To investigate the effect of Angelica polysaccharide (APS) on the differentiation and function of M2 macrophages and underlying molecular mechanism.

Mouse bone marrow derived macrophages (BMDM) and M2 macrophages were induced and treated with APS (0, 80, 160, 320 μg/mL); Mouse peritoneal macrophages were isolated and treated with APS (0, 160 μg/mL). Flow cytometry (FCM) was used to detect mannose receptor(MR), CD11b, F4/80, CD163, and ARG-1 expression levels, apoptosis, and phagocytic ability of M2 macrophages and peritoneal macrophages. Mice were randomly divided into APS gavage group and control group, APS was intragastrically administered to mice, and macrophage MR expression level in blood and spleen were detected by FCM. Fluorescence microscopy was used to observe the morphology of BMDM-differentiated M2 macrophages. RT-qPCR was employed to detect the mRNA expression levels of MR and ARG-1 in M2 macrophages. Immunofluorescence assay was performed to detect the expression of the proteins related to molecular mechanism of differentiation and function of M2 macrophages.

Compared with the 0 μg/mL APS group, the MR expression level in the M2 macrophages was decreased with the increase of APS concentration within a certain concentration range (80~320 μg/mL), and the MR expression level in peritoneal macrophages was also decreased in the 160 μg/mL APS treatment group (P < 0.01). The expression level of macrophage MR was also significantly decreased in peripheral blood and spleen in the APS gavage mice than the control group (P < 0.05). Compared with the 0 μg/mL APS group, the expression levels of CD11b, F4/80, and CD163 in the macrophages were increased in the 80 ~ 320 μg/mL APS treatment groups (P < 0.01). The morphology of macrophage had changed, from mostly spindle-shaped and pseudopodia to mostly round or irregular, and even a few cells with pseudopodia. APS induced apoptosis in M2 macrophages (P < 0.05). Compared with the 0 μg/mL APS group, M2 macrophages treated with 160 μg/mL APS had an increased ability to phagocytose fluorescent microspheres (P < 0.01), but the expression level of ARG-1 was decreased (P < 0.01). The mRNA expression of MR and ARG-1 in M2 macrophages was decreased (P < 0.05). The mean fluorescence intensity of phosphate acidified-signal transducers and activators of transcription 6(p-STAT6)-positive signals in M2 macrophages was significantly reduced in the 160 μg/mL APS-treated group (P < 0.05).

APS has bidirectional regulation on the differentiation and function of M2 macrophages, which may be associated with its downregulation of signal transducers and activators of transcription 6(STAT6) signaling pathway.

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.

To observe the immune regulation of embelin against systemic lupus erythematosus (SLE) in mice, so as to investigate its therapeutic effects on SLE and the possible mechanism.

The mouse model of SLE was established by intraperitoneal injection of 0.5 mL pristane, and urine protein level was detected to confirm the success of modeling. SLE mice then were randomly divided into control group and embelin group (n=5 for each group). The embelin group was given embelin 50 mg/kg by intragastric gavage, 100 μL/time, 3 times a week, while the control group was treated with 1% DMSO+PBS instead. After 1 month of administration, the lymph node size, spleen index and renal index of the mice were measured. The renal pathological changes were observed by HE staining; the serum concentrations of dsDNA, ssDNA and IgG were determined by ELISA; the Th cell subsets and B cell activation indexes were detected by flow cytometry.

As compared with the control group, the embelin group had significantly reduced lymph node size, spleen index, kidney index, and mean glomerular area (P<0.01), while elevated ratios of Th1/Th2 and Treg/Th17 (P<0.05). As for the activation markers, the expression levels of CD69, CD86, MHC-Ⅱ in B cells and CD69, CD154 in Th cells were all obviously decreased (P<0.05). In addition, embelin treatment remarkably lowered the concentrations of dsDNA, ssDNA and IgG in serum (P<0.01).

Embelin plays a therapeutic role in SLE mice by regulating the balance of Th cell subsets and inhibiting the activation of Th and B cells.