目的 观察三七总皂苷(PNS)对巨噬细胞(Mφ)M1极化及炎症因子TNF-α的影响,并初步探究其效应与调节GAS6/MERTK通路表达的相关性。方法 分别建立THP-1来源Mφ单培养体系和THP-1来源Mφ及类风湿关节炎成纤维样滑膜细胞(HFLS-RA)的共培养体系;以LPS+IFN-γ诱导Mφ M1极化,并将细胞分为空白对照组,模型组,5、10、20 μg·mL-1 PNS组;采用CCK-8法筛选PNS最佳给药浓度;流式细胞术检测M1极化比例;实时定量聚合酶链反应(qPCR)检测TNF-α、MERTK受体、GAS6配体表达水平;ELISA法检测TNF-α、GAS6分泌水平;Western blot检测MERTK受体总蛋白及磷酸化水平。结果 在单培养条件下,5~20 μg·mL-1 PNS均可显著抑制M1极化,并上调MERTK表达;在共培养条件下,5~20 μg·mL-1 PNS同样可显著抑制M1极化并上调MERTK表达,与单培养体系不同的是5 μg·mL-1 PNS能显著上调HFLS-RA分泌GAS6水平且5、10 μg·mL-1 PNS处理后MERTK磷酸化水平被显著上调。结论 一定浓度的PNS能显著抑制LPS+IFN-γ诱导的Mφ M1极化及炎症因子TNF-α的表达水平,其作用机制可能与PNS调节GAS6/MERTK通路表达有关。
Abstract
OBJECTIVE To observe the effects of Panax notoginseng saponins (PNS) on the ratio of macrophage M1 polarization and inflammatory factor TNF-α, and preliminarily explore its relationship with regulating the expression of GAS6/MERK pathway. METHODS The single culture of THP-1-derived macrophages and Transwell co-culture system of THP-1-derived macrophages and HFLS-RA were established respectively. LPS+IFN-γ induced the polarization of macrophage M1. Then the cells were divided into control group and PNS intervention groups (5, 10, 20 μg·mL-1). After adding PNS for 48 h, the optimal concentration of PNS was selected by CCK-8 method. The ratio of macrophage M1 polarization was detected by flow cytometry. The expression levels of TNF-α, MERTK receptor and GAS6 ligand were detected by qPCR. The secretion levels of TNF-α and GAS6 were detected by ELISA. Total protein and phosphorylation of MERTK receptor were detected by Western blot. RESULTS In the single culture of THP-1-derived macrophages, 5-20 μg·mL-1 PNS could significantly inhibit macrophages M1 polarization and up-regulate MERTK expression. In Transwell co-culture system, 5-20 μg·mL-1 PNS could also significantly inhibit M1 polarization and up-regulate the expression of MERTK. Compared with the single culture system, 5 μg·mL-1 PNS could significantly up-regulate the secretion level of GAS6 in HFLS-RA, and the phosphorylation level of MERTK was significantly up-regulated after 5 and 10 μg·mL-1 PNS treatment. CONCLUSION Certain concentrations of PNS can significantly inhibit the ratio of macrophage M1 polarization induced by LPS+IFN-γ and the expression level of inflammatory factor TNF-α, and its mechanism may be related with regulating the expression of GAS6/MERK pathway.
关键词
三七总皂苷 /
巨噬细胞 /
成纤维样滑膜细胞 /
GAS6/MERTK通路
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Key words
Panax notoginseng saponin /
macrophage /
fibroblast-like synoviocyte /
GAS6/MERTK pathway
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中图分类号：
R285.5
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脚注
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基金
国家自然科学基金面上项目资助(82174306);国家自然科学基金青年科学基金项目资助(31900283)
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