory cytokines in M1 macrophages.The THP-1 cells were successfully induced to dif-ferentiate into M0 macrophages with PMA treatment.The expression of marker proteins in M1 macrophages increased after LPS and N-Ac-PGP treatment(P<0.05),which demonstrated that M0 macrophages were polarized to M1-type after LPS and N-Ac-PGP treatment.Meanwhile,the expression levels of inflammatory factors TNF-α,IL-1β,IL-6 and IL-23 were up-regulated(P<0.05).In further experiments,the combination of TLR4 inhibitor and N-Ac-PGP reversed the promoting effect of N-Ac-PGP on the polarization of M0 macrophages to M1-type,and also significantly reduced the expression levels of inflammatory cytokines(P<0.05).CONCLUSION:N-Ac-PGP promotes the inflammatory response in COPD by acti-vating TLR4-induced M1 macrophage polarization.

关 键 词：慢性阻塞性肺疾病 N-乙酰化脯氨酸-甘氨酸-脯氨酸 TOLL样受体4 巨噬细胞极化 炎症 

分 类 号：R563[医药卫生—呼吸系统] R363.2[医药卫生—内科学]