There are two major new findings in this study. First, we demonstrated that ACCD could effectively attenuate PM2.5-induced lung inflammation in rats, including a reduction in inflammatory infiltration and cupocytosis of the airways and a decrease in the inflammatory factor (IL-1β) in BALF. Second, by gene microarray technology, we found that the protective effect of ACCD on rats exposed to PM2.5 was closely related to the inflammatory response. Five genes related to inflammation were selected for validation among which three genes, Plcβ-1, Axin2 and Ccbe1, significantly changed between PM2.5 and ACCD treated group. Our results suggest that ACCD can play a protective role against PM2.5-induced lung inflammation, and Plcβ-1, Axin2 and Ccbe1 may be its key targets of action.
PM2.5 exposed is always associated to excessive production of pro-inflammatory cytokines and a pronounced inflammatory responses in the lungs [23]. Our findings are generally concordant with a previous report that characterized the airway inflammatory reaction associated with PM2.5 [24]. Significant inflammatory variations in lung tissue and BALF can be observed rapidly after PM2.5 treatment. IL-1β is considered to be a key pro-inflammatory cytokine that plays an important role in coordinating local and systemic inflammation of infection and inflammation [28, 29]. Several in vitro studies have found that PM2.5 can induce IL-1β secretion from macrophages through multiple pathways [27]. We examined the expression of IL-1β in inflammatory locations. Consistent with previous studies, PM2.5 significantly increased the production of IL-1β in BALF [28].
99 DEGs were identified between PM2.5 group and ACCD group. GO enrichment of DEGs in present study showed that the top 3 terms with the highest number of DEGs in BP were related to lipopolysaccharide, inflammation and inhibition of cell proliferation. Both animal and human research have described that exposure to PM2.5 may cause a respiratory inflammation. However, the inflammatory response is a diversified, comprehensive and complex process. It is associated not merely with genetic members of the inflammatory process, but also with a great number of genes belonging to an atypical inflammatory language. Lipopolysaccharide response and inhibition of cell proliferation belongs to the most basic inflammatory processes, involving multiple aspects such as airway inflammation and goblet cell metaplasia in respiratory diseases. Meanwhile, the lung-protection of ACCD against PM2.5 induced lung inflammation was mainly involved in cancer signaling pathway. Research has shown that there was a significant positive correlation between PM2.5 concentration and cancer mortality [29]. Inflammation, particularly chronic inflammation, is strongly associated with the development of cancer [30]. ACCD may provide the prevention and treatment of inflammation through cancers-associated pathways in PM2.5-induced lung inflammation.
In order to study the potential role of these dysregulated genes in the treatment of PM2.5-induced lung inflammation, we then examined the mRNA (IL6r, Ppbp, Plcβ-1, Axin2, Ccbe1) expression levels through Q-PCR and validated that Plcβ-1, Axin2, and Ccbe1 were the highly downregulated genes treated with ACCD. The results are consistent with the microarray data, further confirming the key dysregulation genes play roles in ACCD to reduce PM2.5-induced lung damage through anti-inflammatory. Phospholipase C-β (PLC-β) is a key player in a signaling cascade that links G protein coupled receptors (GPCR) to an intracellular signaling network [31]. The PLC-β class of phospholipases is composed of four isozymes (β1–β4) encoded by different genes [32]. PLC-β1 is highly expressed in neuronal tissue and may play an important role in neuroendocrine growth factor-stimulated cell proliferation [37, 38]. PLC-β blockade of signaling may be a valuable method to inhibit cell growth and metastasis. The signal transduction role of PLC-β constitutes a possible target for pharmacological interventions to treat disease. In our research, PLC-β1 was upregulated in the model group, which is consistent with previous literature reports.
Axis inhibition protein 2 (AXIN2) is a downstream target of the Wnt signaling pathway, which plays an important role in the regulation of β-catenin stability and its involvement in various cellular functions [39, 40]. Previous studies suggest that AXIN2, one of the downstream targets of T cytokines/lymphopoietic enhancers of the Wnt signaling pathway, may have oncogenic effects [37]. AXIN2 has been shown to be upgraded in various types of cancer [38]. The present study demonstrated that AXIN2 were significantly upregulated after PM2.5-inducing and downregulated by the ACCD treatment. This has not been reported in previous studies on PM2.5 causing lung damage.
Collagen and calcium-binding epidermal growth factor domain calcium-containing protein 1 (CCBE1) can promotes proteolysis of vascular endothelial growth factor (VEGF) [39]. In this study, we reveal CCBE1 overexpressed in PM2.5 induced lung tissue. Nevertheless, it is a future question to explore how CCBE1 transcription is activated and which of the transcription factors augment the transcription of CCBE1 in PM2.5.
Herbs have long been used to prevent or treat diseases and are indeed valuable chemical compounds that have developed into indispensable medicines in medical practice [40]. The extraction of natural compounds from Chinese herbal medicine is increasingly accepted and used in clinical research as a novel drug discovery method. TCM is traditionally used to treat diseases with the characteristics of multi-targeting signaling pathways and even meditate the multi-organ collaborative work in whole body system [41].