Lipopolysaccharide Inhibits Alpha Epithelial Sodium Channel Expression via MiR-124-5p in Alveolar Type 2  Epithelial Cells

Mesenchymal stem cells (MSCs) have been a potential strategy in the pretreatment of pulmonary diseases, while the mechanisms of MSCs-conditioned medium (MSCs-CM) involved with microRNAs on the regulation of lung ion transport are seldom reported. We investigated the role of miR-124-5p in lipopolysaccharide-involved epithelial sodium channel (ENaC) dysfunction and explored the potential target of miR-124-5p. We observed the lower expression of miR-124-5p after the administration of MSCs-CM, and the overexpression or inhibition of miR-124-5p regulated epithelial sodium channel α-subunit (α-ENaC) expression at protein levels in mouse alveolar type 2 epithelial (AT2) cells. We confirmed that α-ENaC is one of the target genes of miR-124-5p through dual luciferase assay and Ussing chamber assay revealed that miR-124-5p inhibited amiloride-sensitive currents associated with ENaC activity in intact H441 monolayers. Our results demonstrate that miR-124-5p can decrease the expression and function of α-ENaC in alveolar epithelial cells by targeting the 3′-UTR. The involvement of MSCs-CM in lipopolysaccharide-induced acute lung injury cell model could be related to the downregulation of miR-124-5p on α-ENaC, which may provide a new target for the treatment of acute lung injury.


Introduction
In addition to inflammation, acute lung injury/acute respiratory distress syndrome (ALI/ARDS) induces extensive capillary damage, leading to noncardiogenic pulmonary edema [1]. erefore, the rate of alveolar fluid clearance (AFC) is a crucial prognostic factor for ALI/ARDS patients, which is mediated by ion transporters, including the alveolar epithelial sodium channel (ENaC) [2,3]. ENaC is a heteromultimeric protein composed of α, β, c, and δ subunits and is essential for the transepithelial absorption of Na + and fluid from alveolar spaces [4,5]. Among all the subunits, only α-ENaC presented Na + -dependent current consistent with active Na + transport in Xenopus laevis oocytes, and in mice with a genetic deficiency for α-ENaC, the newborn were unable to clear airway fluid and died within 40 h of birth [6,7]. ese all suggest the critical importance of α-ENaC required for Na + transport [8].
Here, we aimed to investigate whether miR-124-5p has biologic impacts on the alveolar epithelium, which may exert effects on the pathophysiology of ALI. Mesenchymal stem cells (MSCs) are a subset of cells with a variety of differentiation potential and previous studies have shown the benefits of MSCs-based therapy in respiratory injury [14]. Cell-free MSCs-conditioned medium (MSCs-CM) therapy has been demonstrated to confer pulmonary ischemic tolerance by paracrine factors, and we hypothesized that the administration of MSCs-CM would attenuate ALI through changing the delivery of miR-124-5p into epithelial cells, which will lead to the alteration in the protein expression of ENaC [15]. Using a combination of the LPS-induced ALI cell model, in vitro transfection assays, Western blot, and Ussing chamber, we demonstrated that MSCs-CM participates in the LPS-induced ALI cell model by alleviating the inhibition of miR-124-5p on the expression and function of α-ENaC.
ese findings support a potential therapeutic role for MSCs-CM in ALI through pleiotropic mechanisms.

Animals.
All experiment methods involving C57 mice were performed according to the guidelines of the Animal Care and Use Committee, and all experimental protocols were approved by China Medical University. Mice were kept under pathogen-free conditions.

Preparation of Mouse MSCs-CM.
ree-week-old, weighing 10-13 g pathogen-free C57 male mice were anaesthetized by diazepam (17.5 mg kg − 1 , intraperitoneally) followed by ketamine 6 min later (450 mg kg − 1 , intraperitoneally). e femora were isolated. Bone marrow was collected by gently washing medullary cavity of femora with DMEM/F12 medium (HyClone) supplemented with 10% fetal bovine serum (FBS, Gibco), 10 ng/ml recombinant mouse basic fibroblast growth factor (bFGF, PeproTech), 100 IU penicillin, and 100 μg/ml streptomycin. en, it was cultured in a humidified incubator (5% CO 2 , 37°C) for 24 h. e medium was first changed to remove nonadherent cells and tissues and then replaced every other day. MSCs were passaged after 80% confluent and the cells of the 2 nd and 3 rd passages were used to collect MSCs-CM. At 80% confluence, the medium of MSCs was changed and replaced by FBSdeprived DMEM/F12 medium. MSCs-CM was collected in 24 h and stored at − 80°C freezer after filtering with a 0.22 μm filter.

Isolation and Culture of Mouse Alveolar Type 2 Epithelial
Cells. Isolated lungs from newborn mice (within 24 h) were separated by lobes in cold phosphate buffer saline (PBS). Teased lung tissue was digested with trypsin (Sigma) and collagenase (Sigma) for 30 min, respectively. Cells were filtrated and cultured in 5% CO 2 , 37°C atmosphere in DMEM/F12 containing 10% FBS (Gibco), 100 IU penicillin, and 100 μg/ml streptomycin for 45 min. Unattached cells were collected and the above culture process was repeated 4 times to remove lung fibroblast cells.
en, the cell suspension was transferred on the IgG coated culture dish and incubated for 30 min to remove lymphocytes, macrophages, and neutrophils. Unattached cells were adjusted to 2-3 × 10 6 /ml and the medium was changed after 72 h for the first time and then changed every other day.

Culture of H441 Cells and H441 Monolayers.
Human distal lung epithelial cell line NCI-H441 was obtained from the American Type Culture Collection (ATCC) and cultured as previously described [16]. H441 cells were grown in RPMI medium (ATCC) containing 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g/L glucose, 1.5 g/L sodium bicarbonate, and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). For Ussing chamber assays, cells were seeded on permeable support filters (Costar) at a supraconfluent density (∼5 × 10 6 cells/cm 2 ) and incubated in a humidified atmosphere of 5% CO 2 at 37°C. Dexamethasone (250 nM, Sigma) was supplemented to stimulate ENaC expression. Cells reached confluency in the Costar Transwells 24 h after plating. At this point, media and nonadherent cells in the apical compartment were removed to adapt the cells to the air-liquid interface culture. Culture media in the basolateral compartment were replaced every other day, whereas the apical surface was rinsed with PBS. Transepithelial resistance was measured by an epithelial tissue volt-ohm-meter (World Precision Instruments). Highly polarized tight monolayers with resistance >500 Ω cm 2 were used for measuring short-circuit current (I sc ) levels of transepithelium.

Ussing Chamber Assay.
Measurements of transepithelial I sc and resistance were performed as described previously in H441 monolayers [17]. In brief, H441 monolayers were mounted in Ussing chambers (Physiologic Instrument) and then bathed on both sides with a solution containing (in mM): 120 NaCl, 25 NaHCO 3 , 3.3 KH 2 PO 4 , 0.83 K 2 HPO 4 , 1.2 CaCl 2 , 1.2 MgCl 2 , 10 HEPES sodium salt, and either 10 mannitol for apical compartment or 10 D-glucose for basolateral compartment. e pH of each solution was adjusted to 7.4 and the osmolality was 290-300 mOsm/kg. e solutions of both sides were bubbled with mixed gas containing 95% O 2 and 5% CO 2 at 37°C. I sc was measured with Ag-AgCl electrodes filled with 4% agar in 3 M KCl. e monolayers were short-circuited to 0 mV, and a 10 mV pulse of 1 s was applied every 10 s to monitor the resistance of transepithelium. Acquire and Analyze 2.3 program was used to collect data. ENaC activity is the reduction of I sc after adding amiloride to the apical side (100 μM, amiloridesensitive I sc ).

Western Blot Analysis.
Mouse alveolar type 2 epithelial (AT2) cells were cultured in 6-well plates until 80% confluence and washed three times with PBS and then harvested for immunoblot assays. Proteins were separated on 10% SDS-PAGE gels and transferred onto PVDF membranes (Invitrogen). e blots were incubated in blocking solution containing 20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1% Tween (TBST), and 5% BSA for 1 h at room temperature. en, the membranes were incubated with primary antibody: α-ENaC antibody (PA1-920A, ermo Fisher) and β-actin (sc-47778, Santa Cruz Biotechnology) at 1 : 2000 in TBSTcontaining 5% BSA at 4°C overnight. e specific band between 70 and 100 kDa for α-ENaC protein could be seen, according to the manufacturer's manual and previous studies [18]. Following washing three times with TBST, membranes were incubated with HRP conjugated goat-anti-rabbit or goat-anti-mouse secondary antibody at 1 : 2000 at room temperature for 1 h and then washed for 10 min with TBST three times. Images were developed by an enhanced chemiluminescence (ECL) kit and collected by the ImageJ program.

Real-Time Polymerase Chain Reaction.
Total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions, and spectrophotometry was used to measure the concentration and purity of total RNA. Reverse transcription was performed using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa) and real-time polymerase chain reaction (RT-PCR) with SYBR Premix Ex Taq (TaKaRa) was performed using LightCycler 480 System. e MiR-124-5p level was measured using the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa) and U6 was used as a reference. e following primer sequences were used: miR-124-5p (5′-CGT GTT CAC AGC GGA CCT TGA T-3′), U6 forward (5′-GGA ACG ATA CAG AGA AGA TTA GC-3′) and reverse (5′-TGG AAC GCT TCA CGA ATT TGC G-3′). e data from RT-PCR were analyzed using the 2 − △△CT method.

Transfections.
e negative control, miR-124-5p mimics, miRNA inhibitor negative control, or miR-124-5p inhibitor (GenePharma) was transfected into cells with siRNA-mate (GenePharma) according to the manufacturer's instructions. e final concentration of miR-124-5p mimics was 30 nM and the miR-124-5p inhibitor was 60 nM. All transfection reagents were removed after 6 h and cells were used 48 h after transfection.

Dual Luciferase Reporter Assay.
e luciferase reporter assay was performed to test whether miR-124-5p directly binds to the 3′-UTR of α-ENaC mRNA. e luciferase reporter plasmid, pmirGLO-α-ENaC 3′-UTR, was purchased from GenePharma. Each fragment was of two types: wildtype and mutant. Cotransfection of AT2 cells with the dual luciferase reporter plasmids and miR-124-5p mimics or negative controls were transfected into cells with siRNAmate (GenePharma). After 48 h, luciferase activity was measured using the Dual Luciferase Reporter Assay Kit (Vazyme), according to the manufacturer's instructions.

Statistical Analysis.
All results were presented as mean ± SE. ENaC activity is the difference in the total and amiloride-resistant current fractions. Normality and homoscedasticity test was done by Levene and Shapiro-Wilk test before applying parametric tests. For the comparison of the two groups, we used Student's two-tailed t-test; for the comparison of multiple groups, we performed one-way analysis of variance (ANOVA) followed by Bonferroni's test for all the groups of the experiment. When the data did not pass the normality or homoscedasticity test, we used a nonparametric t-test (Mann-Whitney U test). Variations were considered significant when the P value was less than 0.05. Statistical analysis was performed with Origin 8.0.

MSCs-CM Decreases the Expression of MiR-124-5p in
Mouse AT2 Cells. Previous studies suggested that miR-124-5p involved in the corresponding pathways is related to LPSinduced ALI [11]. In our experiment, we first analyzed the levels of miR-124-5p in mouse AT2 cells after LPS and MSCs-CM administration. We found that exposure to LPS caused a significant increase in miR-124-5p level compared with control which was set as 100% (P < 0.01, n � 4) (Figure 1). Conversely, the levels of miR-124-5p decreased in normal and LPS-treated mouse AT2 cells after the administration of MSCs-CM (P < 0.01 versus control group, and P < 0.05 versus LPS group, n � 4), respectively. ese data proved that the expression level of miR-124-5p increased during ALI and MSCs-CM downregulated the level of miR-124-5p in the LPS-induced ALI cell model.

e Level of MiR-124-5p Is Negatively Correlated with α-ENaC in Mouse AT2
Cells. Previous studies demonstrated that LPS downregulated α-ENaC mRNA expression [19], and we further predicted the binding site between miR-124-5p and α-ENaC. Accordingly, we assumed that LPS may inhibit the expression of α-ENaC protein through miR-124-5p. To test this hypothesis, mouse AT2 cells were transfected with miR-124-5p mimics (Mimic) or inhibitor (Inhibitor), respectively. e effect of miR-124-5p on α-ENaC was examined by Western blot analysis (Figures 2(a) and 2(b)). Transfection of miR-124-5p mimics into mouse AT2 cells resulted in a significant decrease of α-ENaC expression compared with the negative control (NC) group (P < 0.01, n � 4), while the inhibition of miR-124-5p showed no difference (n � 4). ese data suggested that miR-124-5p suppressed the protein expression of α-ENaC. e potential mechanism of ALI induced by LPS may be related to the enhanced miR-124-5p level and sequent inhibition of α-ENaC protein expression.

MiR-124-5p Downregulates α-ENaC Expression by
Binding to the 3′-UTR. Potential miR-124-5p targets were predicted using in silico approaches, and according to the bioinformatic website prediction, we estimated that α-ENaC is a potential target of miR-124-5p. To confirm this finding, a dual luciferase target gene assay was conducted. Wild-type and mutant reporter gene vectors for α-ENaC were constructed, and the sequences of 3′-UTRs of the wild-type and mutant α-ENaC were shown in Figure 3(a). Mouse AT2 cells were cotransfected with the vectors (NC) and miR-124-5p mimics (Mimic). After 24 h, the firefly luciferase activity was measured. Firefly luciferase units were normalized with Renilla luciferase units. We found that the expression of pmirGLO-α-ENaC-WT (Mimic + WT) relative luciferase activity was reduced to 56.53 ± 9.92% by miR-124-5p (P < 0.05, compared with NC + WT), while the expression of pmirGLO-α-ENaC-MT (Mimic + MT) was not suppressed significantly by miR-124-5p (P > 0.05, compared with NC + MT, Figure 3(b)). e above data verified that α-ENaC is one of the target genes of miR-124-5p.

MiR-124-5p Inhibits Amiloride-Sensitive Short-Circuit
Current in H441 Monolayers. Human bronchoalveolar epithelial-derived Clara (H441) cells have been extensively applied in studying the function of ENaC in the lung, and ENaC properties of H441 are similar to those of primary AT2 cells, which could hardly grow into monolayers [20,21]. Amiloride-sensitive AFC reflects ENaC-mediated fluid transport. To further confirm the regulation of miR-124-5p on ENaC activity, we measured I sc in confluent H441 monolayers. As shown in Figure 4(a), amiloride-sensitive I sc (ASI) was defined as the difference between the total current and the amiloride-resistant current, and miR-124-5p decreased ASI from 6.74 ± 0.21 to 3.45 ± 1.24 (μA/cm 2 ) (P < 0.05 versus control, n � 3, Figure 4(b)). ese data indicated that miR-124-5p could inhibit the ion transport of lung through decreasing ENaC activity in alveolar epithelial cells.

MSCs-CM and MiR-124-5p Inhibitor Increase the Protein Expression of α-ENaC in AT2 Cells after LPS Administration.
To further test our hypothesis that MSCs-CM may exert beneficial effects in ALI by inhibiting the LPS-enhanced miR-124-5p expression in AT2 cells, LPS-treated AT2 cells were administrated with MSCs-CM or transfected with miR-124-5p inhibitor negative control (LPS + InNC) or miR-124-5p inhibitor (LPS + Inhibitor), respectively. e α-ENaC expression was examined by Western blot analysis (Figures 5(a) and 5(b)). LPS downregulated α-ENaC expression in AT2 cells compared with the control (CON) group (P < 0.01, n � 4). Incubating with MSCs-CM caused a significant increase of α-ENaC expression compared with LPS alone (P < 0.01, n � 4), and inhibition of miR-124-5p caused a similar increase of α-ENaC expression compared with inhibitor negative control group in LPS-treated AT2 cells (P < 0.01, n � 4). ese data suggested that the potential mechanism of MSCs-CM therapy for ALI induced by LPS may be related to reducing the miR-124-5p level and subsequently increasing α-ENaC protein expression.

MSCs-CM and MiR-124-5p Inhibitor Enhance Amiloride-Sensitive Short-Circuit Currents in H441 Monolayers after LPS
Administration. To test if the above regulation mechanism of MSCs-CM is also related to the function of ENaC, we measured I sc in confluent H441 monolayers in the above groups. As shown in Figures 6(a) and 6(b), LPS decreased ASI from 6.73 ± 0.27 to 2.40 ± 0.50 (μA/cm 2 ) (P < 0.001 versus control, n � 3), indicating that LPS could inhibit the ion transport of lung through decreasing ENaC activity in alveolar epithelial cells. Incubating with MSCs-CM caused a significant increase of ENaC activity compared with LPStreated AT2 cells (P < 0.01, n � 3), and inhibition of miR-124-5p caused a similar increase of ENaC activity compared with inhibitor negative control group in LPS-treated AT2 cells (P < 0.01, n � 3). ese data showed that, compared with LPS alone, the ASI reflecting ENaC activity increased after the transfection of miR-124-5p inhibitor or administration with MSCs-CM in AT2 cells, which nearly recovered to the state of the control group.

Discussion
Pulmonary edema is a major characteristic of LPS-induced ALI/ARDS, which is associated with a leak of fluid into the lung interstitium and the alveolar space. Recovery from ALI requires the removal of fluid from the alveolar space, which contributes to the development, severity, and outcome of pulmonary edema in humans [22]. e AFC process is crucial to efficient gas exchange in the lung, and patients with ALI that have intact AFC have lower morbidity and mortality than those with compromised AFC [23,24]. e active transport of Na + by AT2 cells provides a major driving force for the removal of fluid from the alveolar space. Previous studies revealed that AFC strongly depends on the function of ENaC on the apical surface and Na + /K + -ATPase on the basolateral surface of alveolar epithelial cells [25].
ere were studies revealing that the upregulation of ENaC improved edema fluid reabsorption during pulmonary edema after thiourea-induced lung injury [26]. Because of its role in the regulation of Na + homeostasis, ENaC has been associated with clinical defects of Na + and water transport [27]. Among the four ENaC subunits, altogether three are expressed in mice, named α-, β-, and c-ENaC. Among αβc-ENaC subunits, the α-subunit is believed to form electrically detectable Na + -selective channels, whereas the βand c-subunits serve as accessory regulatory subunits that can increase, by two orders of magnitude, the amplitude of channel activity from the α-subunit alone [16,28,29]. In our previous studies, LPS could reduce the expression of α-and c-ENaC, and we have found that MSCs-CM has similar effects on c-ENaC. Based on several bioinformatic websites,  we have predicted that α-ENaC is a potential target of miR-124-5p, for the seed sequence of miR-124-5p aligns perfectly with the 3′-UTR of α-ENaC mRNA. Accordingly, we chose the α-ENaC subunit as our target for in-depth study. e miRs are an extensive family of short (approximately 22 nucleotides), noncoding RNAs. ey function as posttranscriptional repressors of gene expression by binding predominantly to the 3′-UTR of mRNAs to interfere with protein production [30,31]. ere is currently little information about miRs and Na + regulation [32]. MiR-124-5p has been reported to be related to lung epithelial maturation, but the mechanisms of MSCs-CM involved with miRs on the regulation of lung ion transport are seldom reported [11]. In this study, we first found that the expression of miR-124-5p in mouse AT2 cells decreased after the administration of MSCs-CM, contrary to the effects of LPS alone. A combination of MSCs-CM and LPS could reverse the LPS-induced enhancement of miR-124-5p expression. Consequently, the overexpression or inhibition of miR-124-5p demonstrated the regulation of α-ENaC at the protein level. Western blot analysis showed a marked decrease of α-ENaC protein expression in mouse AT2 cells transfected by miR-124-5p. Next, we confirmed that α-ENaC is one of the target genes of miR-124-5p by Luciferase reporter assay. To test the effects of miR-124-5p on ion transport function, I sc was measured by Ussing chamber assay, and we revealed that miR-124-5p inhibited amiloride-sensitive currents associated with ENaC activity in intact H441 monolayers. Finally, the protein expressions of α-ENaC and ENaC activity were performed, and as expected, both MSCs-CM and miR-124-5p inhibitor caused a significant increase of ENaC expression and activity, which offset the effects of LPS alone. Taken together, our data showed that the level of miR-124-5p increased in the LPS-induced ALI cell model, which could bind 3′-UTR of α-ENaC mRNA and led to the reduction of its expression and function in alveolar epithelial cells, causing the formation of pulmonary edema (Figure 7). Meanwhile, MSCs-CM could increase ENaC expression and activity through reducing the miR-124-5p level to treat LPS-induced ALI.
For recording I sc , the data from primary AT2 cells could be more convincing, but it is a pity that primary AT2 cells from mice are hard to form tight monolayers in the airliquid culture mode and it is nearly impossible to record the I sc in these cells for the technique difficulties [33,34]. H441 is human Clara-like airway epithelial cells, which can easily form monolayers at the air-liquid interface and exhibit a number of recognized morphological structures and functional similarities to primary human AT2 cells [20,21,35,36]. erefore, we recorded the I sc in H441 monolayers in our electrophysiological experiment but applied the other experiments in mouse AT2 cells.
MSCs have been a potential strategy in the pretreatment of pulmonary diseases and MSCs-CM may be a preferable therapeutic option for ALI [37]. MiR-124-3p transferred by MSCs-derived exosomes had been demonstrated to inhibit purinergic receptor P2X ligand-gated ion channel 7P2X7 expression, thus improving oxidative stress injury and suppressing inflammatory response in traumatic ALI [38,39]. In our experiment, we focused on miR-124-5p and found the inhibition of miR-124-5p on α-ENaC by binding the 3′-UTR. MSCs-CM may contain some paracrine factors and inhibit miR-124-5p accordingly by affecting the microenvironment in AT2 cells, and the potential mechanism of MSCs-CM therapy for LPS-induced ALI may be related to reducing the miR-124-5p level and subsequently increasing ENaC protein expression and activity, which benefits the resolution of edema in ALI. However, how the expression level of miR-124-5p was downregulated in MSCs-CM was still a direction that awaits our further investigation.

Conclusions
We demonstrate that MSCs-CM may involve in the LPSinduced ALI cell model by the regulation of miR-124-5p, which can decrease the expression and activity of α-ENaC in alveolar epithelial cells through targeting the 3′-UTR. is is a novel mechanism for MSCs-CM and may provide a new target for the treatment of ALI.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.