Pulmonary fibrosis (PF) is a senescence-associated disease with poor prognosis. Currently, there is no effective therapeutic strategy for preventing and treating the disease process. Mounting evidence suggests that arachidonic acid (ARA) metabolites are involved in the pathogenesis of various fibrosis. However, the relationship between the metabolism of ARA and PF is still elusive. In this study, we observed a disorder in the cyclooxygenase-2/cytochrome P450 (COX-2/CYP) metabolism of ARA in the lungs of PF mice induced by bleomycin (BLM). Therefore, we aimed to explore the role of COX-2/CYP-derived ARA metabolic disorders in PF. PTUPB, a dual COX-2 and soluble epoxide hydrolase (sEH) inhibitor, was used to restore the balance of COX-2/CYP metabolism. sEH is an enzyme hydrolyzing epoxyeicosatrienoic acids derived from ARA by CYP. We found that PTUPB alleviated the pathological changes in lung tissue and collagen deposition, as well as reduced senescence marker molecules (p16 Ink4a and p53-p21 Waf1/Cip1 ) in the lungs of mice treated by BLM. In vitro, we found that PTUPB pretreatment remarkably reduced the expression of senescence-related molecules in the alveolar epithelial cells (AECs) induced by BLM. In conclusion, our study supports the notion that the COX-2/CYP-derived ARA metabolic disorders may be a potential therapeutic target for PF via inhibiting the cellular senescence in AECs.

Necroptosis is the major cause of death in alveolar epithelial cells (AECs) during acute lung injury (ALI). Here, we report a previously unrecognized mechanism for necroptosis. We found an accumulation of mitochondrial citrate (citratemt) in lipopolysaccharide (LPS)-treated AECs because of the downregulation of Idh3α and citrate carrier (CIC, also known as Slc25a1). shRNA- or inhibitor–mediated inhibition of Idh3α and Slc25a1 induced citratemt accumulation and necroptosis in vitro. Mice with AEC-specific Idh3α and Slc25a1 deficiency exhibited exacerbated lung injury and AEC necroptosis. Interestingly, the overexpression of Idh3α and Slc25a1 decreased citratemt levels and rescued AECs from necroptosis. Mechanistically, citratemt accumulation induced mitochondrial fission and excessive mitophagy in AECs. Furthermore, citratemt directly interacted with FUN14 domain-containing protein 1 (FUNDC1) and promoted the interaction of FUNDC1 with dynamin-related protein 1 (DRP1), leading to excessive mitophagy-mediated necroptosis and thereby initiating and promoting ALI. Importantly, necroptosis induced by citratemt accumulation was inhibited in FUNDC1-knockout AECs. We show that citratemt accumulation is a novel target for protection against ALI involving necroptosis.

Epoxyeicosatrienoic acids (EETs) derived from arachidonic acid exert anti‐inflammation effects. We have reported that blocking the degradation of EETs with a soluble epoxide hydrolase (sEH) inhibitor protects mice from lipopolysaccharide (LPS)‐induced acute lung injury (ALI). The underlying mechanisms remain essential questions. In this study, we investigated the effects of EETs on the activation of nucleotide‐binding domain leucine‐rich repeat‐containing receptor, pyrin domain‐containing‐3 (NLRP3) inflammasome in murine macrophages. In an LPS‐induced ALI murine model, we found that sEH inhibitor 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl), TPPU, profoundly attenuated the pathological injury and inhibited the activation of the NLRP3 inflammasome, characterized by the reduction of the protein expression of NLRP3, ASC, pro‐caspase‐1, interleukin precursor (pro‐IL‐1β), and IL‐1β p17 in the lungs of LPS‐treated mice. In vitro, primary peritoneal macrophages from C57BL/6 were primed with LPS and activated with exogenous adenosine triphosphate (ATP). TPPU treatment remarkably reduced the expression of NLRP3 inflammasome‐related molecules and blocked the activation of NLRP3 inflammasome. Importantly, four EETs (5,6‐EET, 8,9‐EET, 11,12‐EET, and 14,15‐EET) inhibited the activation of NLRP3 inflammasome induced by LPS + ATP or LPS + nigericin in macrophages in various degree. While the inhibitory effect of 5,6‐EET was the weakest. Mechanismly, EETs profoundly decreased the content of reactive oxygen species (ROS) and restored the calcium overload in macrophages receiving LPS + ATP stimulation. In conclusion, this study suggests that EETs inhibit the activation of the NLRP3 inflammasome by suppressing calcium overload and ROS production in macrophages, contributing to the therapeutic potency to ALI.

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Background. Epoxyeicosatrienoic acids (EETs), the metabolite of arachidonic acid by cytochrome P450 (CYP), reportedly serve as a vital endogenous protective factor in several chronic diseases. EETs are metabolized by soluble epoxide hydrolase (sEH). We have observed that prophylactic blocking sEH alleviates bleomycin- (BLM-) induced pulmonary fibrosis (PF) in mice. However, the underlying mechanism and therapeutic effects of EETs on PF remain elusive. Objective. In this study, we investigated the effect of CYP2J2/EETs on the activation of murine fibroblasts and their mechanisms. Results. we found that administration of the sEH inhibitor (TPPU) 7 days after the BLM injection also reversed the morphology changes and collagen deposition in the lungs of BLM-treated mice, attenuating PF. Fibroblast activation is regarded as a critical role of PF. Therefore, we investigated the effects of EETs on the proliferation and differentiation of murine fibroblasts. Results showed that the overexpression of CYP2J2 reduced the cell proliferation and the expressions of α-SMA and PCNA induced by transforming growth factor- (TGF-) β1 in murine fibroblasts. Then, we found that EETs inhibited the proliferation and differentiation of TGF-β1-treated-NIH3T3 cells and primary murine fibroblasts. Mechanistically, we found that 14,15-EET disrupted the phosphorylation of Smad2/3 murine fibroblasts by activating PPARγ, which was completely abolished by a PPARγ inhibitor GW9662. Conclusion. our study shows that EETs inhibit the activation of murine fibroblasts by blocking the TGF-β1-Smad2/3 signaling in a PPARγ-dependent manner. Regulating CYP2J2-EET-sEH metabolic pathway may be a potential therapeutic option in PF.