The transition of AECs into mesenchymal cells has been reported to cause and/or aggravate PF . In this study, the direct effects of PTUPB on the TGF-β1-induced EMT were investigated. We found that PTUPB restored the phenotype changes, reduced the migration ability, and inhibited the collagen synthesis of TGF-β1-stimulated AECs by disrupting the TGF-β1-Smad2/3 pathway. We demonstrate for the first time that PTUPB blocks TGF-β1-induced EMT in AECs via inhibition of the TGF-β1-Smad2/3 signaling pathway.
ARA is one of the most abundant polyunsaturated fatty acids in the body . ARA is involved in a variety of biological processes, such as angiogenesis, cell migration, and apoptosis . It has been found that inhibiting sEH could increase endogenous EETs content and reduce the EMT process [23, 32]. 14,15-EET and its synthetic analog EET-A could decrease expression of the EMT inducer factors, ZEB1 and Snail1, prevent the decrease expression of E-cadherin, and reduce expression of mesenchymal/myofibroblast markers in the UUO model . However, another ARA pathway, COX-2 metabolism, promotes EMT. COX-2 inhibitor-induced EMT reversal with restored E-cadherin expression has been observed in several cancer cells [33, 34]. The COX-2 metabolite PGJ2 induces EMT by up-regulating the expression of snails . It can be seen that different metabolites of ARA play different roles in the process of EMT. We found that the protein expression of sEH and COX-2 increased significantly during the TGF-β1-induced EMT process, which was manifested by the disorder of CYP/COX-2 metabolism in ARA.
Studies have found a common phenomenon about the three metabolic pathways of ARA: inhibition of any one of these pathways may shunt ARA to the other pathway, thereby reducing efficacy and causing adverse reactions [36–38]. For example, NSAIDs may have anti-inflammatory effects by inhibiting COX, but their side effects may lead to an increased risk of stroke and kidney failure . At the same time, selective inhibition of COX-2 reduces the levels of endothelin PGI2 and the platelet aggregator TXA2, which increases the risk of cardiovascular disease . Therefore, the development of bimolecular inhibitors targeting ARA metabolism has become increasingly important. It has long been found that drugs targeting a single molecule can produce other toxicity and drug resistance, while drugs targeting multiple molecules are less likely to develop resistance and have better therapeutic effects . PTUPB is a novel COX-2 and sEH dual inhibitor , and we demonstrated that PTUPB could suppress the PF , acute lung injury , non-alcoholic fatty liver disease , and sepsis . However, the direct effects of PTUPB on TGF-β1-induced EMT in AECs are unknown. In the present study, PTUPB significantly improved E-cadherin expression, decreased α-SMA expression, reduced excessive extracellular matrix deposition in BLM-treated mice. TIMPs serve an important role in controlling tissue organization and fibrosis following injury . We found that PTUPB decreased the expression of Timp1 mRNA in BLM-treated PF mice lung tissue, which may be one of the reasons for decreased collagen synthesis.
Further, in vitro EMT models of MLE-12 and A549 cells were induced by exogenous TGF-β1. We found that PTUPB attenuated TGF-β1-induced the acquisition of mesenchymal markers (such as α-SMA), prevented TGF-β1-induced the loss of epithelial markers (such as E-Cadherin), decreased TGF-β1-induced the enhancement of migration ability, reduced TGF-β1-induced the accumulation of collagen synthesis. These results suggest that the regulation of COX-2/CYP metabolism in AECs alleviates TGF-β1-induced EMT. Our results support the hypothesis that inhibition of COX-2/sEH by PTUPB potently inhibits the progression of EMT. In short, our findings indicate that a COX-2 and sEH dual inhibitor shows pivotal therapeutic potential for EMT.
TGF-β1-activated Smads play an important role in the process of EMT . The combination of activated Smad2 or Smad3 and Smad4 can transcriptionally regulate the occurrence of EMT, while blocking the expression of Smad2 or Smad3 can reduce TGF-β1-induced EMT . TGF-β1 activates TβRI by acting on the receptor complex and directly phosphorylates the C-terminal of Smad2 and Smad3. After phosphorylation, Smad2, Smad3, and Smad4 form trimer, which are transported to the nucleus, bind to DNA-binding transcription factors, and cooperatively regulate the transcription of target genes . Our study found that PTUPB significantly reduced TGF-β1-induced phosphorylation of Smad2 and Smad3 in A549. Meanwhile, PTUPB also reduced the phosphorylation level of Smad3 induced by TGF-β1 in MLE12 and tended to decrease the phosphorylation level of Smad2 induced by TGF-β1 in MLE12. From the multiple of Smad2/3 phosphorylation change, we believe that PTUPB mainly inhibited the phosphorylation level of Smad3 in AECs. Whether the reduction of EMT by PTUPB is related to the downstream transcription of the TGF-β1-Smad pathway is unclear. ZEB and SNAIL are transcription factors activated by the TGF-β1-Smad signaling pathway . Our results show that PTUPB reduced the expressions of ZEB1 mRNA and SNAIL1 mRNA. These data indicate that PTUPB could inhibit activation of the TGF-β1-Smad2/3 pathway, therefore suppressing TGF-β1-induced EMT. However, we do not yet know the effect of PTUPB on the TGF-β receptor, which will focus on our further research.
In conclusion, our findings demonstrate that the disorder in the COX-2/CYP metabolism of ARA plays a role in TGF-β1-induced EMT. PTUPB could alleviate EMT, and the mechanism is related to the inhibition of TGF-β1-Smad2/3 pathway activation (Figure 7). This study might promote the application of PTUPB in PF treatment.