COX-2/sEH Dual Inhibitor PTUPB Inhibits Epithelial-Mesenchymal Transformation of Alveolar Epithelial Cells by Suppressing TGF-β1-Smad2/3 Signaling Pathway


 Background: Arachidonic acid (ARA) metabolites are involved in the pathogenesis of epithelial-mesenchymal transformation (EMT). However, the role of ARA metabolism in the progression of EMT in pulmonary fibrosis (PF) has not been fully elucidated. The purpose of this study was to investigate the role of cytochrome P450 oxidase (CYP)/ soluble epoxide hydrolase (sEH) and cyclooxygenase-2 (COX-2) metabolic disorders of ARA in EMT during PF.Methods: A signal intratracheal injection of bleomycin (BLM) was given to induce PF in C57BL/6J mice. A COX-2/sEH dual inhibitor PTUPB was used to establish the function of CYPs/COX-2 dysregulation to EMT in PF mice. In vitro experiments, murine alveolar epithelial cells (MLE12) and human alveolar epithelial cells (A549) were used to explore the roles and mechanisms of PTUPB on transforming growth factor (TGF)-β1-induced EMT. Results: PTUPB treatment reversed the increase of mesenchymal marker molecule α-smooth muscle actin (α-SMA) and the loss of epithelial marker molecule E-Cadherin in lung tissue of PF mice. In vitro, COX-2 and sEH protein levels were increased in TGF-β1-treated alveolar epithelial cells (AECs). PTUPB decreased the expression of α-SMA and restored the expression of E-cadherin in TGF-β1-treated AECs, accompanied by reduced migration and collagen synthesis. Moreover, PTUPB alleviated the activation of the TGF-β1-Smad2/3 pathway induced by TGF-β1 in AECs.Conclusion: PTUPB inhibits TGF-β1-induced EMT via inhibition of the TGF-β1-Smad2/3 pathway, which holds great promise for the clinical treatment of PF.


Introduction
Pulmonary brosis (PF) is a prototype of chronic, progressive, and brotic lung disease. An altered extracellular matrix replaces healthy tissue and alveolar architecture is destroyed, which leads to the decreased lung compliance, disrupted gas exchange, and ultimately respiratory failure and death [1].
Although pirfenidone and nintedanib have been authorized by the Food and Drug Administration [2], they only slow down lung function decline in patients with mild and moderate disease [3]. Therefore, it's urgent to develop an effective treatment for PF.
Epithelial-mesenchymal transition (EMT) is a reversible process in which epithelial cells lose their cellular polarity and obtain migration characteristics through down-regulation of E-cadherin-mediated cell adhesion [4]. EMT is involved in wound healing, brosis, embryonic development, and cancer metastasis [5]. Most investigators concur that alveolar type II epithelial cells undergo EMT during PF development [6,7]. Studies have shown that pulmonary broblasts are derived from various routes, of which about onethird are derived from alveolar type II epithelial cells via EMT [8]. Transforming growth factor (TGF)-β1 is the most studied and key EMT inducer [9]. TGF-β1 activates its downstream Smad signaling pathway and plays an important role in the process of brosis [10]. TGF-β1 binds to its receptor to trigger intracellular signaling and phosphorylates Smad2 and Smad3. Phosphorylated Smad2 and Smad3 are transported to the nucleus and regulate the transcription of target genes [11]. Consequently, blocking EMT of alveolar epithelial cells (AECs) might be a promising strategy for the treatment of PF.
Our previous study suggested that the expressions of sEH and COX-2 are signi cantly increased in the lungs of PF mice induced by bleomycin (BLM) [24]. A compound that concurrently inhibits both COX-2 and sEH is called 4-(5-phenyl-3-{3-[3-(4-tri uoromethylphenyl)-ureido]-propyl}-pyrazol-1-yl)benzenesulfonamide (PTUPB), which prevents the release of PGs and increases the blood levels of EETs [25]. PTUPB is more potent in suppressing in ammatory pain and tumor growth than celecoxib, t-AUCB (an inhibitor of sEH), or the combination of celecoxib and t-AUCB [25,26]. We have shown that PTUPB can alleviate acute lung injury [27], non-alcoholic fatty liver disease [28], and sepsis [29] in mice. What's more, we have found that PTUPB signi cantly attenuates BLM-induced PF in mice [24]. However, it is not clear whether PTUPB can inhibit EMT. The present study was aimed at investigating the effect of PTUPB on TGF-β1-mediated pulmonary EMT.

Animal
C57BL/6J mice (adult male, 6-8 weeks) were obtained from Hunan SJA Laboratory Animal Co., Ltd (Hunan, China). Mice were placed in speci c pathogen free conditions for a 12 h day-night cycle. Mice have free access to food and water.

Murine model of PF and treatment
Mice were randomly divided into four groups: the control group, PTUPB group, BLM group, and BLM+PTUPB group. For PF induction, mice received an intratracheal injection of BLM (1.5 mg/kg; Nippon Kayaku, Tokyo, Japan) dissolved in 50 µL saline. At the same time, mice in the control and PTUPB groups received 50 µL saline intratracheally. Mice in the PTUPB and BLM+PTUPB groups were subcutaneously injected with PTUPB (5 mg/kg/day) dissolved in PEG400 on the 7th day after BLM injection. PTUPB was given by Bruce D. Hammock at UC Davis Comprehensive Cancer Center, University of California [25].
PEG400 was subcutaneously injected for the control group and BLM groups. Twenty-one days after the BLM injection, mice were sacri ced. All surgeries were performed under anesthesia.

Pulmonary histopathology analysis
The left lung tissue was placed in a tube lled with 4% paraformaldehyde (Servicebio, Wuhan, China, G1101), followed by conventional para n embedding. Para n-embedded sections were made. Hematoxylin-eosin staining (HE) was used to observe the morphological changes of lung tissue of mice, and Masson staining was used to observe the collagen deposition.

Immuno uorescent staining
The lung tissue sections were dewaxed and hydrated. EDTA buffer was used for antigen repair under high temperature and pressure conditions. 3% H 2 O 2 was dropped on the sample for 10 min to achieve the

Scratch wound healing assay
A549 cells were cultured in 12-well plates with 2% bovine calf serum. Assigned areas of the cell surface were scratched with a 200-µL tip and washed with phosphate buffer solution three times [45]. Cells were pre-treated with PTUPB (1 µM) for 1 h, followed by TGF-β1 (10 ng/mL; Novus Biologicals, Littleton, CO, USA). After 48 h of TGF-β1 treatment, the ability of cells to migrate to the scratch area was assessed by measuring the width of the scratch and calculating the difference from the initial width. Photographs were taken with a microscope (Nikon).
The results were detected at 450 nm with a microplate analyzer (Thermo Fisher Scienti c, Waltham, MA, USA).

The quantitative real-time PCR analysis
Total RNA from right middle lung tissue or cells was extracted with RNAiso Plus (Takara, Kusatsu, Japan). Total RNA (1 µg) was reverse transcribed using PrimeScript RT reagent Kit (Takara). Real-time PCR was carried out to detect mRNA expression levels as described in our previous study [46]. Relative expression of genes was computed by the 2 −ΔΔCT method according to our previous study [47]. The sequence of primers used in this study is shown in Table1. Table 1 Sequences of speci c primers were used in this study.

Western blot
Protein from right lower lung tissue or cells was extracted according to our previous research [24].

Statistical analyses
All data were presented as means ± standard deviation. Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software, Inc, San Diego, CA, USA). Multiple group comparisons were made using a one-way analysis of variance. Tukey's test was used as a post hoc test to make pairwise comparisons. Differences between two groups were determined by unpaired t-test. All experiments were independently repeated three times. P < 0.05 was considered statistically signi cant.

PTUPB reduces PF in mice induced by BLM
In this study, a COX-2/sEH dual inhibitor PTUPB (5 mg/kg, s.c. once a day) was employed on the 7th day after BLM administration. Through HE staining and Masson staining, we found that PTUPB treatment for 14 days also signi cantly reduced BLM-induced lung histological changes and collagen deposition in lungs (Figure 1a). PTUPB signi cantly decreased Collagen I protein (Figure 1b-c) and the expression of tissue inhibitors of metalloproteinase 1 (Timp1) mRNA (Figure 1d). At the same time, we found that PTUPB signi cantly reduced α-SMA expression and restored E-cadherin expression in the lungs (Figure  1e-g). These results suggest that the reduction of PF by PTUPB is related to the reduction of EMT.
3.2 COX-2 and sEH expression are increased in TGF-β1treated AECs The protein expressions of COX-2 and sEH were detected in TGF-β1-treated A549 and MLE-12 cells. We found that both COX-2 and sEH protein levels were increased in TGF-β1-treated A549 (Figure 2a

Prophylactic treatments of PTUPB suppresses the TGF-β1-induced EMT in AECs
Then, we wondered whether PTUPB suppressed the EMT induced by TGF-β1 in vitro. We observed that PTUPB alone did not affect the EMT of A549 cells ( Figure S1). Further, we found that the treatment with TGF-β1 (10 ng/mL) for 12 h signi cantly increased the mRNA expression of actin alpha 2 (ACTA2) (encoding α-SMA) and Vimentin, indicating the occurrence of EMT, which was effectively suppressed by the pre-treatment with PTUPB in A549 cells (Figure 3a-b). We found that PTUPB (1 µM) was the most effective inhibition concentration. In addition, western blotting results showed that the pre-treatment with PTUPB (1 µM) reduced α-SMA protein expression and restored E-cadherin protein expression induced by TGF-β1 (10 ng/mL) (Figure 3c-h). Collectively, these results provide strong evidence that PTUPB directly suppresses the EMT induced by TGF-β1 in AECs.

Prophylactic treatments of PTUPB inhibits the migration induced by TGF-β1 in A549 cells
We further investigated the effect of PTUPB on TGF-β1-induced cell migration with the scratch woundhealing assay. The results showed that TGF-β1 treatment (10 ng/mL) for 48 h signi cantly promoted the migration of A549 cells. PTUPB could signi cantly reduce this effect (Figure 4a-b). In order to con rm that PTUPB inhibits cell migration but not cell proliferation, we further evaluated proliferation with CCK-8. Results showed that this effect didn't attribute to the alteration of cell proliferation (Figure 4c). Taken together, these results indicate that PTUPB suppresses cell migration by inhibiting EMT in AECs.

Prophylactic treatments of PTUPB inhibits the collagen synthesis induced by TGF-β1 in AECs
The collagen synthesis can directly re ect the severity of PF. We found that the gene expression of COL1A1 and bronectin (FN) was signi cantly increased in A549 cells stimulated by TGF-β1, which was effectively suppressed by the pre-treatment with PTUPB (Figure 5a-b). TGF-β1 treatment also induced the increase of protein expression of Collagen I in A549 cells and MLE-12 cells (Figure 5c-f). Pre-treatment with PTUPB restored these changes induced by TGF-β1. Altogether, these results indicate that PTUPB inhibits the TGF-β1-induced collagen synthesis in AECs.

Prophylactic treatments of PTUPB disrupts TGF-β1-Smad2/3 signaling pathway in AECs
To elucidate the mechanism under the effect of PTUPB on EMT, we focused on the classical TGF-β1-Smad2/3 signaling pathway. We found that treatment with PTUPB strongly reduced the phosphorylation of Smad2 and Smad3 in A549 cells induced by TGF-β1 treatment for 30 min (Figure 6a-c). Meanwhile, PTUPB was also observed to reduce the phosphorylation Smad3 in MLE12 cells induced by TGF-β1 (Figure 6d-f). At this time, the total protein of Smad2/3 in MLE12 cells and A549 cells did not change ( Figure S2). Then we found that treatment with PTUPB suppressed the gene expression of the downstream targets of TGF-β1-Smad2/3 signaling, including ZEB1 and SNAIL1 (Figure 6g-h). These data indicate that PTUPB blocks the TGF-β1 signaling pathway through the inhibition of TGF-β1-Smad2/3 activation in AECs.

Discussion
The transition of AECs into mesenchymal cells has been reported to cause and/or aggravate PF [6]. 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 rst 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 [30]. ARA is involved in a variety of biological processes, such as angiogenesis, cell migration, and apoptosis [31]. 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/myo broblast markers in the UUO model [23]. 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 [35]. 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 signi cantly 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 e cacy and causing adverse reactions [36][37][38]. For example, NSAIDs may have anti-in ammatory effects by inhibiting COX, but their side effects may lead to an increased risk of stroke and kidney failure [39]. 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 [37]. 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 [40]. PTUPB is a novel COX-2 and sEH dual inhibitor [25], and we demonstrated that PTUPB could suppress the PF [24], acute lung injury [27], non-alcoholic fatty liver disease [28], and sepsis [29]. However, the direct effects of PTUPB on TGF-β1-induced EMT in AECs are unknown. In the present study, PTUPB signi cantly 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 brosis following injury [41]. 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 ndings 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 [42]. 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 [43]. 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 [42]. Our study found that PTUPB signi cantly 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 [44]. 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 ndings 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.

Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Con ict of Interests
The authors declared no con ict of interests.  b-c, n=6). The mRNA expressions of Timp1, Acta2, and Cdh1; were detected by real-time PCR (d-f, n=5-6). The deposition of α-SMA and E-cadherin were detected by immuno uorescence (e, bar=100 μm). Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey's test was used as a post hoc test to make pairwise comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 2
COX-2 and sEH expression are increased in TGF-β1-treated AECs. COX-2 and sEH protein expressions in A549 cells (a-c) and MLE-12 cells (d-f) were detected using western blot (n=3). Data shown are from a representative experiment with biological triplicates. Data are expressed as the mean ± SD. Differences between two groups were determined by unpaired t-test. *P < 0.05. representative experiment with biological triplicates. Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey's test was used as a post hoc test to make pairwise comparisons. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Figure 4
Prophylactic treatments of PTUPB inhibits the migration induced by TGF-β1 in A549 cells. Scratch wound healing assay showed that PTUPB (1 μM) inhibited the migratory ability of the A549 cells under the stimulation of TGF-β1 (10 ng/mL) (a-b, n=3, bar=500 px). PTUPB treatment didn't affect the proliferation of A549 under low-serum condition (c, n=5). Data shown are from a representative experiment with biological triplicates. Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey's test was used as a post hoc test to make pairwise comparisons. * P < 0.05 and ** P < 0.01. biological triplicates. Data are expressed as the mean ± SD. Differences among multiple groups were performed using ANOVA. Tukey's test was used as a post hoc test to make pairwise comparisons. * P < 0.05, ** P < 0.01, and *** P < 0.001.