芹菜素对小鼠RAW264.7巨噬细胞极化和炎症反应的作用及其机制

doi:10.13481/j.1671-587X.20230301

摘要/Abstract

摘要：

目的探讨不同浓度芹菜素（API）对氧化低密度脂蛋白（ox-LDL）诱导的小鼠单核巨噬细胞RAW264.7炎症反应和极化的作用，并阐明其可能机制。方法将RAW264.7细胞分为RAW264.7组（不做任何处理）和RAW264.7+API组（2、4、8、16 和32 μmol·L^－1API）。药物处理细胞24 h后，CCK-8法检测各组细胞增殖率，选取API实验浓度。将RAW264.7细胞分为RAW264.7组（正常RAW264.7细胞）、RAW264.7+ox-LDL组（0.08 g·L^－1ox-LDL诱导24 h）、RAW264.7+ox-LDL+低剂量API组（2 μmol·L^－1API和0.08 g·L^－1ox-LDL诱导24 h）和RAW264.7+ox-LDL+高剂量API组（8 μmol·L^－1API和0.08 g·L^－1ox-LDL诱导24 h），药物处理细胞24 h，采用油红O染色观察各组RAW264.7细胞中泡沫细胞形态表现，酶联免疫吸附测定（ELISA）法检测各组细胞培养上清液中肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）、白细胞介素4（IL-4）和白细胞介素10（IL-10）水平，Western blotting法检测各组细胞中核转录因子κB（NF-κB）p65、磷酸化NF-κB（p-NF-κB）p65、诱导型一氧化氮合成酶（iNOS）、信号转导与转录激活因子6（STAT6）、磷酸化STAT6（p-STAT6）和精氨酸酶1（Arg-1）蛋白表达水平。结果 CCK-8法检测，随着API的浓度增加，RAW264.7细胞增殖率降低（P<0.05），选择无毒的低浓度（2 μmol·L^－1）和高浓度（16 μmol·L^－1）API作为后续实验API浓度。油红O染色，RAW264.7组极少数RAW264.7细胞被油红O染色；RAW264.7+ox-LDL组较多细胞被染成暗红色，胞内脂质明显增加，表明成功建立了RAW264.7源性泡沫细胞；RAW264.7+ox-LDL+低剂量API组和RAW264.7+ox-LDL+高剂量API组少量细胞被油红O染色。ELISA法检测，与RAW264.7组比较，RAW264.7+ox-LDL组、RAW264.7+ox-LDL+低剂量API组和RAW264.7+ox-LDL+高剂量API组细胞培养上清液中TNF-α和IL-1β水平升高（P<0.05），IL-4和IL-10水平降低（P<0.05）；与RAW264.7+ox-LDL组比较，RAW264.7+ox-LDL+低剂量API组和RAW264.7+ox-LDL+高剂量API组RAW264.7细胞培养上清液中TNF-α和IL-1β水平降低（P<0.05），IL-4和IL-10水平升高（P<0.05）。Western blotting法检测，与RAW264.7组比较，RAW264.7+ox-LDL组、RAW264.7+ox-LDL+低剂量API组和RAW264.7+ox-LDL+高剂量API组RAW264.7细胞中iNOS和p-NF-κB p65蛋白表达水平升高（P<0.05），Arg-1和p-STAT6蛋白表达水平降低（P<0.05）；与RAW264.7+ox-LDL组比较，RAW264.7+ox-LDL+低剂量API组和RAW264.7+ox-LDL+高剂量API组RAW264.7细胞中iNOS和p-NF-κB p65蛋白表达水平降低（P<0.05），Arg-1和p-STAT6蛋白表达水平升高（P<0.05）；与RAW264.7+ox-LDL+低剂量API组比较，RAW264.7+ox-LDL+高剂量API组RAW264.7细胞中iNOS和p-NF-κB p65蛋白表达水平降低（P<0.05），Arg-1和p-STAT6蛋白表达水平升高（P<0.05）。结论 API能抑制ox-LDL诱导的RAW264.7细胞中泡沫细胞形成和调控巨噬细胞向M2极化，改善炎症反应，其机制可能与NF-κB和STAT6通路有关。

关键词: 芹菜素, 动脉粥样硬化, 核因子κB, 信号转导与转录激活因子6, 巨噬细胞极化

Abstract:

Objective To discuss the effects of different concentrations of apigenin （API） on the inflammatory and polarization response of the mouse mononuclear macrophage cells RAW264.7 induced by oxidized low-density lipoprotein （ox-LDL），and to clarify their possible mechanisms. Methods The RAW264.7 cells were divided into RAW264.7 group （without any treatment）and RAW264.7+API group （2， 4， 8， 16 and 32 μmol·L^－1 API）.After the cells were treated for 24 h.CCK-8 assay was used to detect the proliferation rates of the cells in various groups.The experimental concentration of API was selected.The RAW264.7 cells were divided into RAW264.7 group（normal RAW264.7 cells）， RAW264.7+ox-LDL group（induced with 0.08 g·L^－1 ox-LDL for 24 h）， RAW264.7+ox-LDL+low dose of API group（induced with 2 μmol·L^－1API and 0.08 g·L^－1 ox-LDL for 24 h） and RAW264.7+ox-LDL+high dose of API group. Oil red O staining was used to observe the morphology of the foam cells in various groups. The levels of tumor necrosis factor-α（TNF-α），interleukin-1β（IL-1β）， interleukin-4（IL-4）， and interleukin-10（IL-10） in the cell culture supernant were detected by enzyme-linked immunosorbent assay（ELISA） method. The expression levels of neclear factor-κB（NF-κB） p65， phosphorylated NF-κB（p-NF-κB） p65，inducible nitric oxide synthase（iNOS）， signal transducer and activator of transcription 6（STAT6）， phosphorylated STAT-6（p-STAT6）， and arginase-1（Arg-1） in the cells in various groups were detected by Western blotting method. Results The results of CCK-8 assay showed that the proliferation rates of the cells were significantly decreased with the increasing of the concentrations of API （P<0.05）.The non-toxic low concentration （2 μmol·L^－1） and high concentration （8 μmol·L^－1） of API were selected to use in the follow-up experiment.The Oil red O staining results showed that few RAW264.7 cells were stained with Oil red O； a lot of RAW264.7 cells in RAW264.7+ox-LDL group were stained as dark red，and the lipids in the cells were increased，indicating the foam cell model was successfully established； a little of RAW264.7 cells in RAW264.7+ox-LDL+low dose of API group and RAW264.7+ox-LDL+high dose of API group were stained with Oil red O.The ELISA results showed that compared with RAW264.7 group， the levels of TNF-α and IL-1β in the cell culture supernatant in RAW264.7+ox-LDL group were significantly increased （P<0.05）， while the levels of IL-4 and IL-10 were significantly decreased （P<0.05）； compared with RAW264.7+ox-LDL group， the levels of TNF-α and IL-1β in the cell culture supernatant in RAW264.7+ox-LDL+low dose of API group，the levels of IL-4 and IL-10 were significantly increased （P<0.05）.The Western blotting results showed that compared with RAW264.7 group， the expression levels of iNOS and p-NF-κB p65 proteins in the RAW264.7 cells in RAW264.7+ox-LDL group were significantly increased （P<0.05）， and the expression levels of Arg-1 and p-STAT6 proteins were significantly decreased （P<0.05）； compared with RAW264.7+ox-LDL group， the expression levels of iNOS and p-NF-κB p65 proteins in the RAW264.7 cells in RAW264.7+ox-LDL+low dose of API group and RAW264.7+ox-LDL+high dose of API group were decreased（P<0.05），and the expression levels of Arg-1 and p-STAT6 proteins were increased（P<0.05）；compared with RAW264.7+ox-LDL+low dose of API group，the expression levels of iNOS and p-NF-κB p65 proteins in the RAW264.7 cells in RAW264.7+ox-LDL+high dose of API group were decreased（P<0.05），and the expression levels of Arg-1 and p-STAT6 proteins were increased（P<0.05）. Conclusion API can inhibit the foam cell formation of RAW264.7 cells induced by ox-LDL and improve the inflammatory response by regulating the polarization from macrophages into M2， and their mechanisms may be related to NF-κB and STAT6 pathways.

Key words: Apigenin, Atherosclerosis, Nuclear factor-κB, Signal transducer and activator of transcription 6, Macrophage polarization

中图分类号:

R543.5

李海涛, 李沁, 蔡飞, 胡国富, 滕云飞. 芹菜素对小鼠RAW264.7巨噬细胞极化和炎症反应的作用及其机制[J]. 吉林大学学报(医学版), 2023, 49(3): 549-556.

Haitao LI, Qin LI, Fei CAI, Guofu HU, Yunfei TENG. Effect of apigenin on polarization and inflammation of mouse RAW264.7 macrophages and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2023, 49(3): 549-556.

图/表 5

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