Background
As a lung disease, pulmonary fibrosis (PF) is chronic, progressive and interstitial [
1]. The main histopathological features of lung fibroblasts include fibroblasts losing control of proliferation and excessive accumulation of the extracellular matrix (ECM) deposited by muscle fibroblasts (differentiated from lung fibroblasts) [
2]. Although the pathogenesis of PF has been extensively studied over the past decade, the exact cause of PF has not been elucidated. Our previous studies exploring the role of interleukin-27 (IL-27) in PF have demonstrated that IL-27 attenuates PF both in vivo and in vitro [
3,
4], but the underlying mechanism by which IL-27 attenuates PF is unclear.
Autophagy is an intracellular degradation pathway that is essential for cell homeostasis and is evolutionarily conserved [
5]. Some studies have shown that autophagy may be involved in PF diseases [
6,
7]. In lung epithelial and lung fibroblast cells, reduced autophagic pathways were found in patients with idiopathic pulmonary fibrosis (IPF) [
8]. In addition, autophagy is also involved in regulating the formation of ECM [
9]. Del Principe et al. [
10] found that autophagy deficiency promotes ECM deposition in lung fibroblasts and accelerates the fibrosis process. In addition, IL-27 can induce autophagy in macrophages [
11]. However, it is not clear whether IL-27 can attenuate PF by inducing autophagy.
Increasing evidence shows that long noncoding RNAs (lncRNAs) have an important influence on PF [
12,
13]. Gokey et al. [
14] showed that in IPF epithelial cells, the most improved lncRNA is lncRNA maternally expressed gene 3 (MEG3), which plays an important role in regulating the function of basic progenitor cells. This may contribute to the organizational remodeling of IPF. In addition, Gao et al. [
15] showed that MEG3 attenuates nickel oxide nanoparticle (NiO NP)-induced PF through regulation of Hedgehog signaling pathway-mediated autophagy. In eukaryotes, as a widely found family of serine/threonine protein kinases, MAPKs include p38, extracellular signal-regulated kinases (ERK), and c-Jun NH2-terminal kinase (JNK) [
16]. ERK and p38 have also been shown to be involved in autophagy [
17]. A recent study showed that the use of ERK and p38 pathway inhibitors significantly inhibited the expression of α-smooth muscle actin (α-SMA) and collagen production mediated by TGF-β1, thereby suppressing fibroblast differentiation and ECM production [
18]. However, it is not known whether MEG3 induces autophagy by inhibiting the ERK/p38 pathway to attenuate PF.
Epigenetic changes affected by the environment and aging have an important effect on IPF [
19,
20]. To date, changes, including DNA methylation, histone modification and noncoding RNA expression, are considered epigenetic modifications [
21]. As the main epigenetic modification pathway in mammals, DNA methyltransferase 1 (DNMT1) is responsible for maintaining the methylation of related genes during DNA replication, thus affecting the expression of related genes [
22]. DNMT1 is associated with the development of PF. DNMT1 is significantly highly expressed in silica and BLM-induced IPF, and inhibition of DNMT1 attenuates the extent of PF [
23,
24]. In addition, DNMT1 inhibits MEG3 expression by mediating methylation of the MEG3 promoter region [
25,
26]. However, whether DNMT1 affects the course of PF by influencing the promoter methylation of MGE3 is not known.
In this study, we sought to confirm that the attenuating effect of IL-27 on PF is produced through the induction of autophagy, which is regulated through the DNMT1/lncRNA MEG3/ERK/p38 axis. Our data suggest that hypomethylation of the MEG3 promoter can weaken PF because IL-27 can inhibit the methylation of the MEG3 gene mediated by DNMT1, which inhibits the ERK/p38 pathway to induce autophagy.
Materials and methods
BLM-induced PF
Six- to eight-week-old male C57BL/6 mice were obtained from the Animal Experiment Center of Kunming Medical University. Mice used for experiments were housed in a specific pathogen-free (SPF) environment. As previously mentioned [
3], after a week of adaptive feeding, C57BL/6 mice were randomly divided into three groups: normal control group (given phosphate-buffered saline (PBS) buffer); BLM group (5 mg/kg BLM was dissolved in PBS and given to the mice through intratracheal instillation for a single time); IL-27 group (IL-27 recombinant protein was injected subcutaneously after BLM solution was given for a single time; 1 μg per mouse for 7, 14 and 28 days). All group mice were euthanized and sacrificed on days 7, 14 and 28 of treatment (five mice were sacrificed at each time period in each group). Lung tissues were collected for subsequent analysis. During the construction of the PF model, BLM administration caused death in mice, but the fatality rate was low, approximately 4%. All procedures of this study were performed according to the Helsinki Declaration of the World Medical Association, and the program was approved by the Ethics Committee of Kunming Medical University.
Cell culture and treatment
The MRC-5 (human lung fibroblast-derived) cell line was purchased from the Chinese Academy of Sciences Cell Bank and cultured in minimum essential medium (MEM) containing 10% fetal bovine serum, penicillin (100 μg/mL) and streptomycin 100 (μg/mL). The cell lines were placed in a humidified atmosphere containing 5% CO2 at 37 °C. After pretreatment with PD98059 (1 μmol/L, TOCRIS, Bristol, UK), SB203580 (100 nmol/L, TOCRIS, Bristol, UK), or 3-methyladenine (20 mmol/L, 3-MA, Absin, Shanghai, China) for 2 h, MRC-5 cells were exposed to IL-27 (100 ng/mL, eBioscience, California, USA) and/or TGF-β1 (40 ng/mL, eBioscience, CA, USA) for 48 h.
Cell transfection
In 6-well plates, cells were inoculated to 90% confluence (approximately 1 × 105 cells/well) before transfection. Subsequently, 1 µg of plasmids (DNMT1, MEG3, sh-MEG3, sh-DNMT1 and the corresponding negative control plasmids, Sangon Biotech, Shanghai) were transfected into MRC-5 cells by Lipofectamine™ 2000 (Life Technologies, USA). Subsequently, the cells were placed at 37 °C and cultured under 5% CO2, and the transfection efficiency was measured for subsequent experiments.
Tissue preparation and fibrosis assessment
After the mice were euthanized and sacrificed, part of the right lung tissues of mice were dissected and soaked in 4% paraformaldehyde for 2 days. Then, the sample was sliced into 5-µm-thick paraffin sections after dehydration and embedding. Subsequently, to evaluate alveolitis and PF, hematoxylin and eosin (HE) and Masson staining were used according to the methods in a previous report [
27].
Enzyme-linked immunosorbent assay (ELISA)
In this study, part of the right lung tissues of mice were taken using a tissue homogenizer, and PBS was added to 5 times the volume to fully grind on ice to collect tissue homogenates. The hydroxyproline (HYP) content was measured using an ELISA kit. The ELISA kit was purchased from CUSABIO and used according to the manufacturer's instructions.
Methylation-specific PCR (MSP)
Consistent with the abovementioned method [
26], the methylation level of the MEG3 gene promoter region was determined by a DNA methylation detection kit in this study. The PCR product was purified by a DNA purification kit, and the concentration of DNA was determined by a Nanodrop2000. The purified DNA was reacted with CT conversion reagent for desulfurization and purified again. Subsequently, the purified DNA was collected for MSP amplification. The procedure was as follows: predenaturation at 95 °C for 10 min, denaturation at 95 °C for 45 s, and methylation at 95 °C for 35 cycles. Finally, the PCR products were detected by agarose gel electrophoresis, stained with ethidium bromide and analyzed by an image analysis system.
Chromatin immunoprecipitation
According to previous reports [
26], 4% formaldehyde was used to fix cells, and the cells were disrupted by ultrasonication. After anti-DNMT1 antibody was added to the lysate, it was incubated for 2 h at 4 °C with rotation (100 rpm/min) to interact with the MEG3 promoter. Subsequently, 10 μL of Protein A agarose/SaLmon sperm DNA was added to the lysate and incubated for 2 h to precipitate the protein‒DNA. The beads were washed with NETN buffer three times to remove nonspecific binding. Subsequently, the complex rich in the MEG3 promoter was decrosslinked. Finally, the MEG3 promoter fragment was collected and purified for RT‒qPCR analysis.
RT‒qPCR
In this study, part of mouse right lung tissue was homogenized, total RNA Extractor (Sangon Biotech) was used to extract total RNA from the lung tissues and MRC-5 cells, 1 μL of RNA samples was taken, RNA integrity detection was conducted on 1% agarose gel electrophoresis, and 1 μL of RNA samples was taken after dilution to measure the OD value through the ratio of OD260/OD280 to identify total RNA purity. A cDNA synthesis kit (Vazyme, Nanjing, China) was used to reverse transcribe 2 μg of mRNA into cDNA, and then the cDNA was used as the amplification template in a SYBR reaction system (Vazyme, Nanjing, China). Amplification procedure conditions: predenaturation at 94 °C for 2 min, 40 cycles at 94 °C for 15 s, and annealing at 60 °C for 30 s [
28]. All primers used in this study were designed with Premier 5.0. The internal controls were U6 and GAPDH. The confidence of the PCR results was assessed by the dissociation curve and cycle threshold (CT) values. The results were calculated by the 2
−ΔΔCt method after being repeated at least 3 times.
Ethynyl-2′-deoxyuridine (EdU) assay
In this study, an EdU proliferation test was used to evaluate the proliferation ability of MRC-5 cells by an EdU kit (RIBOBIO, Guangzhou, China). First, the EdU reagent was diluted according to the instructions, and then an appropriate amount was added to the cells and incubated for 2 h. The solution was discarded, PBS was used to wash the cells, and paraformaldehyde solution (4%) was used to fix the cells for 30 min. Then, the paraformaldehyde solution was discarded, and the cells were incubated with 2 mg/mL glycine solution on a decolorizing shaker for 5 min. Next, the fixative was poured off, and the cells were washed with PBS. The cells were then treated with 0.5% Triton X-100 and incubated on a shaker for 10 min. The cells were then washed with PBS once before adding previously prepared Apollo staining solution and destaining. The cells were incubated in the dark on a shaker for 30 min. The staining solution was discarded, the cells were rinsed with PBS, and DAPI was added for nuclear staining. Cells were then incubated in the dark for 30 min, and the staining solution was discarded. After washing with PBS, microscopic photography was performed under a fluorescence microscope.
Western blot analysis
In this study, part of mouse right lung tissue was homogenized, the proteins from lung tissues and MRC-5 cells were extracted utilizing RIPA lysis buffer (Sangon Biotech, Shanghai), and a lysate containing phenylmethanesulfonyl fluoride (PMSF) was added. 50 μL of RIPA lysis and extraction buffer was added to the cultured cells and ground tissues, which were placed on ice for 20 min and centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a precooled EP tube, and a BCA assay (Sangon Biotech, Shanghai) was used to determine the total protein concentration. Proteins were denatured by 2 × SDS loading buffer, and the denatured protein was stored at − 80 °C. The target bands were transferred to polyvinylidene fluoride (PVDF) membranes by taking 50 μg for 10% SDS–polyacrylamide gel electrophoresis (SDS‒PAGE) and using skim milk powder (5%) to block the PVDF membrane for 2 h. PVDF membranes were incubated with the following Abcam antibodies for 12 h at 4 °C: p38 (1:1000), IL-27 (1:1000), α-SMA (1:5000), p-ERK (1:1000), fibronectin (FN; 1:500), collagen I (COL I; 1:5000), LC3B (1:1000), Beclin1 (1:2000), p-p38 (1:1000), collagen III (COL III; 1:1000), ERK (1:1000), and β-actin (1:5000). The secondary antibodies were added, and TBST buffer was used to wash the PVDF membranes. β-Actin was used as a control. Subsequently, chemiluminescent reagents were added, and the bands were analyzed for grayscale values using ImageJ software. Each experiment was repeated 3 times independently.
Immunofluorescence assay
In this study, we used 24-well sterile slides to culture MRC-5 cells for 24 h. After allowing the cells to fuse to 60–70%, IL-27 (100 ng/mL) and/or TGF-β1 (40 ng/mL) were used to treat the cells for 48 h. Immunofluorescence assays were performed according to previous studies [
29]. When incubation was complete, cells were washed three times with prechilled PBS before fixation with immunostaining fixative (Beyotime) for 30 min. Subsequently, cells were incubated with Triton X-100 (Beyotime) permeabilization buffer for 15 min and then blocked with QuickBlock™ (Beyotime) for 30 min at 37 °C. Then, primary antibodies against α-SMA, FN, COL1, LC3B and Beclin1 were incubated with the cells at 4 °C for 12 h. Subsequently, the appropriate fluorescein-conjugated secondary antibody was added to the cells and incubated. The cell nuclei were stained with DAPI. The samples were observed with a confocal microscope and photographed for analysis.
Statistical analysis
GraphPad 8.0 was used to analyze and prepare graphs in this study. Experiments were set up with 3–5 samples/replicates per experiment/group/condition. Data are given as the mean ± SD. In statistical comparisons, Student’s t test was used when there were only two groups of differences. Moreover, one-way analysis of variance (ANOVA) followed by Tukey’s posttest for multiple comparisons was used to determine significant differences for groups of three or more. P values < 0.05 were considered statistically significant.
Discussion
Consistent with previous findings [
3], our results showed that the level of IL-27 in BLM-induced PF mice first increased and then gradually decreased. The attenuating effect of IL-27 on PF may be produced by inhibiting DNMT1-mediated methylation of the MEG3 promoter region, thereby upregulating MEG3 expression and inhibiting the ERK/p38 signaling pathway to induce autophagy.
Autophagy is a self-degrading process [
34]. During PF, overactivation of lung fibroblasts becomes an important pathogenic process [
35]. Regardless of the type of fibrosis, including PF, hepatic fibrosis and renal fibrosis, excessive deposition of ECM is a key disease feature [
36‐
38]. The excessive activation of pulmonary fibroblasts synthesizes ECM components, especially collagen, which in turn promotes fibrosis [
39]. In addition, impaired autophagic flux has been observed in TGF-β1-stimulated lung fibroblasts [
40]. More importantly, a necessary and sufficient condition for the maintenance of normal lung fibroblast fate is the promotion of autophagy [
41]. Therefore, the induction of autophagy in lung fibroblasts could somehow attenuate the course of PF. In this study, in TGF-β1-induced MRC-5 cells, the autophagy-related marker proteins LC3 and Beclin1 were significantly reduced, and IL-27 treatment-induced cellular autophagy and the proliferation viability of MRC-5 cells were inhibited. IL-27-induced cellular autophagy and protection against PF were reversed when the autophagy inhibitor 3-MA was used. In addition, as a multieffect cytokine, IL-27 is related not only to autophagy but also to oxidative stress and inflammatory reactions and plays a corresponding role in many diseases [
42,
43]. Both inflammation and oxidative stress are crucial in inducing the progression of PF, and inhibiting inflammation and oxidative stress can reduce BLM-induced PF [
44,
45]. In this study, combined with the previous discussion, we found that IL-27 can promote autophagy in MRC-5 cells and reduce the expression of TGF-β1-induced fibrosis markers. This expression was partially restored by the addition of 3-MA. However, whether IL-27 participates in inflammatory reactions, oxidative stress or other possible mechanisms is unknown and is worthy of further exploration in future experiments.
The MAPK signaling pathway plays a key role in fibrosis in many major organs, such as myocardial fibrosis, renal fibrosis, and PF [
46]. Epithelial mesenchymal transition (EMT) is a key step in fibrosis [
47]. The MAPK signaling pathway has been found to mediate paraquat-induced EMT in alveolar epithelial cells to promote PF [
48]. In addition, Wang et al. [
49] showed that the phosphorylation levels of JNK, p38 and ERK were significantly elevated in both BLM-induced PF and TGF-β1-stimulated MRC-5 cells and that the MAPK signaling pathway plays a key role in the inhibition of PF. Similarly, Li et al. [
50] showed that p38 phosphorylation was significantly increased in TGF-β1-stimulated human embryonic lung fibroblasts (HLFs) and that the use of p38 MAPK inhibitors inhibited TGF-β1-stimulated HLF proliferation, induced HLF autophagy, and attenuated PF [
51]. In MRC-5 cells, we found that the levels of ERK and p38 induced by TGF-β1-induced phosphorylation were increased, and IL-27 treatment reversed this process. Meanwhile, the use of ERK and p38 inhibitors further promoted the protective effect of IL-27 on PF.
DNA methylation is associated with the pathogenesis and progression of PF [
52]. The prevalence of DNA methylation in PF has been confirmed by DNA methylation analysis and targeted gene studies [
53]. DNMT1 is one of the most important DNA methylation transferases [
54]. It can influence the growth of fibrosis by influencing the hypermethylation of specific genes [
55]. Studies declared that the downregulated MEG3 could be an important indicator of fibrotic diseases, and further mechanism analysis revealed that the overexpressed MEG3 alleviated the progression of distinct organ fibrosis [
56]. DNMT1 regulates MEG3 expression by altering the methylation level of the MEG3 promoter in TGFβ1-induced renal fibrosis, thereby affecting the development of renal fibrosis [
57]. In addition, Gao et al. [
15] showed that MEG3 attenuates nickel oxide nanoparticle (NiO NP)-induced PF through regulation of Hedgehog signaling pathway-mediated autophagy. However, whether DNMT1 mediates MEG3 promoter hypermethylation to affect the development of PF is unclear. In TGF-β1-induced MRC-5 cells, DNA hypermethylation occurred, and DNMT1 expression was upregulated and MEG3 was downregulated after TGF-β1 stimulation, a result that was reversed by treatment with IL-27. Furthermore, we demonstrated that DNMT1 affects MEG3 expression by mediating MEG3 promoter hypermethylation, and IL-27 inhibits DNMT1-mediated MEG3 promoter hypermethylation, thereby upregulating MEG3 expression and thus inhibiting the ERK/p38 pathway to attenuate TGF-β1-induced PF in vitro.
In conclusion, our study shows that IL-27 upregulates MEG3 expression by inhibiting DNMT1-mediated lncRNA MEG3 promoter methylation, which in turn inhibits the ERK/p38 signaling pathway to induce autophagy to attenuate BLM-induced PF, that provides help to elucidate the potential mechanism by which IL-27 attenuates PF.
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