The acute airway inflammation induced by PM2.5 exposure and the treatment of essential oils in Balb/c mice

Abstract

PM_2.5 is the main particulate air pollutant whose aerodynamic diameter is less than 2.5 micron. The inflammation of various respiratory diseases are associated with PM_2.5 inhalation. Pro-inflammatory cytokine IL-1β generated from effected cells usually plays a crucial role in many kinds of lung inflammatory reactions. The exacerbation of Th immune responses are identified in some PM_2.5 related diseases. To elucidate the underlying mechanism of PM_2.5-induced acute lung inflammation, we exposed Balb/c mice to PM_2.5 intratracheally and established a mice model. Acute lung inflammation and increased IL-1β expression was observed after PM_2.5 instillation. Regulatory factors of IL-1β (TLR4/MyD88 signaling pathway and NLRP3 inflammasome) participated in this lung inflammatory response as well. Treatment with compound essential oils (CEOs) substantially attenuated PM_2.5-induced acute lung inflammation. The decreased IL-1β and Th immune responses after CEOs treatment were significant. PM_2.5 may increase the secretion of IL-1β through TLR4/MyD88 and NLRP3 pathway resulting in murine airway inflammation. CEOs could attenuate the lung inflammation by reducing IL-1β and Th immune responses in this model. This study describes a potentially important mechanism of PM_2.5-induced acute lung inflammation and that may bring about novel therapies for the inflammatory diseases associated with PM_2.5 inhalation.

Figure 1. Mice lung tissues pathohistological changes, total cells and differential cells count of BALF after administration by PM_2.5 or saline (bar = 50 μm).

Mice lung tissues were obtained on day 3, 7, 14 after administration by PM_2.5. Samples were stained using H&E. Histopathological lesions and changes were assessed by histological analyses of six random fields per sample by optical microscope. PM_2.5 significantly increased cells infiltration and altered alveolar walls structure (A). PM_2.5 facilitated the accumulation of inflammatory cells in BALF. Total cells (B), neutrophils (C), lymphocytes (D) and macrophages (E) in BALF were counted using Giemsa staining. Results are represented as means ± SEM (**P < 0.01, *P < 0.05 versus the control group).

Figure 2. PM_2.5 upregulated IL-1β mRNA level of lung tissues and IL-1β protein level of BALF.

IL-1β mRNA expression of lung tissues (A) was assayed by real-time RT-PCR using the ΔCt method. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading controls for mRNA expressions. Levels of IL-1β (B) in BALF were detected by ELISA. PM_2.5 promoted the generation and secretion of IL-1β in lung and BALF respectively. Data are expressed as mean ± standard error of the mean (**P < 0.01, *P < 0.05 versus the control group).

Figure 3. PM_2.5 increased mRNA expression of TLR4/MyD88 pathway and NLRP3 inflammasome.

mRNA levels were assayed by real-time RT-PCR using the ΔCt method. GAPDH was used as loading controls for mRNA expressions. PM_2.5 increased the expression of TLR4 (A), MyD88 (B), Nf-κB (C), NLRP3 (D) and P2 × 7 (E) of lung tissues. Data are expressed as mean ± standard error of the mean (**P < 0.01, *P < 0.05 versus the control group). PM_2.5 increased TLR4 (F) and NLRP3 (G) protein expression in lung tissues at all time-points, especially at bronchiole section.

Figure 4. Mice lung tissues pathohistological changes, total cells and differential cells count in BALF after saline or CEO treatment upon PM_2.5 exposure (bar = 50 μm).

Mice lung tissues were obtained on day 3, 7, 14 after administration by PM_2.5. Samples were stained using H&E. Histopathological lesions and changes were assessed by histological analyses of six random fields per sample by optical microscope. CEOs significantly reduced cells infiltration and alveolar walls alteration time-dependently (A). CEOs decreased the accumulation of inflammatory cells of BALF. Total cells (B), neutrophils (C), lymphocytes (D) and macrophages (E) of BALF were counted using Giemsa staining. Results are represented as means ± SEM (**P < 0.01, *P < 0.05 versus PM_2.5 + saline group).

Figure 5. CEOs downregulated IL-1β mRNA level of lung tissues and IL-1β protein level of BALF respectively.

IL-1β mRNA expression of lung tissues (A) was assayed by real-time RT-PCR using the ΔCt method. GAPDH was used as loading controls for mRNA expressions. IL-1β level (B) of BALF was detected by ELISA. Data are expressed as mean ± standard error of the mean (*P < 0.05 versus the PM_2.5 + saline group).

Figure 6. CEOs downregulated mRNA expression of TLR4/MyD88 pathway and NLRP3 inflammasome.

mRNA levels were assayed by real-time RT-PCR using the ΔCt method. GAPDH was used as loading controls for mRNA expressions. CEOs downregulated mRNA expression of TLR4 (A), MyD88 (B), Nf-κB (C), NLRP3 (D) and P2 × 7 (E) of lung tissues. Data are expressed as mean ± standard error of the mean (**P < 0.01, *P < 0.05 versus the PM_2.5 + saline group). CEOs reduced TLR4 (F) and NLRP3 (G) protein expression in lung tissues at all time-points, especially at bronchiole section.

Figure 7. CEOs reduced T-bet, GATA3, ROR-γt and Foxp3 mRNA expression of spleen tissues and IFN-γ and IL-4 protein expressions of serum.

mRNA levels were assayed by real-time RT-PCR using the ΔCt method. GAPDH was used as loading controls for mRNA expressions. CEOs significantly reduced expression of the key transforming growth factors of Th1 (A), Th2 (B), Th17 (C) and Treg cells (D). Cytokine levels of serum were detected by ELISA. Mice IFN-γ (E) and IL-4 (F) protein expressions were decreased after CEOs administration. Data are expressed as mean ± standard error of the mean (**P < 0.01, *P < 0.05 versus the PM_2.5 + saline group).