Lipoxin A₄ ameliorates lipopolysaccharide-induced lung injury through stimulating epithelial proliferation, reducing epithelial cell apoptosis and inhibits epithelial–mesenchymal transition

LXA4 and LY294002 (PI3K inhibitor) were obtained from Cayman Chemical Company (Ann Arbor, MI, USA). LPS (Escherichia coli serotype 055: B5), Sis3 (smad3 inhibitor) and SP-C antibody were purchased from Sigma (St Louis, MO, USA), BOC-2 (N-t- BOC-PHE-LEU-PHE-LEU-PHE; Gene Script USA Inc., Piscataway, NJ, USA) and BML-111 (Enzo Life Sciences, NY, United States) were purchased from Shang Hai Bo Yun. Antibody against anti-alpha smooth muscle actin (α-SMA) antibody, Vimentin and the secondary antibodies were obtained from Abcam Company (Cambridge, UK). Antibodies against E-cadherin and N-cadherin were from Cell Signaling Technology Company (Boston, USA). Recombinant Human TGF-β₁ (HEK293 derived) was purchased from Peprotech Company (Rocky Hill, USA). DMEM and FBS were purchased from Life Technologies BRL (Grand Island, NY). Protein levels were determined using a Bicinchoninic acid kit (Thermo Scientific).

Human alveolar type II (HAT II) cells were isolated from lungs of grossly normal appearance after lung tumor resection. The cells were isolated in accordance with approval from the local research ethics committees at the University of Wenzhou Medical University (Wen Zhou, China). Primary human AT II cells were extracted according to the methods described previously (see online supplement) [20].

C57BL/6 J mice at 6–8 weeks of age were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd. The animals were acclimatized for 7 days prior to experimental use. Mice were caged with free access to food and fresh water in a temperature-controlled room (22–24 °C) on a 12-h light/dark cycle. Mice (male; ethics code: 2015048) were randomized into 5 groups of 6 mice per group: control group, LPS group (24 h, 48 h, 72 h), LPS + LXA4 group. For the induction of ARDS, mice were anaesthetised and instilled by intra-tracheal (IT) route as a model of direct lung injury with LPS (10 mg/kg dissolved in 30ul N.S) for 24 h, 48 h or 72 h. No treatment control mice were anaesthetised and instilled by intra-tracheal (IT) route with physiological saline. In LPS + LXA4 group, LXA4 was administrated by intraperitoneal injection at 1 μg/per mouse 10 min after intra-tracheal (IT) LPS administration. Mice were subsequently sacrificed by using cervical dislocation, lungs were removed and washed with sterile PBS and stored in 4% paraformaldehyde for HE and immunofluorescence, or at − 80 °C for Western blot, in tube for wet/dry ratio.

Lung tissue were fixed and stained as the method described in the online Supplementary Information.

Total RNA samples in HAT II cells were isolated using TRIzol reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s protocol. The cDNA of mRNA was synthesized using the reverse transcription kit (Bio-Rad, USA). The expression of mRNA was detected using SYBR green super-mix PCR kit (Bio-Rad) by qPCR (ABI7500, Applied Biosystems). The gene-specific primers used are listed in Table 1 and mRNA normalized to GAPDH, was calculated using the 2^-ΔΔCt method.

Cells or lung sections were washed in ice-cold PBS and harvested using RIPA buffer supplemented with protease inhibitors. The resulting supernatant fraction was homogenized in 1x SDS–PAGE sample buffer and boiled for 5 min at 99 °C. For the immunoblotting, protein lysates were electrophoresed via 10% SDS-PAGE gel and then transferred to to polyvinylidene difloride membranes. Membranes were blocked and incubated with the indicated primary antibody (Ab) overnight at 4 °C. Bound primary Abs were incubated with appropriate secondary Abs for 1 h. The proteins were detected using chemiluminescence reagents (Thermo Scientific). Images were scanned with a UVP imaging system and analyzed using an Image Quant LAS 4000 mini system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

Apoptosis of HAT II cells was assessed using flow cytometry. HAT II cells were left in serum free media for 24 h before exposure to LPS (1 μg/ml) with or without LXA4100nM for 24 h. After treatment with LPS and LXA4, HAT II cells were harvested and suspended in the binding buffer supplied in the Annexin V-FITC/Propidium iodide (PI) Apoptosis Detection Kit, and were then stained with Annexin V-FITC and Propidium iodide (PI) according to the manufacturer’s instruction (BD Biosciences, USA). The cytometric data were analyzed with FlowJo software.

The present study adopted randomized, blinded methods. The randomization list of animals was computer-generated by the statistician using SAS/STAT software.

Data are presented as mean ± SD or mean ± SEM. All data were analyzed using one-way ANOVA, followed by a Tukey test for post hoc comparisons. P < 0.05 was considered significant. Statistical analyses were performed using Prism 6.0 software (Graph Pad Software, San Diego, CA).

As shown in Additional file 1: Figure S1, intratracheal instillation of LPS (10 mg/kg) in mice induced lung injury with characteristic neutrophil accumulation, septal thickening, interstitial fluid accumulation, and alveolar hemorrhage at 24 h (Additional file 1: Figure S1B), 48 h (Additional file 1: Figure S1C) and 72 h (Additional file 1: Figure S1D). Treatment with LXA4 attenuated LPS-induced lung injury (Additional file 1: Figure S1E). The lung injury score was consistent with the histopathological changes (Additional file 1: Figure S1F). The Wet/Dry (W/D) ratio increased after LPS treatment, and LXA4 reversed the W/D ratio induced by LPS at 72 h (Additional file 1: Figure S1G), suggesting that LXA4 can alleviate lung permeability damage induced by LPS. The proliferation and apoptosis of AT II cells in the intratracheal LPS murine model of ALI/ARDS were observed by lung specimen immunofluorescence double staining of SP-C (a type II cell marker) and PCNA, SP-C and TUNEL respectively. LPS inhibited the proliferation of AT II cell (SP-C/PCNA double positive cells) and LXA4 abrogated the inhibition of LPS on AT II cells proliferation at 24 h (Fig. 1a, b). Meanwhile, apoptosis of AT II cells was calculated by the co-dying of SP-C and TUNEL. As shown in Fig. 1c and d, LPS increased AT II cell apoptosis, and LXA4 reduced apoptosis of AT II cell induced by LPS at 24 h.

Apoptosis is accompanied by caspase-3 cleavage, so cleaved caspase-3 was measured by both immunofluorescence and western blotting in different groups. Our results demonstrated that LPS increased the expression of cleaved caspase-3 in lung tissue, and LXA4 inhibited LPS-stimulated cleaved caspase-3 expression at 24 h in lung tissue (Fig. 2).

As shown in Fig. 1, LXA4 stimulated HAT II cell proliferation and reduced HAT II cell apoptosis in the intratracheal LPS murine model of ALI/ARDS. Next, we investigated whether LXA4 could also stimulate HAT II cell proliferation and reduce HAT II cell apoptosis in vitro. As shown in Fig. 3a and c, LPS increased HAT II cell apoptosis, and treatment with LXA4 reduced apoptosis of HAT II cell induced by LPS at 24 h. LPS inhibited the proliferation of HAT II cells, while LXA4 promoted their proliferation. (Fig. 3c).

To observe the process of EMT in the LPS-induced lung injury model, we performed immunofluorescence staining of the EMT markers including E-cadherin, α-SMA, N- cadherin and vimentin. We found that LPS reduced the expression of epithelial cell markers E-cadherin in a time dependent manner, while LXA4 promoted the expression of E-cadherin in the lung tissue (Fig. 4a, b). In contrast, LPS increased the expression of mesenchymal cell markers including N- cadherin, α-SMA and vimentin in a time dependent manner, but LXA4 down-regulated the expression of mesenchymal cell markers stimulated by LPS (Fig. 4c-h). To determine whether AT II cells undergo EMT process in LPS-induced lung injury, lung specimens SP-C (a type II cell marker) and α-SMA double staining immunofluorescence were observed. We found that SP-C/α-SMA double positive cells were increased after LPS treatment (Fig. 4i, j). However, treatment with LXA4 significantly decreased SP-C/α-SMA double positive cells in the intratracheal LPS murine model of ALI/ARDS (Fig. 4i, j).

To investigate if TGF-β₁ could induce EMT in HAT II cells, HAT II cells were incubated with TGF-β₁ (0 ng/ml, 5 ng/ml, 10 ng/ml, 20 ng/ml) for 48 h or with TGF-β₁ 10 ng/ml for 0 h, 24 h, 48 h and 72 h. We found that the mRNA levels of epithelial markers, including CDH-1 (Fig. 5a), SP-C (Fig. 5b) and AQP-5 (Fig. 5c) in different TGF-β₁ concentration groups, were all reduced by treatment with TGF-β₁, and 10 ng/ml TGF-β₁ group was lower than other groups. The mRNA levels of mesenchymal markers including CDH-2 (Fig. 5d), snail (Fig. 5e), α-SMA (Fig. 5f), and fibronectin (Fig. 5g), were promoted as the TGF-β₁ concentration increased. There was no significant difference in the mRNA levels between treatment with 10 ng/ml and 20 ng/ml TGF-β₁. After HAT II cells were treated with TGF-β₁ 10 ng/ml for 0 h, 24 h, 48 h and 72 h, the mRNA expression of CDH-1 (Fig. 5h), SP-C (Fig. 5i) and AQP-5 (Fig. 5j) were inhibited by TGF-β₁ at different time points, and the mRNA expression of CDH-1, AQP-5 at 48 h were lower than at other time points, while the mRNA expression of SP-C reached the lowest level at 72 h. However, the mRNA expression of mesenchymal markers, including CDH-2 (Fig. 5k), Snail (Fig. 5l), α-SMA (Fig. 5m), fibronectin (Fig. 5n) were higher at 72 h than other time points, but there was no significant difference in the mRNA levels between treatment with TGF-β₁ for 48 h and 72 h. Based on these results, it is reasonable to establish the vitro EMT model with the concentration of TGF-β₁ 10 ng/ml for 48 h.

To investigate the effect of LXA4 on EMT induced by TGF-β₁, Realtime-PCR and western blotting analyses were applied respectively. As shown in Fig. 6a-g, LXA4 promoted the mRNA expression of epithelial cell markers (CDH1, SP-C and AQP-5) in a does-dependent manner, while inhibiting the mRNA expression of mesenchymal cell markers, including CDH2, Snail, fibronectin and α-SMA in a does-dependent manner. Furthermore, the effects of LXA4 (100 nM) on the TGF-β₁-induced CDH1 (E-cadherin), α-SMA, CDH2 (N-cadherin) protein expression of HAT II cells were confirmed by western blot (Fig. 6h-k).

To identify the involvement of ALX in LXA4 blockade of TGF-β₁ induced EMT, HAT II cells were pre-treated with ALX ligands including BOC-2 10 μM (the LXA4 receptor antagonist) and BML-111 10 μM (the LXA4 receptor agonist) separately for 30 min. The effect of LXA4 on EMT was abrogated by the preincubation of HAT II cells with BOC-2 (Fig. 7a-d). While BML-111 promoted the effects of LXA4 on TGF-β₁-induced EMT in HAT II cells (Fig. 7e-h). These results suggest that the effects of LXA4 on TGF-β₁-induced EMT are mediated via activation of ALX.

To confirm the involvement of the Smad2/3 and PI3K/Akt pathways in LXA4 blockade of TGF-β₁-induced EMT in primary HAT II cells, HAT II cells were pre-treated with 10 μM Sis3 (a specific Smad3 inhibitor) and 10 μM LY294002 (PI3Kinhibitor) for 30 min prior to TGF-β₁ and/or LXA4 administration. Sis3 and LY294002 treatment abolished the inhibitory effect of LXA4 on the EMT in HAT II cells (Fig. 8a-h). To further determine the activity of the PI3K/AKT and SMAD signaling pathways in primary HAT II cells stimulated by TGF-β₁ after treatment with LXA4, the phosphorylation of AKT and Smad in HAT II cells were measured. The expressions of p-AKT and p-Smad were stimulated by TGF-β₁ in primary HAT II cells and significantly downregulated by LXA4 (Fig. 8i-k). The agonist and antagonists had no effect on cell viability (Additional file 1: Figure S2).