A COX-2/sEH dual inhibitor PTUPB alleviates lipopolysaccharide-induced acute lung injury in mice by inhibiting NLRP3 inflammasome activation

Abstract

Rationale: Dysregulation of arachidonic acid (ARA) metabolism results in inflammation; however, its role in acute lung injury (ALI) remains elusive. In this study, we addressed the role of dysregulated ARA metabolism in cytochromes P450 (CYPs) /cyclooxygenase-2 (COX-2) pathways in the pathogenesis of lipopolysaccharide (LPS)-induced ALI in mice. Methods: The metabolism of CYPs/COX-2-derived ARA in the lungs of LPS-induced ALI was investigated in C57BL/6 mice. The COX-2/sEH dual inhibitor PTUPB was used to establish the function of CYPs/COX-2 dysregulation in ALI. Primary murine macrophages were used to evaluate the underlying mechanism of PTUPB involved in the activation of NLRP3 inflammasome in vitro. Results: Dysregulation of CYPs/COX-2 metabolism of ARA occurred in the lungs and in primary macrophages under the LPS challenge. Decrease mRNA expression of Cyp2j9, Cyp2j6, and Cyp2j5 was observed, which metabolize ARA into epoxyeicosatrienoic acids (EETs). The expressions of COX-2 and soluble epoxide hydrolase (sEH), on the other hand, was significantly upregulated. Pre-treatment with the dual COX-2 and sEH inhibitor, PTUPB, attenuated the pathological injury of lung tissues and reduced the infiltration of inflammatory cells. Furthermore, PTUPB decreased the pro-inflammatory factors, oxidative stress, and activation of NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome in LPS-induced ALI mice. PTUPB pre-treatment remarkably reduced the activation of macrophages and NLRP3 inflammasome in vitro. Significantly, both preventive and therapeutic treatment with PTUPB improved the survival rate of mice receiving a lethal dose of LPS. Conclusion: The dysregulation of CYPs/COX-2 metabolized ARA contributes to the uncontrolled inflammatory response in ALI. The dual COX-2 and sEH inhibitor PTUPB exerts anti-inflammatory effects in treating ALI by inhibiting the NLRP3 inflammasome activation.

Keywords: COX-2/sEH dual inhibitor; NLRP3 inflammasome; acute lung injury; oxidative stress.

Figure 1

Dysregulation of CYPs/COX-2 metabolism of ARA occurs in the lungs and macrophages under the LPS challenge. Cyp2j9 was the most abundant CYP isoform expressed in the lungs, whereas Cyp2c29 and Cyp2c44 mRNA were undetectable (A, n = 6). Cyp2j9, Cyp2j6, and Cyp2j5 mRNAs were robustly decreased at 12 h after LPS administration (5 mg/kg, i.t.) in the lungs (B, n = 6). Western blotting and RT-qPCR results showing increased sEH and COX-2 proteins, and Cox-2 mRNA at 12 h after LPS administration. (C-F, n = 4-8). Cyp2j6 was the most abundant CYP isoform expressed in primary murine macrophages after treatment with LPS (100 ng/mL), whereas Cyp2c29 and Cyp2c44 mRNA were undetectable (G, n = 3). Cyp2j6 and Cyp2j9 mRNA were remarkably decreased at 6 h after LPS administration (H, n = 3). Expression of sEH and COX-2 protein and Cox-2 mRNA were detected by Western blotting and RT-qPCR (I-L, n = 3). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 2

PTUPB attenuates the pathological lung injury and rescues the respiratory function of ALI mice. C57BL/6 mice were subcutaneously injected with PTUPB 1 h before the LPS administration. Twelve hours after LPS injection (5 mg/kg, i.t.), lung histopathology was performed with H&E staining (A). Inflammation score was measured independently by three pathologists blinded to the experiment (B, n = 6). Activity of LDH in the serum was assayed (C, n = 7-8). Respiratory function was detected by Buxco, including airway resistance (D, n = 5-8), lung compliance (E, n = 5-8), and pulmonary ventilation (F, n = 5-8). Data are mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 3

PTUPB reverses the infiltration of inflammatory cells and oxidative stress in ALI mice. C57BL/6 mice received LPS injection (5 mg/kg, i.t.) with or without PTUPB pre-treatment (5 mg/kg) for 1 h. Twelve hours after the LPS injection, the total cells (A), macrophages (B), and neutrophils (C) in the BALF were counted (n = 6-10). MPO activity was detected to quantify the infiltration of neutrophils in the lungs (D, n = 7-10). MDA and total ROS in the lungs were detected 12 h after the LPS administration (E-F, n = 6-9). mRNA expression of Nox2 in the lungs was detected by RT-qPCR (G, n = 5-8). Total SOD activity in serum was detected 12 h after the LPS administration (H, n = 6-10). Protein expression of Nrf2 in the lungs was assayed by Western blotting (I-J, n = 4-8). Data are mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 4

PTUPB attenuates the production of pro-inflammatory factors in the lungs of ALI mice. C57BL/6 mice received LPS injection (5 mg/kg, i.t.) with or without PTUPB pre-treatment (5 mg/kg) for 1 h. Twelve hours after the LPS administration, mRNA expression of Tnf-α (A, n = 6-7), Mcp-1 (D, n = 6-7), and Trem-1 (G, n = 5-7) in the lungs was detected by RT-qPCR. Protein content of TNF-α in lung tissue (B, n = 6-8) and BALF (C, n = 6-8), MCP-1 in serum (E, n = 5-8) and BALF (F, n = 6-8) was assayed by ELISA. Expression of TREM-1 in lung tissue was assayed by Western blotting (H-I, n = 4). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 5

PTUPB inhibits the activation of NLRP3 inflammasome in the lung of ALI mice. C57BL/6 mice received LPS injection (5 mg/kg, i.t.) with or without PTUPB pre-treatment (5 mg/kg) for 1 h. mRNA expression of Nlrp3 (A, n = 6-7), pro-caspase-1 (B, n = 6-7), Asc (C, n = 6-7), and pro-Il-1β (D, n = 6-7) in the lungs was detected by RT-qPCR 12 h after the LPS injection. Protein expression of NLRP3 and pro-IL-1β (E-G, n = 4) in the lungs was detected by Western blotting. mRNA expression of Nf-κb/p65 (H, n = 6-9) in the lungs was detected by RT-qPCR 12 h after the LPS injection. Protein expression of caspase-1p10 and IL-1βp17 in the lungs was detected by Western blotting (I-K, n = 4). Concentration of IL-1β in lung tissue was detected by ELISA (L, n = 5-8). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Figure 6

Prophylactic and therapeutic treatment of PTUPB prevents death from LPS administration in mice. PTUPB (5 mg/kg, s.c.) was administered to mice 1 h before (A) or 6 h after (B) induction of ALI by a lethal dose of LPS (25 mg/kg, i.t.). The mortality of the mice was monitored every 6 h, and the percent survival rate was expressed as a Kaplan-Meier survival curve (n = 20 per group). *** P < 0.001.

Figure 7

PTUPB reduces the inflammation and priming of NLRP3 inflammasome in primary murine macrophages. Primary murine macrophages were treated with serial concentrations of PTUPB (10, 100, 1000, and 10000 nM) for 6 h and the LDH activity in the supernatant of cell culture was detected (A). Macrophages were treated with LPS (100 ng/mL) for 6 h with or without PTUPB pre-treatment (10, 100, and 1000 nM) for 1 h and activity of LDH in the supernatant was detected (B). Expression of Tnf-α (C), Mcp-1 (E), Nlrp3 (G), pro-caspase-1 (H), and Nf-κb/p65 (K) mRNAs in macrophages was detected by RT-qPCR. Concentration of TNF-α (D) and MCP-1 (F) in the supernatant of cell culture was detected by ELISA (n = 3). Protein expression of NLRP3 and IκBα in macrophages was detected by Western blotting (I-J, n = 4; L-M, n = 3). Data are expressed as the mean ± SD. ** P < 0.01, and *** P < 0.001.

Figure 8

PTUPB inhibits the activation of NLRP3 inflammasome in primary murine macrophages. To evaluate the effects of PTUPB on the activation of NLRP3 inflammasome, PTUPB (1000 nM) was added 1 h before LPS-priming (100 ng/mL) for 135 min. Subsequently, cells were stimulated with ATP (2.5 mM) for 45 min. Protein expression of caspase-1 p10 and IL-1β p17 in macrophages was detected by Western blotting (A-C, n = 3). Interaction between endogenous NLRP3 and ASC was analyzed by IP (D). The ROS was analyzed by a ROS kit (E-F). Data are expressed as the mean ± SD. * P < 0.05, ** P < 0.01, and *** P < 0.001.