三七总皂苷对巨噬细胞M1极化的影响及机制初探

Preliminary Exploration of the Effect of Panax notoginseng Saponins on Macrophage M1 Polarization and Its Mechanism

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MφM1极化,并将细胞分为空白对照组,模型组,5、10、20μg·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,5~20μg·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,5~20μg·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5μg·mL^(-1)PNS能显著上调HFLS-RA分泌GAS6水平且5、10μg·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MφM1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。

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-γinduced the polarization of macrophage M1.Then the cells were divided into control group and PNS intervention groups(5,10,20μg·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-αand 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,5-20μg·mL^(-1) PNS could significantly inhibit macrophages M1 polarization and up-regulate MERTK expression.In Transwell co-culture system,5-20μg·mL^(-1) PNS could also significantly inhibit M1 polarization and up-regulate the expression of MERTK.Compared with the single culture system,5μg·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 10μg·mL^(-1) PNS treatment.CONCLUSION Certain concentrations of PNS can significantly inhibit the ratio of macrophage M1 polarization induced by LPS+IFN-γand the expression level of inflammatory factor TNF-α,and its mechanism may be related with regulating the expression of GAS6/MERK pathway.