基于PPARγ信号通路研究香烟烟雾提取物干预不同时间下对肺泡巨噬细胞胞葬及吞噬功能影响的分子机制  

Mechanism of cigarette smoke extract stimulate alveolar macrophages at different times influencing efferocytosis and phagocytosis function based on PPARγsignaling pathway

作　　者：周哲旭 陈星[1] 王省 尚艺婉 刘洋[1] 陈玉龙[1] ZHOU Zhexu;CHEN Xing;WANG Xing;SHANG Yiwan;LIU Yang;CHEN Yulong(Henan Key Laboratory of Traditional Chinese Medicine(TCM)Syndrome and Prescription in Signaling,Henan International Joint Laboratory of TCM Syndrome and Prescription in Signaling,Academy of Chinese Medical Sciences,Henan University of Chinese Medicine,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学中医药科学研究院,河南省中医方证信号传导重点实验室,河南省中医方证信号传导国际联合重点实验室,郑州450046

出　　处：《中国免疫学杂志》2024年第7期1364-1372,共9页Chinese Journal of Immunology

基　　金：国家自然科学基金(81873285);河南省特色骨干学科中医学学科建设项目(重点项目)(STG-ZYXKY-2020022)。

摘　　要：目的:观察香烟烟雾提取物(CSE)干预不同时间下肺泡巨噬细胞胞葬及吞噬功能的变化,探讨其对肺部疾病炎症反应的影响及分子机制。方法:大鼠肺泡巨噬细胞NR8383采用10%CSE分别干预6 h、12 h、24 h、48 h,分为6 h空白对照组、6 h-10%CSE组、12 h空白对照组、12 h-10%CSE组、24 h空白对照组、24 h-10%CSE组、48 h空白对照组、48 h-10%CSE组。10%CSE干预12 h后联用PPAR抑制剂/激动剂,分为PPAR抑制剂组、PPAR激动剂组。Alamar Blue法检测PPAR激动剂对NR8383细胞增殖及毒性作用;流式细胞术检测NR8383细胞胞葬、吞噬功能及M1、M2极化分型;酶联免疫吸附实验检测TNF-α、TGF-β1、MFG-E8含量;免疫印迹法检测PPARγ信号通路及下游分子CD36蛋白表达;qPCR检测PPARγ、CD36 mRNA的表达。结果:10%CSE干预6 h后,NR8383细胞吞噬及胞葬功能均有所升高,PPARγ表达下调,CD36 mRNA表达增加,TNF-α、TGF-β1、MFG-E8表达均升高,但无明显极化方向;干预12 h,NR8383细胞吞噬功能及胞葬率显著降低,PPARγ、CD36表达显著下调,TNF-α表达增强,TGF-β1、MFG-E8表达降低,向M1型巨噬细胞极化;干预24 h后,NR8383细胞胞葬率降低,但吞噬大肠杆菌的功能增强,PPARγ表达下调,CD36蛋白表达降低,TNF-α表达降低但差异无统计学意义,TGF-β1、MFG-E8表达仍处于降低状态,有明显的M1型极化倾向;48 h后NR8383细胞胞葬率仍旧降低,但吞噬能力显著提升,PPARγ表达显著降低,CD36表达显著增加,TNF-α表达降低,TGF-β1、MFG-E8表达增加,巨噬细胞向M1、M2方向极化均增加。选择10%CSE干预12 h联合PPAR激动剂、抑制剂后发现,PPAR激动剂增强NR8383细胞胞葬及吞噬能力,上调PPARγ、CD36表达,抑制炎症因子TNF-α表达,促进抑炎因子TGF-β1、胞葬辅助因子MFG-E8表达。结论:随着CSE干预时间的变化,肺泡巨噬细胞从早期炎症反应的活化状态,逐渐进展为慢性炎症反应状态,进而导致肺泡巨噬细胞胞葬及吞噬功�Objective:To observe the stimulation of cigarette smoke extract(CSE)at different times influencing the alveolar macrophage efferocytosis and phagocytosis function,and to explore the effect of CSE on inflammatory response in lung diseases and molecular mechanism.Methods:Rat alveolar macrophages NR8383 were intervened with 10%CSE for 6 h,12 h,24 h,48 h,divided into 6 h blank control group,6 h-10%CSE group,12 h blank control group,12 h-10%CSE group,24 h blank control group,24 h-10%CSE group,48 h blank control group,48 h-10%CSE group.10%CSE intervention 12 h in combination with PPAR inhibitor/agonist,divided into PPAR inhibitor group and PPAR agonist group.Alamar Blue colorimetry was used to detect the proliferation and toxicity of PPAR agonist on NR8383 cells.Flow cytometry was used to detect the efferocytosis and phagocytosis of NR8383 cells,and M1 and M2 polarizations.The contents of TNF-α,TGF-β1 and MFG-E8 were detected by enzyme-linked immunosorbent assay.Western blot was used to detect the expressions of PPARγand CD36 protein.mRNA expressions of PPARγand CD36 were detected by qPCR.Results:After 6 hours of 10%CSE stimulation,the phagocytosis and efferocytosis of NR8383 cells were increased,the expression of PPARγwas down-regulated,the expression of CD36 mRNA was increased,and the expressions of TNF-α,TGF-β1 and MFG-E8 were increased,but there was no obvious polarization direction.After 12 hours CSE stimulation,the efferocytosis and phagocytosis functione of NR8383 cells were significantly decreased,the expressions of PPARγand CD36 were significantly down-regulated,the expression of TNF-αwas increased,the expressions of TGF-β1 and MFG-E8 were decreased,and the cells were polarized to M1-type macrophages.After 24 hours of intervention,the efferocytosis rate of NR8383 cells were decreased,but the phagocytosis function of E.coli was increased,the expression of PPARγwas down-regulated,the expressions of CD36 protein was decreased,the expression of TNF-αwas decreased,while there was no statistical differen

关 键 词：香烟烟雾提取物 肺泡巨噬细胞 胞葬功能 吞噬功能 过氧化物酶体增殖物激活受体Γ 

分 类 号：R563[医药卫生—呼吸系统]