Aerosol Inhalation of Luteolin-7-O-Glucuronide Exerts Anti-Inflammatory Effects by Inhibiting NLRP3 Inflammasome Activation

Abstract

Background: Luteolin-7-O-glucuronide (L7Gn) is a flavonoid isolated from numerous traditional Chinese herbal medicines that exerts anti-inflammatory effects. Previous research has revealed that aerosol inhalation is the most straightforward way of administration for the delivery of respiratory agents. Thus far, the impact of aerosol inhalation of L7Gn on lung inflammation and the underlying mechanisms remain unknown. Methods: The real-time particle size for L7Gn aerosol inhalation was detected by the Spraytec spray droplet size measurement system, including transmission and size diameters. The acute lung injury (ALI) rat model was induced by aerosol inhalation of LPS to evaluate the protective effect of L7Gn. The inhibitory effect of NLRP3 inflammasome activation assays was conducted in LPS-induced MH-S cells. Elisa, Western blotting, and RT-PCR were utilized to investigate the expression of NLRP3 inflammasome-relevant proteins and genes. Results: In this study, we found that inhalation of L7Gn aerosol significantly reduced pulmonary injury by inhibiting inflammatory infiltration and enhancing lung function. Meanwhile, the NLR family pyrin domain containing 3 (NLRP3) inflammasome was activated dramatically, accompanied by upregulated expression of IL-1β and IL-18, both in the ALI rat model and in LPS-induced MH-S cells. Moreover, L7Gn was found to significantly downregulate the expression of NLRP3, ASC, caspase-1, and cleaved caspase-1, which are critical components of the NLRP3 inflammasome, as well as the expression of IL-1β and IL-18. Conclusions: Based on our findings, L7Gn could exert anti-inflammatory effects by inhibiting NLRP3 inflammasome activation, which may emerge as potential therapeutic agents for the treatment of ALI.

Keywords: Inflammation inhibition; NLRP3 inflammasome; acute lung injury; aerosol delivery; luteolin-7-O-glucuronide.

Figure 1

The structure of L7Gn (A), the compound of interest, and the experimental design of the study (B).

Figure 2

The parameter of real-time particle size of L7Gn. (A) The histogram of particles diameter; (B) Real-time transmission and percentiles of inhalation of aerosolized L7Gn, Trans (purple line) Dv(10) (dark green line), Dv(50) (light green line), and Dv(90) (green line).

Figure 3

The effect of L7Gn on pulmonary function in LPS-induced ALI rats. Pulmonary function was measured quantitatively by assaying for TV, MV, Cdyn, and Ve (A–D). All the data presented as mean ± SEM. n = 5. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 4

L7Gn inhibits LPS-induced pulmonary damage in rats. Tissue sections of a representative lung section from each group: normal group (A,a), LPS + vehicle group (B,b), LPS + L7Gn 10 min group (C,c), LPS + L7Gn 20 min group (D,d), and LPS + dexamethasone group (E,e), which were stained with hematoxylin and eosin (H&E) ((A–E), 25×; (a–e), 100×), bronchiolar inflammation (red arrow), and interstitial thickening (green arrow).

Figure 5

L7Gn inhibited NLRP3 inflammasome activation in LPS-induced rats. Sections of lung tissue were immunostained with CD68 (green) and Caspase-1 (red) antibodies shown as representative images (A); Representative Western blotting of NLRP3, Caspase-1, and ASC (A): The relative protein expression of NLRP3, Caspase-1, and ASC normalized to that of β-actin. (B–E). All data are presented as mean ± SEM., n = 3. * p < 0.05; **, p < 0.01.

Figure 6

L7Gn decreased the levels of inflammatory cytokines in LPS-induced bronchiolar inflammation. The levels of IL-1β in BALF (A) and lung homogenates (B) and IL-18 in BALF (C) and lung homogenates (D) were determined using ELISA kits. All data are presented as mean ± SEM., n = 6. * p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 7

L7Gn inhibited NLRP3 inflammasome activation in LPS-induced MH-S cells. Representative Western blotting of NLRP3, caspase-1, IL-1β, ASC, IL-18, cleaved IL-1β, and cleaved caspase-1 (A). The relative protein expression of NLRP3, caspase-1, IL-1β, ASC, IL-18, cleaved IL-1β, and cleaved caspase-1 was normalized to that of β-actin (B–H). Relative mRNA expression of IL-1β and IL-18 (I,J). All data are presented as mean ± SEM., n = 3. * p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.