Stemona alkaloids suppress the positive feedback loop between M2 polarization and fibroblast differentiation by inhibiting JAK2/STAT3 pathway in fibroblasts and CXCR4/PI3K/AKT1 pathway in macrophages

Abstract

This study aimed to investigate the interaction between macrophages and fibroblasts in pulmonary fibrosis and the effects of total alkaloids of Stemona tuberosa (STA, 9 alkaloids with relative content of 91.2%) on them. The culture medium of LPS- or IL-4-induced macrophages was used as conditioned medium (CM) to co-culture with fibroblasts to study the effect of macrophages on the differentiation of fibroblasts. Similarly，the CM of TGF-β1-induced fibroblasts was co-culture with macrophages to study the effect of fibroblasts on the polarization of macrophages. The results showed that the TGF-β1 level in IL-4-induced (M2) rather than LPS-induced (M1) macrophages was significantly high (p < 0.001), and the SDF-1 level in TGF-β1-induced fibroblasts (MF) was significantly high (p < 0.001). The expressions of α-SMA and Col-1 in M2-CM-induced fibroblasts and Arg-1 and CXCR4 in MF-CM-induced macrophages were significantly increased (p < 0.01). STA effectively decreased the expressions of α-SMA (p < 0.05, 0.01 at 10, 100 μg/mL), Col-1 (p < 0.05, 0.05, 0.01 at 1, 10, 100 μg/mL), Arg-1 (p < 0.01 at 1, 10, 100 μg/mL) and CXCR4 (p < 0.01, 0.001 at 10, 100 μg/mL), which were consistent with the experimental results in vivo. These results suggested that there was a positive feedback loop between M2 polarization and fibroblast differentiation in pulmonary fibrosis. Further studies showed that the transcription of sdf-1 gene in MF was initiated by JAK2/STAT3 pathway and the M2 polarization was promoted by SDF-1/CXCR4/PI₃K/AKT1 pathway. STA blocked the feedback loop by suppressing JAK2/STAT3 pathway in fibroblasts and CXCR4-PI₃K/AKT1 pathway in macrophages.

Keywords: Fibroblast; Macrophage; Pulmonary fibrosis; SDF-1; Stemona tuberosa; TGF-β1.