According to recent clinical studies, both long and short period exposure to PM2.5 could increase the prevalence and mortality of respiratory diseases[2–4]. In our clinical application, we discovered that MGMD and YPFP could relieve the respiratory symptoms caused by hazy weather. However, the mechanism is still unclear. We designed this research to explore the effect and underlying mechanism of MGMD and YPFP against the PM2.5-induced lung injury. We established an animal model of PM2.5-induced lung injury by intratracheal instillation of PM2.5 suspensions and treated with MGMD and YPFP. The results of HE staining demonstrated that PM2.5 could induce the damage of airway epithelium, destruction of pulmonary alveoli, and inflammatory cell infiltration in lung tissues of rats. After treatment with MGMD or YPFP, we found that lung injury could be attenuated to a certain extent.
To further investigate the types of inflammatory cells, we applied immunohistochemistry and discovered that PM2.5 significantly increased neutrophil, macrophage, and T lymphocyte infiltration surrounding the airway, which the specific markers MPO, CD68, CD4, and CD8 were stained positive respectively. The inflammation can be staged into acute inflammation, subacute inflammation, and chronic inflammation[23]. Acute inflammation is characterized by neutrophils and monocyte infiltration, which initiated by rolling along and sticking to the epithelium. After been stimulated by PM2.5, the airway epithelial cells and alveolar macrophages secreted adhesion molecule, chemokines, and cytokines. In this study, we detected that PM2.5 could dramatically increase ICAM-1 expression, especially in airway epithelium by morphology. We achieved the same result in the protein level of total lung tissues. ICAM-1 can lead to leukocyte adhesion and migration into airway submucosa and alveolar interstitium by binding to the ligands (e.g., LFA-1, Mac-1, integrins, and fibrinogen). Moreover, the overexpression of ICAM-1 may increase the risk of common colds, for it is also the binding site for rhinovirus[24]. MCP-1, primarily expressed by macrophages and epithelial cells is another inflammatory mediator recruiting monocytes. In the early stage of exposure to PM2.5, MCP-1 elevated in the lung tissue and BALF[25]. The current study demonstrated that PM2.5 upregulated the MCP-1 expression in the airway epithelium and lung tissues. The same trends of IL-1, TNF-α, and IL-17A, the typical pro-inflammatory cytokines, could be found in BALF. The overexpression of NE in the lung tissues of model rats also indicated the augment and activation of neutrophils. This evidence demonstrated that PM2.5 could initiate acute inflammation. Except for the acute inflammatory cells, we also detected macrophages and lymphocytes infiltration and gland proliferation the characteristic in chronic inflammation. Therefore, PM2.5-induced inflammatory response was the result of the combination of acute and chronic inflammation.
Both MGMD and YPFP could reduce neutrophil, monocyte, and lymphocytes infiltration for the expression of MPO, CD68, CD4, and CD8 were obviously inhibited. In terms of pro-inflammatory mediators, the results of IHC and Western Blot demonstrated that MGMD and YPFP effectively downregulated the expression of MCP-1 in lung tissues. In the study of ICAM-1, another inflammatory medium, YPFP remarkably reduced the over-expression of ICAM-1 stimulated by PM2.5, but no significant inhibitory effect was found in MGMD group. Furthermore, the levels of IL-1β and IL-17A in BALF and NE in lung tissue showed that YPFP was more potent in inhibiting lung tissue inflammation than MGMD.
In addition to type 1 inflammation, HE-staining showed that there was a mass of eosinophils in model rats’ lung tissues, which was the symbol of type 2 inflammation. Furthermore, IgE and IL-4 closely related to type 2 inflammation exhibited higher levels in BALF after treated with PM2.5 suspensions. These mediators are essential for airway remodeling and abnormal mucus secretion. Previous evidence indicated that PM2.5 exacerbated accumulation of eosinophils and neutrophils into the airway submucosa[26, 27]. Another study reached the same conclusion that PM2.5 caused significant increase of eosinophils and neutrophils in BALF over time[28]. Therefore, we believed the PM2.5-induced inflammatory injury was the combined action of type 1 and type 2 inflammation. It could be found that both MGMD and YPFP had the inhibitory effects on PM2.5-induced eosinophil proliferation. The results indicated that MGMD and YPFP effectively decreased the levels of IL-4 and IgE in BALF, and the effect of MGMD on IL-4 was more potent. It was well acknowledged that the high affinity Fc receptor (FcεRI) played a mediating role in allergic inflammation. FcεRI mainly expressed on mast cell could bind to IgE and activate the release of histamine, prostaglandins, and leukotrienes which was known to exacerbate allergic response[29]. We detected that FcεRI protein in lung decreased markedly compared with model rats after MGMD and YPFP treatment.
It is considered that TLR2-MyD88-NFκB played an essential role in promoting inflammation. Several studies showed that PM2.5 could work as pathogen-associated molecular patterns (PAMP) combining with pattern recognition receptors (PRR) like TLR2 on the surface of the airway epithelial cells and alveolar macrophages. TLR2 delivered the activated signals to downstream MyD88 and then induced the phosphorylation of NFκB, which caused inflammatory cytokines synthesis. This signal transduction was generally recognized as an important mechanism of PM2.5-triggered inflammatory injury[26]. However, in the current study, the levels of TLR2, MyD88, and NFκB phosphorylation of each group are consistent with no significance. The reason for this is because, except for NFκB related pathway, there are numerous approaches promoting inflammation, such as MAPKs, Smads, and Nrf2 signalling pathways[30–32].
MUC5AC, the primary component of airway mucus, is synthesized and released by goblet cells in the epithelium. We detected by AB-PAS staining that exposure to PM2.5 could significantly exacerbate the goblet cell hyperplasia and MUC5AC release in the airway. The results of MUC5AC protein and mRNA expression in lung tissues came out the same trend. After treatment with MGMD and YPFP, we found that the therapy could reduce the goblet cell proliferation and inhibit MUC5AC expression. Moreover, the effects of YPFP was more significant than that of MGMD. In terms of the mechanism, it has been considered that EGFR-PI3K-AKT signalling pathway is tightly associated with regulating MUC5AC mRNA expression. PM2.5 and PM2.5-induced inflammatory mediators cause excess MUC5AC synthesis via the cascade of EGFR-PI3K-AKT signalling pathway[18]. Western Blot bands manifested remarkable increase in the ratio of phosphorylation of PI3K and AKT. However, phosphorylation of EGFR showed no difference in each group, indicating that EGFR is not the main signal receptor that causes the cascade of PI3K-AKT in this study. It has been investigated that receptor tyrosine kinase (RTK), G protein coupled receptor (GPCR) and T cell receptor can also bind to extracellular stimulation signals and activate the PI3K-AKT signalling pathway[33]. According to the results of Western Blot, YPFP had the effect of simultaneously inhibiting the phosphorylation of PI3K and AKT, whereas MGMD only showed a significant difference in AKT. After synthesized, MUC5AC protein was packed tightly in granule in the cytoplasm of goblet cell and remained stable until activated by stimuli like NE and MCP-1. MGMD and YPFP could downregulate the expression of NE and MCP-1, but IHC images showed that YPFP had more inhibitory effect on NE and thereby had advantage on suppressing MUC5AC release in turn.
According to TCM theory, the pathogenesis of PM2.5-induced respiratory symptom is “wind evil invading the lung”. YPFP, a classic formula, can dispel wind-evil and consolidate superficies. In recent researches, YPFP was discovered to be effective in restricting inflammation, alleviating allergic response, repairing tight junction of epithelia barrier[34–36]. GMD which comes from experience prescription is mainly applied in various allergic diseases. Previous study suggested that the mechanism of GMD was associated with reducing the production of IgE and suppressing inflammatory cell infiltration[20]. In clinical application, GMD with Qianhu and Jiegeng was more potent in treating allergic symptom for the two herbs could recover the lung function of dispersing and descending. Recent researches also explored that Qianhu and Jiegeng could significantly attenuate eosinophilic airway inflammation, downregulate the expression of inflammatory cytokines, and inhibit the activity of Th2 cells[37, 38].