Pyrroloquinoline Quinone Administration Alleviates Allergic Airway Inflammation in Mice by Regulating the JAK-STAT Signaling Pathway

Abstract

The current asthma therapies are inadequate for many patients with severe asthma. Pyrroloquinoline quinone (PQQ) is a naturally-occurring redox cofactor and nutrient that can exert a multitude of physiological effects, including anti-inflammatory and antioxidative effects. We sought to explore the effects of PQQ on allergic airway inflammation and reveal the underlying mechanisms. In vitro, the effects of PQQ on the secretion of epithelial-derived cytokines by house dust mite- (HDM-) incubated 16-HBE cells and on the differentiation potential of CD4+ T cells were investigated. In vivo, PQQ was administered to mice with ovalbumin- (OVA-) induced asthma, and lung pathology and inflammatory cell infiltration were assessed. The changes in T cell subsets and signal transducers and activators of transcription (STATs) were evaluated by flow cytometry. Pretreatment with PQQ significantly decreased HDM-stimulated thymic stromal lymphopoietin (TSLP) production in a dose-dependent manner in 16-HBE cells and inhibited Th2 cell differentiation in vitro. Treatment with PQQ significantly reduced bronchoalveolar lavage fluid (BALF) inflammatory cell counts in the OVA-induced mouse model. PQQ administration also changed the secretion of IFN-γ and IL-4 as well as the percentages of Th1, Th2, Th17, and Treg cells in the peripheral blood and lung tissues, along with inhibition the phosphorylation of STAT1, STAT3, and STAT6 while promoting that of STAT4 in allergic airway inflammation model mice. PQQ can alleviate allergic airway inflammation in mice by improving the immune microenvironment and regulating the JAK-STAT signaling pathway. Our findings suggest that PQQ has great potential as a novel therapeutic agent for inflammatory diseases, including asthma.

Figure 1

Cell viability and cytokine production in 16-HBE cells treated with different doses of PQQ. (a) 16-HBE cell viability was reduced after administration of PQQ in a dose-dependent manner. Cell viability was measured by CCK-8 assay after 24 h and 48 h of incubation with PQQ. (b) Cytokine production by 16-HBE cells after 2 h of pretreatment with PQQ and subsequent incubation with HDMs. The columns and error bars represent the means and SEMs (n = 3 per group). ^∗P < 0.05, ^∗∗P < 0.01; ns: not statistically significant. Similar results were obtained in three independent experiments.

Figure 2

PQQ administration inhibits Th2 cell differentiation. CD4+ T cells were primed under Th2 conditions. PQQ and DXM were added. Five days later, the cells were stimulated with PMA and ionomycin. The Th2 cytokines were measured using an ELISA kit. The columns and error bars represent the means and SEMs (n = 3 per group). ^∗P < 0.05, ^∗∗P < 0.01; ns: not statistically significant. Similar results were obtained in three independent experiments.

Figure 3

PQQ administration attenuates airway inflammation in an OVA-induced mouse model. (a) Protocols for establishment of the OVA-induced allergic asthma mouse model and administration of PQQ. (b) Representative photomicrographs of lung sections stained with H&E and examined at 100x magnification. (c) Lung pathological scores of peribronchial and perivascular inflammatory cell infiltration in asthmatic model mice and PQQ-/DXM-treated asthmatic model mice. (d) Serum IgE levels in asthmatic model mice and PQQ-/DXM-treated asthmatic model mice. The columns and error bars represent the means and SEMs (n = 5 per group). ^∗P < 0.05, ^∗∗P < 0.01. Similar results were obtained in at least three independent experiments.

Figure 4

PQQ administration reduces BALF inflammatory cell counts and changes IFN-γ and IL-4 secretion. (a) Total cell numbers in BALF were counted with an Automatic Blood Cell Analyzer and specific differential cell counts in BALF were tested by flow cytometer. (b) Levels of released IFN-γ in BALF as measured by ELISA. (c) Levels of released IL-4 in BALF measured by ELISA. The columns and error bars represent the means and SEMs (n = 5 per group). ^∗P < 0.05, ^∗∗P < 0.01. Similar results were obtained in at least three independent experiments.

Figure 5

Effects of PQQ treatment on Th1, Th2, Th17, and Treg cells in peripheral blood and lung tissues of asthmatic model mice. (a and b) The gating strategy of flow cytometry for blood and tissue. (c and d) Representative scatter diagrams in flow cytometry and quantitative analysis of the percentages of CD4 + IFN-γ + T cells in blood and lung tissues. (e and f) Representative scatter diagrams in flow cytometry and quantitative analysis of the percentages of CD4 + IL-4+ T cells in blood and lung tissues. (g and h) Representative scatter diagrams in flow cytometry and quantitative analysis of the percentages of CD4 + RORγt+ T cells in blood and lung tissues. (i and j) Representative scatter diagrams in flow cytometry and quantitative analysis of the percentages of CD4 + CD25 + FoxP3+ T cells in blood and lung tissues. The columns and error bars represent the means and SEMs (n = 5 per group). ^∗P < 0.05, ^∗∗P < 0.01. Similar results were obtained in at least three independent experiments.

Figure 6

Effects of PQQ treatment on the pSTAT1, pSTAT3, pSTAT4, and pSTAT6 signaling pathways in lung tissue and T cells. The phosphorylated STAT1, STAT3, STAT4, and STAT6 levels in lung tissue (a–d), in T cells (M-P), and STAT1, STAT3, STAT4, and STAT6 levels in lung tissue (e–h) and in T cells (Q-T), which are presented as the geometric mean fluorescent intensity (GMFI) values measured by flow cytometry (colored lines). The gray peak is the geometric mean fluorescence intensity of the blank control. The quantitative analysis of the phosphorylated and total STAT proteins in mouse lung tissue (i–l) and in T cells (u–x). The columns and error bars represent the means and SEMs (n = 3 − 5 per group). ^∗∗P < 0.01, ^∗P < 0.05. Similar results were obtained in at least three independent experiments.