Saikosaponin A affects differentiation and function of M1/M2 macrophages by regulating NF-κB and STAT6 signaling pathways

To investigate the effect of Saikosaponin A (SSA) on the differentiation, apoptosis and function of mouse bone marrow derived macrophages (BMDM)-derived M1/M2 macrophages, and to explore its molecular mechanism.

BMDM was induced to differentiate into M1/M2 macrophages in vitro, and SSA was added at the same time: CCK-8 assay was used to detect the viability of BMDM and M1/M2 macrophages. The morphology of M1/M2 macrophages was observed by inverted fluorescence microscopy. Flow cytometry (FCM) and ELISA were used to detect the levels of surface markers and cytokines in M1/M2 macrophages. Real-time fluorescent quantitative PCR (qPCR) was used to detect the mRNA levels of IL-6, TNF-α and arginase-1(Arg-1). FCM was used to detect the phagocytosis of peritoneal macrophages to fluorescent microsphere particles. Immunofluorescence (IF) assay and Western blotting were used to detect the molecular mechanism of SSA regulating M1/M2 macrophages.

No significant effect on viability of M1/M2 macrophages was observed at SSA concentration of 10.0 mg/L, and obvious inhibition was seen at a concentration of 15.0 mg/L (P<0.01). Treatment of 10.0 mg/L SSA induced obvious morphologic changes in M1/M2 macrophages, with M1 macrophages in irregular shape, a few having pseudopods, and some showing unclear boundaries; while some M2 macrophages presenting round or irregular (P<0.001) with unclear boundaries. SSA treatment also resulted in significantly decreased proportion of M1/M2 macrophages after BMDM differentiation (P<0.05), with reduced contents of IL-6 and TNF-α secreted by M1 macrophages and their mRNA levels (P<0.05), but increased secretion of Arg-1 and mRNA levels by M2 macrophages (P<0.05). SSA treatment also inhibited the phagocytosis ability of peritoneal macrophages to fluorescent microsphere particles (P<0.01) in a concentration-dependent manner. SSA decreased the phosphorylation of NF-kappaB (p-NF-κB)(P<0.01) and enhanced the phosphorylation of signal transducer and activator of transcription 6(p-STAT6) in M2 macrophages (P<0.05).

SSA may affect the differentiation and function of M1/M2 macrophages by regulating NF-κB and STAT6 signaling pathways.