Fraxinol attenuates LPS-induced acute lung injury by equilibrating ACE-Ang II-AT1R and ACE2-Ang (1-7)-Mas and inhibiting NLRP3

Abstract

Context: Acute lung injury (ALI) is a serious heterogenous pulmonary disorder. Fraxinol was selected for this study since it is a simple coumarin compound, not previously investigated in ALI.

Objectives: This study investigates the ALI therapeutic effect and mechanisms of fraxinol.

Materials and methods: Male BALB/c mice were treated with fraxinol (20, 40, and 80 mg/kg) following intranasal injection of lipopolysaccharide (LPS; 10 μg in 50 μL). The mice in control group were intratracheally injected with 50 μL phosphate buffered saline (PBS). Raw264.7 cells were treated with fraxinol by 100 ng/mL LPS for 6 h, then treated by different concentrations of fraxinol (5, 10, and 25 μM) for 48 h. Cells in control group were treated with PBS.

Results: Fraxinol with doses of 20, 40, and 80 mg/kg significantly attenuated LPS-induced lung injury in mice (lung injury score, 10.4, 31.2, 50.3%). Fraxinol attenuated the apoptosis and nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing-3 (NLRP3) activation induced by LPS (apoptosis, 18.3, 30.2, 55.6%; NLRP3, 30.0, 47.7, 63.6%). The anti-apoptosis and anti-inflammation effects of fraxinol were also confirmed in Raw264.7 cells (apoptosis, 38.8, 55.3, 68.9%; NLRP3, 20.6, 55.7, 73.9%).

Discussion and conclusion: The anti-ALI effects of fraxinol maybe by equilibrating ACE-Ang II-AT1R and ACE2-Ang (1-7)-Mas axis and inhibiting NLRP3 inflammasome. Our research provides a candidate drug in the treatment of ALI.

Keywords: Inflammation; apoptosis; renin-angiotensin system.

Figure 1.

Network pharmacology analysis of Chinese medicine to pneumonia. (A) The effective chemical components corresponding to inflammation lung disease are predicted through TCMSP and CTD database. (B) The structure of fraxinol. (C) The target proteins corresponding to pneumonia and fraxinol are predicted by CTD, malacards, disgenet, and Swiss Target Prediction databases, respectively. (D) The related signalling pathways were predicted through overlapping analysis of the pneumonia signalling and target proteins.

Figure 2.

Fraxinol attenuates LPS-induced ALI in mice. (A,B) Histological changes were evaluated by using H&E staining. Bar = 20 μm. (C) The lung W/D ratio was used to assess the pulmonary edoema. (D,E) Apoptotic cells were measured by TUNEL assay. Bar = 20 μm. (F) The level of MPO activity. (G) Effect of fraxinol on total proteins in BALF. (H,I) TNF-α and IL-1β levels in BALF were measured by ELISA kits. *p < 0.05 vs. Control group; ^#p < 0.05 vs. LPS group.

Figure 3.

Fraxinol alleviates LPS-induced ALI by equilibrating ACE-Ang II-AT1R and ACE2-Ang (1-7)-Mas in mice. (A–C) The protein expression of ACE and AT1R was measured using Western blot assay. (D) The level of Ang II was detected by ELISA kit. (E–G) The protein expression of ACE2 and MasR was measured using Western blot assay. (H) The level of Ang (1–7) was detected by ELISA kit. *p < 0.05 vs. control group; ^#p < 0.05 vs. LPS group.

Figure 4.

Fraxinol ameliorates LPS-induced NLRP3 inflammasome in mice. (A–E) The protein expression of NLRP3, ASC, pro-caspase-1, and cleaved caspase-1 was measured using Western blot assay. *p < 0.05 vs. Control group; ^#p < 0.05 vs. LPS group.

Figure 5.

Fraxinol ameliorates LPS-induced Raw264.7 cell inflammation and apoptosis. (A,B) Effects of fraxinol on LPS-induced TNF-α and IL-1β levels in Raw264.7 cells were detected by ELISA kits. (C,D) Effects of fraxinol on LPS-induced apoptosis in Raw264.7 cells were detected by flow cytometry. *p < 0.05 vs. Control group; ^#p < 0.05 vs. LPS group; ⁺p < 0.05 vs. LPS + Fraxinol (25 μM) group.

Figure 6.

Fraxinol equilibrates ACE-Ang II-AT1R and ACE2-Ang (1-7)-Mas axis in Raw264.7 cells. (A–C) The protein expression of ACE and AT1R was measured by Western blot assay. (D–F) The protein expression of ACE2 and MasR was measured by Western blot assay. *p < 0.05 vs. Control group; ^#p < 0.05 vs. LPS group; ⁺p < 0.05 vs. LPS + Fraxinol (25 μM) group.

Figure 7.

Fraxinol inhibits LPS-induced Raw264.7 cell inflammation and apoptosis via regulation of NLRP3. (A–E) The protein expression of NLRP3, ASC, pro-caspase-1, and cleaved caspase-1 was measured by Western blot assay. (F,G) Effects of fraxinol and nigericin on LPS-induced TNF-α and IL-1β levels in Raw264.7 cells were detected by ELISA kits. (H,I) Effects of fraxinol and nigericin on LPS-induced apoptosis in Raw264.7 cells were detected by flow cytometry. *p < 0.05 vs. Control group; ^#p < 0.05 vs. LPS group; ⁺p < 0.05 vs. LPS + Fraxinol (25 μM) group.