Through previous experiments, we found that LMWF effectively alleviated the symptoms of PF and significantly reduced collagen deposition in mice. However, the specific mechanism of LMWF on PF is not clear. Although it has been confirmed that LMWF could block the TGF-β/Smad signaling pathway and Nrf-2 to inhibit PF (Wu et al., 2021; Dong et al., 2022), the exact mechanism is still to explore.
In the early stage of PF, a variety of injuries and stimuli can recruit circulating monocytes to differentiate into macrophages after entering the diseased tissue (Cheng et al., 2021; Rao et al., 2021). Macrophage differentiation can form two subpopulations with different inflammatory states. Classically activated M1 macrophages and alternatively activated M2 macrophages are the two subgroups of polarization. They have different gene expression profiles and protein markers. In the early stage of tissue injury, i.e., in acute inflammatory stage, necrotic cells recruit proinflammatory monocytes to aggregate, differentiate into classically activated M1 macrophages, clear necrotic tissue, and express a large number of proinflammatory cytokines, such as TNF-α and IL-6. Subsequently, the proinflammatory signal is inhibited and the cells are more inclined to differentiate into alternatively activated M2 macrophages, expressing high levels of anti-inflammatory cytokines and growth factors, such as IL-10 and TGF-β. M2 macrophages promote the differentiation and proliferation of epithelial and endothelial cells, restore the tissue morphological structure, promote the phenotypic transformation of fibroblasts into myofibroblasts, synthesize and secrete extracellular matrix, and promote tissue repair (Wang et al., 2021); however, if M2 macrophages are continuously activated, they promote myofibroblasts to further secrete extracellular matrix and lead to fibrosis (Zhu et al., 2017).
To clarify the mechanism by which LMWF inhibits PF through macrophage polarization, we detected the expression levels of M2 macrophage biomarkers, such as arginase-1, Fizz-1, YM-1, and TGF-β, in lung by western blot, qPCR and Immunohistochemistry. We found that LMWF effectively regulated the expression levels of these biomarkers. This shows that LMWF could inhibit the development of pulmonary fibrosis by regulating the polarization of M2 macrophages. In the late stage of pulmonary fibrosis, M2 macrophages is dominant, the most critical factor is TGF-β. The LMWF could effectively block its secretion, it is also important for inhibiting the process of pulmonary fibrosis. In addition, inflammation is the early clinical manifestation of pulmonary fibrosis, and the main clinical manifestation in the later stage is collagen deposition, which is also why we detected the expression of inflammatory factors in this experiment. We found that inflammatory factors and M1 macrophage biomarkers were also highly expressed in qPCR results, and LMWF could effectively reduce their expression. This suggested whether LMWF inhibits pulmonary fibrosis through the early anti-inflammatory effect and regulating the M2 macrophages polarization in the late stage.
Then we verified the mechanism by in vitro experiments. First, we induced MH-S cells with IL-4 to construct M2 macrophage polarization model. In the experiment, we found that the morphological changes of cells were obvious after IL-4 treatment. Subsequently, the Western blot and qPCR results showed that the expression of M2 macrophage biomarkers increased, while LMWF could effectively reduce its expression, especially TGF-β. This also showed that LMWF could regulate the polarization of M2 macrophages and the results of in vivo experiments were verified. In addition, we found collagen, fibronectin and α-SMA expression increased after IL-4 induction and decreased after LMWF treatment.
Secondly, we induced MH-S cells by LPS combined with IFN-γ to construct M1 macrophage polarization model. Through experiments, we found that LMWF could reduce the secretion of inflammatory factors (iNOS and TNF-α,) and reduce the expression of inflammation. This shows that LMWF could effectively block the M1 macrophages polarization and inhibit the development of inflammation through its anti-inflammatory effect, which is also consistent with our previous experimental research (Dong et al., 2022). Combined with in vitro experimental studies, we confirmed that LMWF could regulate the polarization of M1/M2 macrophages, inhibited the development of inflammation, and blocked the process of fibrosis. However, we also found that LMWF did not act macrophage at the same time, but could regulate the inflammatory response in the early stage, inhibit the secretion of inflammatory factors, and block the polarization of M2 macrophages and inhibit TGF-β in the late stage to inhibit TGF- β Induce fibrosis symptoms.
Finally, we summarized the results of this experiment: previous in vivo experimental studies found that LMWF could regulate M2 macrophage polarization and reduce inflammatory response, and then in vitro experiments found that LMWF could inhibit pulmonary fibrosis by regulating M1/M2 macrophage polarization.