关键词: 细粒棘球蚴抗原B, 巨噬细胞极化, STAT6, PPAR-γ

Abstract:

Objective To investigate the regulatory effect of native antigen B (nAgB) from Echinococcus granulosus on the polarization of RAW264.7 macrophages in vitro and its potential involvement in the Signal Transducer and Activator of Transcription 6 (STAT6)/peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling pathway. Methods nAgB was extracted and purified under sterile conditions. RAW264.7 cells were treated with nAgB at concentrations of 0, 1, 4, 16, and 64 μg/ml. Cell viability was assessed at 0, 6, 12, 24, and 48 hours using the CCK8 assay, and cell proliferation was evaluated at 24 hours via 5-ethynyl-2-deoxyuridine (EdU) staining. Cells were divided into 6 groups: control group, M1-polarized group, M2-polarized group, nAgB group, nAgB-treated M1-polarized group, and nAgB-treated M2-polarized group. The nAgB, nAgB-treated M1-polarized, and nAgB-treated M2-polarized groups were treated with 1 μg/ml nAgB. After 1 hour of intervention, the M1-polarized and nAgB-treated M1-polarized groups were stimulated with 500 ng/ml lipopolysaccharide (LPS) and 100 ng/ml interferon-γ (IFN-γ), while the M2-polarized and nAgB-treated M2-polarized groups were stimulated with 100 ng/ml interleukin-4 (IL-4) and 100 ng/ml IL-13. Cells and culture supernatants were collected from all groups. The relative mRNA transcription levels of macrophage polarization markers, STAT6, and PPAR-γ were detected by qPCR. The relative protein expression levels of relevant markers were measured by Western blotting, and the concentrations of related cytokines in the supernatant were determined by ELISA. Results CCK8 assay results at 24 hours showed that cell viability in the 1, 4, 16, and 64 μg/ml nAgB-treated groups (1.41 ± 0.09, 1.62 ± 0.08, 1.78 ± 0.04, 1.90 ± 0.04, respectively) was significantly higher than in the 0 μg/ml group (1.07 ± 0.05) (t = 8.67, 14.57, 27.43, 31.87; all P < 0.01). EdU assay results indicated that the cell proliferation capacity in the 1, 4, 16, and 64 μg/ml nAgB-treated groups (0.65 ± 0.01, 0.78 ± 0.02, 0.89 ± 0.02, 0.99 ± 0.02, respectively) was significantly higher than in the 0 μg/ml group (0.47 ± 0.02) (t = 16.21, 20.58, 33.47, 39.43; all P < 0.01). qPCR results showed that the relative mRNA levels of inducible nitric oxide synthase (iNOS), IL-6, and IL-1β in the nAgB-treated M1-polarized group (3.23 ± 0.92, 1.56 ± 0.58, 19.87 ± 0.35) were significantly lower than those in the M1-polarized group (10.62 ± 1.68, 3.97 ± 0.25, 24.40 ± 0.03) (t = 6.69, 6.62, 22.07; all P < 0.01). Conversely, the relative mRNA level of Arg-1 in the nAgB-treated M2-polarized group (29.30 ± 2.92) was significantly higher than that in the M2-polarized group (14.94 ± 0.77) (t = 8.23, P < 0.01). The relative mRNA levels of STAT6 and PPAR-γ in the nAgB group (59.12 ± 3.03, 7.82 ± 0.50) were significantly higher than those in the control group (26.38 ± 1.89, 3.71 ± 0.17) (t = 15.90, 13.40; both P < 0.01), in the nAgB-treated M1-polarized group (40.73 ± 2.91, 4.19 ± 0.88) were higher than in the M1-polarized group (17.93 ± 1.90, 1.76 ± 0.08) (t = 11.37, 4.75; both P < 0.01), and in the nAgB-treated M2-polarized group (140.50 ± 5.64, 11.67 ± 0.80) were higher than in the M2-polarized group (37.55 ± 5.92, 6.87 ± 0.28) (t = 21.82, 9.84; both P < 0.01). Western blotting results showed that the relative protein expression of iNOS in the nAgB-treated M1-polarized group (0.60 ± 0.02) was significantly lower than in the M1-polarized group (1.02 ± 0.03) (t = 21.86, P < 0.01), while the expression of CD206 in the nAgB-treated M2-polarized group (1.03 ± 0.04) was significantly higher than in the M2-polarized group (0.84 ± 0.02) (t = 7.78, P < 0.01). The relative protein levels of p-STAT6 and PPAR-γ in the nAgB group (0.76 ± 0.03, 0.77 ± 0.02) were significantly higher than in the control group (0.55 ± 0.05, 0.37 ± 0.00) (t = 6.11, P < 0.05; t = 40.16, P < 0.01), in the nAgB-treated M1-polarized group (0.60 ± 0.01, 0.42 ± 0.04) were higher than in the M1-polarized group (0.39 ± 0.05, 0.18 ± 0.01) (t = 6.64, 10.06; both P < 0.01), and in the nAgB-treated M2-polarized group (1.12 ± 0.11, 0.94 ± 0.02) were higher than in the M2-polarized group (0.86 ± 0.05, 0.66 ± 0.00) (t = 3.71, 28.18; both P < 0.01). ELISA results revealed that the secretion of TGF-β1 and IL-10 in the nAgB group [(70.27 ± 4.57), (167.00 ± 29.27) pg/ml] was significantly higher than in the control group [(29.87 ± 2.24), (50.17 ± 8.99) pg/ml] (t = 13.76, 6.61; both P < 0.01). In the nAgB-treated M1-polarized group, the levels of TNF-α and IL-1β [(523.20 ± 6.72), (387.80 ± 3.84) pg/ml] were significantly lower, while the levels of TGF-β1 and IL-10 [(47.15 ± 0.98), (137.30 ± 9.80) pg/ml] were significantly higher, compared to the M1-polarized group, [(995.70 ± 9.92), (680.90 ± 3.33) pg/ml] and [(18.05 ± 0.57), (21.66 ± 0.07) pg/ml], respectively (t = 68.32, 99.90, 44.41, 20.44; all P < 0.01). Similarly, nAgB-treated M2-polarized group,the levels of TNF-α and IL-1β [(398.50 ± 2.57), (85.18 ± 5.14) pg/ml] were significantly lower, while the levels of TGF-β1 and IL-10 [(293.20 ± 15.09), (341.20 ± 77.94) pg/ml] were significantly higher, compared to the M2-polarized group [(578.70 ± 12.36), (157.60 ± 14.25) pg/ml] and [(167.20 ± 20.34), (72.44 ± 5.28) pg/ml], respectively (t = 24.71, 8.28, 8.62, 5.82; all P < 0.01). Conclusion nAgB promotes macrophage polarization towards the M2 phenotype and suppresses the M1 response. This immunomodulatory effect is potentially mediated through the activation of the STAT6/PPAR-γ signaling pathway.

Key words: Echinococcus granulosus antigen B, Macrophage polarization, STAT6, PPAR-γ