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Silencing SMAD4 inhibits inflammation and ferroptosis in asthma by blocking the IL-17A signaling pathway

Respiratory Research volume 25, Article number: 429 (2024) Cite this article

Abstract

Background

The TGF-β/SMAD signaling pathway is crucial in the pathogenesis of asthma. However, SMAD family member 4 (SMAD4), a key mediator of TGF-β, its roles and underlying mechanisms in asthma remain unclear.

Methods

The in vivo and in vitro roles of SMAD4 in asthma were investigated through an ovalbumin (OVA)-induced mouse model and an interleukin-13 (IL-13)-induced cell model. The molecular mechanism of SMAD4 influenced asthma was examined using transcriptome sequencing, followed by feedback experiments involving recombinant human interleukin 17 A (rhIL-17 A), an IL-17 A signaling pathway activator.

Results

SMAD4 was highly expressed in the asthma models. SMAD4 silencing alleviated damage to lung tissue and decreased inflammatory infiltration. Expression levels of Caspase-3, IgG, and inflammatory factors were reduced after silencing SMAD4. Silencing SMAD4 suppressed ferroptosis. Silencing SMAD4 also enhanced IL-13-induced BEAS-2B cell proliferation and suppressed apoptosis. Furthermore. IL-17 A signaling pathway was promoted in the asthma models, as evidenced by elevated IL-17RA, IL-17 A, and Act1 protein levels. SMAD4 silencing inhibited the expression levels of these IL-17 A pathway-associated proteins. Moreover, rhIL-17 A treatment notably reversed the impacts of SMAD4 silencing on asthma in the IL-13-induced cell model and OVA-induced mouse model, indicating that silencing SMAD4 inhibited inflammation and ferroptosis in asthma by blocking the IL-17 A signaling pathway.

Conclusion

Silencing SMAD4 prevents inflammation and ferroptosis in asthma by inhibiting the IL-17 pathway, which provides a novel potential approach for asthma therapy.

Introduction

Asthma, a Th2-cell dependent and IgE-mediated allergic disorder, is characterized by airway inflammation, hyperresponsiveness, and remodeling [1]. More than 300 million people worldwide suffer from asthma, with symptoms including recurrent wheezing, breathing shortness, and chest tightness. In severe cases, asthma can lead to death, posing a serious threat to life and global socio-economic status [2, 3]. Genetics and environmental exposures that affect the immune response are the two major risk factors for severe asthma [4]. Corticosteroids and bronchodilators are the first-line drugs for asthma intervention, and most patients can control their symptoms well with these medications [5]. However, asthma is a heterogeneous lung disease with multiple phenotypes and unique endotypes. This diversity leads to varying responses to treatment and refractory symptoms still occur in patients with severe disease [6]. Therefore, exploring the pathogenesis of asthma is crucial for developing personalized and precise treatments.

SMAD proteins are intracellular signaling mediators of the transforming growth factor β (TGFβ) superfamily and play important roles in immune response, fibrosis, and tumorigenesis [7]. It is reported that inhibiting the TGF-β/SMAD signaling pathway can alleviate asthma [8, 9].SMAD family member 4 (SMAD4), a key element of the TGF-β pathway, forms a complex with receptor-activated SMADs and translocates to the nucleus, where it modulates the transcription of different target genes and impacts a range of biological processes [10]. Several previous studies have revealed that SMAD4 plays an important role in tumor development [11, 12]. Recently, the function of SMAD4 in asthma has attracted increasing attention. Li et al. found that overexpression of TRIM33 reduces SMAD4 expression and inhibits Wnt/β-catenin activation, which regulates asthma inflammation and airway remodeling [13]. Wang et al. has revealed that tectorigenin is effective in preventing ovalbumin (OVA)-induced pulmonary fibrosis and airway inflammation in guinea pigs by modulating the TGF-β1/SMAD signaling pathway, which involved a decrease in SMAD4 expression [14]. However, the specific role and molecular mechanisms of SMAD4 in asthma are still unclear.

Ferroptosis, a newly discovered type of regulated cell death, is defined by iron accumulation and lipid peroxidation [15]. Ferroptosis is implicated in numerous lung disorders, including chronic obstructive pulmonary disease, cystic fibrosis, and obstructive sleep apnea [16]. Furthermore, increasing studies indicate that ferroptosis has a pivotal role in the development of asthma [17, 18]. Iron metabolism, lipid metabolism, reactive oxygen species (ROS) production, and amino acid metabolism are biological processes associated with ferroptosis, and they are all related to asthma [19]. Genes linked to ferroptosis are implicated in asthma and manage the immune microenvironment [20]. SMAD has an important correlation with ferroptosis. TGF-β-SMAD signaling pathway has been proven to affect ferroptosis in cancer cells [21]. AAV9-HGF combined with a TGF-β/SMAD inhibitor reduces silicosis fibrosis by suppressing ferroptosis [22]. However, it is unknown whether SMAD4 influences the development of asthma by regulating ferroptosis.

Interleukin 17 (IL-17), also referred to as IL-17 A, is a pro-inflammatory cytokine produced by T helper 17 (Th17) cells. IL-17 signaling through the IL-17RA/IL-17RC receptor complex triggers the expression of chemokines and cytokines [23, 24]. IL-17 A contributes to the development of multiple chronic inflammatory diseases such as asthma [25]. Increased levels of IL-17 are found in lung biopsy specimens and sputum from asthmatic patients, correlating with severe asthma [26]. It is reported that UBD is implicated in neutrophilic asthma by driving the activation of the IL-17 signaling cascade [27]. Furthermore, anti-inflammatory steroids like glucocorticoids are the preferred treatment for managing airway inflammation and are highly effective for treating persistent asthma; however, Th17 cell-mediated airway inflammation remains resistant to steroids, leaving a subset of asthmatics unresponsive to this therapy [28]. Therefore, it is vital to examine the interplay between SMAD4, ferroptosis, and IL-17 signaling pathways to gain a comprehensive understanding of the potential role of the IL-17 signaling pathway in asthma. This research sought to understand the role and molecular pathways involving SMAD4 in asthma. OVA-induced mice model and IL-13-induced human bronchial epithelial cell line (BEAS-2B cells) model are commonly used to study asthma [29,30,31]. In this study, based on the OVA-induced mice model and IL-13-induced BEAS-2B cell model, we found that by blocking the IL-17 A signaling pathway, silencing SMAD4 mitigated inflammation and ferroptosis in asthma, offering a new approach for treating asthma.

Materials and methods

Animal treatment

Female BALB/c mice (aged 6–7 weeks, 18–20 g) were purchased from the SPF Biotechnology Co. (Beijing, China) and kept in specific pathogen-free and controlled temperature (24 ± 1 °C) conditions. The mice were maintained under a 12-h light/dark cycle and supplied with a normal diet and drinking water. After one week of housing, mice were allocated randomly to four groups (six mice in each group): a sham-treatment group (control), an OVA model group (OVA), an OVA model treated with short hairpin RNA-negative control group (OVA + LV-shNC), and an OVA model treated with sh-SMAD4 (OVA + LV-sh SMAD4). Three days before the OVA model establishment, 200 µl (2 × 107 TU/mL) of sh-NC or sh-SMAD4 was injected into the tail vein of mice [32]. For OVA model induction, mice received an intraperitoneal injection of 125 µl of OVA antigen suspension, which contained 50 µl aluminum hydroxide (Sigma-Aldrich, St Louis, MO, USA), 5 µl 1% OVA (grade V; Sigma-Aldrich), and 70 µl phosphate-buffered saline (PBS). On days 8–21, the mice were kept in sealed containers and then exposed to 20 ml 5% aerosolized OVA (grade II, Sigma-Aldrich) for 30 min. In the control group, mice were treated with an identical volume of PBS in the same way. Apart from asthma-related symptoms, the mice had no other abnormalities. On day 22, mice were euthanized by inhalation of isoflurane. Blood samples from mice were obtained from the right ventricle and then bronchoalveolar lavage fluid (BALF) was retrieved by instilling cold sterile saline (600 µl each time/ three times). Following centrifugation of blood and BALF samples at 3,000 rpm for 10 min at 4 °C, the supernatants were harvested for subsequent measurements. A total of 1 mg of lung tissue was homogenized in 5 ml of ice-cold PBS using a glass homogenizer, centrifuged at 5000 rpm for 10 min at 4 °C, and the supernatant was obtained for further examination.

Hematoxylin–eosin (HE) staining

After being fixed in 4% paraformaldehyde, the lung tissues underwent dehydration, were embedded in paraffin, and sectioned to a thickness of 5 μm. The sections were cleared using xylene and then rehydrated by anhydrous ethanol I and II, 95, 90, 80, and 70% anhydrous ethanol. Following a rinse with distilled water, the sections were immersed in Harris hematoxylin for 5 min. Next, they were differentiated using 1% hydrochloric acid for a few seconds, followed by ammonia treatment to restore the blue color, and counterstained with eosin for 3 min. After hyalinization, sections were sealed with neutral gum, and the histological images were obtained using a light microscope (Olympus, Tokyo, Japan).

Immunohistochemistry

Paraffin-embedded sections were dewaxed, rehydrated, and treated with 10 mmol/L EDTA for antigen repair. Endogenous peroxidase was inactivated by incubating the slices with 0.3% H2O2 at 37 °C. Sections were blocked with normal goat serum for 60 min and then incubated with anti-Caspase-3 antibody (ab184787, 1:1000, Abcam, Cambridge, USA) overnight at 4℃. Then, the sections were treated with a secondary antibody (Abcam) in the dark at 37℃ for 60 min. After performing three washes with PBS, the sections were stained with 3,3-diaminobenzidine (DAB) to develop the staining. Following HE staining, the sections underwent decolorization, dehydration, and sealing, and were subsequently observed with a microscope (Olympus).

Enzyme-linked immunosorbent assay (ELISA)

Inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), IL-4, and interferon gamma (IFN-γ) levels in lung homogenates, BALF, and cell supernatant, and lgG level in serum were detected using ELISA kits (Esebio Biotechnology Co., Ltd., Shanghai, China) following the manufacturer’s guidelines. Briefly, Following the addition of the sample and standard to 96-well plates, 50 µl of the antibody cocktail was subsequently added to each well for 60-min incubation. After washing, tetramethylbenzidine was introduced and terminated using 100 µl of termination solution. The absorbance was assessed at 450 nm with a microplate reader (DALB, Shanghai, China).

Fe2+, MDA, GSH, and ROS measurements

The ferrous iron (Fe2+), malondialdehyde (MDA), glutathione (GSH), and ROS levels were examined in lung homogenates, BALF, and cell supernatant. Briefly, MDA level was quantified with a lipid peroxidation MDA assay kit (S0131S; Beyotime, Beijing, China), Fe2+ content was evaluated utilizing an iron content assay kit (ab83366; Abcam, UK), GSH level was measured using GSH assay kit (S0138S; Beyotime), and ROS level was detected by ROS detection kit (S0033, Beyotime). All procedures followed the manufacturer’s guidelines.

DCFH-DA staining to detect intracellular ROS

Cells were seeded into 12-well plates and DCFH-DA staining was performed when the cell density reached about 80%. After washing twice with PBS, DCFH-DA (10 µM) was introduced into each well and allowed to incubate in the dark for 15 min. Cells were rinsed three times with serum-free medium, followed by measurement of DCF fluorescence using a fluorescence microscope (Nikon, Japan).

Western blot

Protein extraction from lung tissues and cells was conducted employing the RIPA lysis buffer containing a protein inhibitor (Solarbio, Beijing, China). Subsequently, 20 µg of protein was loaded onto the SDS-PAGE gel and then transferred to polyvinylidene difluoride (PVDF; Roche, Basel, Switzerland) membranes. Following blocking with 5% BSA for 2 h at room temperature, the PVDF membranes were incubated with primary antibodies, including SMAD4 (ab155282, Abcam), ACSL4 (ab155282, Abcam), GPX4 (ab125066, Abcam), xCT (ab307601, Abcam), IL-17RA (ab263908, Abcam), IL-17 A (ab79056, Abcam), and Act1 (ab137395, Abcam) with dilutions of 2000, overnight at 4 °C. The following day, PVDF membranes were exposed to HRP-conjugated secondary antibodies for 1.5 h, and proteins were visualized utilizing the ECL reagent and quantified with ImageJ software.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

Total RNA was isolated from lung tissues and cells. The cDNA synthesis was executed with the HiScript III RT SuperMix for qPCR kit (Vazyme). Using SYBR Green qPCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), qPCR was conducted on the 7500 Real-Time PCR System. (Thermo Fisher Scientific). The GAPDH was applied as an internal control. Relative mRNA expression levels were assessed by 2−∆∆Ct method. The primer sequences for qRT-PCR are shown in Supplementary Table 1.

Cell culture and treatment

BEAS-2B cells were acquired from Icellbioscience Biotechnology Co., Ltd. (Shanghai, China). BEAS-2B cells were cultured with RPMI-1640 medium containing 10% fetal bovine serum (Thermo Fisher Scientific) at a 37 °C environment with 5% CO2. To construct an in vitro model of asthma, BEAS-2B cells were induced with the IL-13 (10 ng/ml, Thermo Fisher Scientific) for 24 h [29]. Then, the SMAD4 small interfering RNA target sequences (si-SMAD4-1, si-SMAD4-2, and si-SMAD4-3; RIBOBIO, Guangzhou, China) were transfected into BEAS-2B cells with Lipofectamine 3000 (Thermo Fisher Scientific) in line with the manufacturer’s protocols. siRNA of scrambled sequence (si-NC) was employed as a negative control. After 48 h of transfection, cells were harvested and transfection efficiency was quantified by qRT-PCR. Additionally, cells were treated with recombinant human interleukin 17 A (rhIL-17 A; 50 ng/mL; R&D Systems, Minneapolis, MN, USA) for 24 h to activate the IL-17 signaling pathway [33].

Cell counting kit-8 (CCK-8) assay

BEAS-2B cells were inoculated into 96 plates and incubated for 24, 48,72, and 96 h. After that, 10 ul of CCK-8 reagent (C0039, Beyotime) was introduced into each well and incubated at 37 ℃ for 2 h. Then, absorbance at 450 nm was recorded using a microplate reader (DALB).

Flow cytometry

After washing once with pre-cooled phosphate buffer, BEAS-2B cells were resuspended in 300 µl of 1× binding buffer. 5 µl of Annexin V-FITC was supplemented and incubated in the dark for 15 min, followed by fluorescent labeling of the cell suspension with 5 µl of propidium iodide (PI) solution (Beyotime, C1062L) for 5 min. Apoptosis of cells was evaluated with a FACScan flow cytometer (Becton, Dickinson and Company, New Jersey, USA).

The 5-ethynyl-2′-deoxyuridine (EdU) assay

BEAS-2B cells were inoculated in 96-well plates with EdU medium dilution (C0081S, Beyotime) and then cultivated for 2 h. Subsequently, nuclei were restained by 5 µg/mL Hoechst 33,342. Photographs were taken under a fluorescence microscope (Nikon), and proliferation cells were calculated.

Transcriptome sequencing

Three lung tissue samples from OVA + LV-shNC and OVA + LV-shSMAD4 groups were sent to OE Biotech, Inc. (Shanghai, China) to perform transcriptome sequencing. Total RNA was harvested with TRIzol reagent (Thermo Fisher Scientific) and 2 µg of RNA was taken from each sample for sequencing libraries. The mRNA was cleaved into small fragments of 300 bp in length. mRNA sequencing was implemented on the Illumina Novaseq 6000 platform. HISAT2 V.2.0.5 was employed to align paired-end clean reads to mm10. The differentially expressed genes (DEGs) were identified by the DESeq2 package in R software (version 3.6.3) with |log2FoldChange| > 1 and P < 0.05. The volcano map and heatmap of DEGs were generated with the ggplots2 package and Pheatmap package, respectively. The Gene Ontology (GO) and Kyoto Encyclopedia of Genomes (KEGG) functional enrichment analysis were performed using the clusterProfiler package in R software. The cutoff criterion for significant enrichment was set at P < 0.05.

Statistical analysis

GraphPad Prism 7.0 (GraphPad Software, USA) was used for statistical analyses. Data were shown as mean ± SD. To compare two groups, the t-test was utilized, and one-way analysis of variance (ANOVA) was used for comparisons involving multiple groups. Tukey’s test was then applied for pairwise comparisons post-ANOVA. P < 0.05 was deemed statistically significant.

Results

SMAD4 silencing mitigated lung injury and inflammation in OVA-induced asthmatic mice

First, we examined the expression level of SMAD4 in the asthma mice model. Western blot analysis revealed that protein level of SMAD4 was increased in the asthma model relative to the controls. Then, the role of SMAD4 was explored by constructing an SMAD4-silenced asthma mouse model. Compared to the OVA + LV-shNC group, SMAD4 protein level was reduced in the OVA + LV-sh SMAD4 group, indicating that SMAD4 was effectively knocked down (Fig. 1A). Then, OVA-specific antibody IgG level in mice serum were detected using ELISA assay in different groups. The data revealed that the IgG level was higher in the OVA and OVA + LV-shNC groups than in the control group. At the same time, SMAD4 knockdown significantly reduced the level of IgG compared with the OVA + LV-shNC group (Fig. 1B). Histopathological changes in lungs of mice were explored by HE staining. The results indicated that the alveolar tissue structure and cellular hierarchy of mice were clear, with neatly arranged cells and no inflammatory cell infiltration in the control group. In contrast, the OVA model group showed a marked increase in inflammatory cells, disorganized cellular arrangement, unclear hierarchy, and condensed, solidified nuclei. Notably, silencing of SMAD4 significantly attenuated these symptoms (Fig. 1C). Immunohistochemistry results showed that Caspase-3 expression was enhanced in OVA-induced mice in contrast to controls, which was significantly reversed by SMAD4 silencing (Fig. 1D). Furthermore, the expression levels of pro-inflammatory factors TNF-α, IL-4, and IFN-γ in BALF and lung tissues of mice were elevated. SMAD4 silencing reduced these inflammatory factors levels (Fig. 1E and F). The above results suggest that SMAD4 silencing attenuated lung histopathology and inflammatory response in OVA-induced asthmatic mice.

Fig. 1
figure 1

SMAD family member 4 (SMAD4) was highly expressed in asthma and silencing SMAD4 ameliorated lung injury and inflammation levels in ovalbumin (OVA)-induced asthma mice. A. Western blot was used to detect the protein expression level of SMAD4 in control, OVA, OVA + LV-shNC, and OVA + LV-shSMAD4 groups. B. In mouse serum, IgG expression levels were measured by enzyme-linked immunosorbent assay (ELISA) assay in the four groups. C. Hematoxylin–eosin (HE) staining of mouse lung tissue was performed to assess histopathologic changes. D. Caspase-3 expression level in lung tissues of mice were assessed by immunohistochemistry. E. Expression levels of inflammatory factors, including tumor necrosis factor alpha (TNF-α), interleukin-4 (IL-4), and interferon gamma (IFN- γ) in mouse bronchoalveolar lavage fluid (BALF) and lung tissues were detected by ELISA assay. Scale: 100 μm, Magnification: 200x. N = 6. **P < 0.01 vs. Control; ##P < 0.01 vs. OVA + LV-shNC

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SMAD4 silencing inhibited ferroptosis in OVA-induced asthmatic mice

Previous study indicated that OVA induction promotes ferroptosis in mice [34]. Therefore, this study further examined ferroptosis-related factor levels in OVA-induced asthmatic mice. The results showed that OVA-induced mice exhibited elevated levels of Fe2+, MDA, and ROS and decreased GSH level in BALF and lung tissue compared with controls. Silencing of SMAD4 suppressed Fe2+, MDA, and ROS levels, and promoted GSH level in BALF and lung tissue of OVA-induced asthmatic mice (Fig. 2A and B). Furthermore, based on the western blot results, we found that the protein level of the ACSL4 was elevated, whereas GPX4 and xCT protein levels were reduced in lung tissues of OVA-induced asthmatic mice in comparison with controls, which was significantly reversed by SMAD4 silencing (Fig. 2C).

Fig. 2
figure 2

Silencing of SMAD4 inhibited ferroptosis in OVA-induced asthma mice model. A. ELISA was employed to quantify Fe2+, malondialdehyde (MDA), reactive oxygen species (ROS), and glutathione (GSH) levels in BALF (A) and lung tissues (B). C. Ferroptosis-associated protein expression levels, including Acyl-CoA synthetase long-chain family 4 (ACSL4), glutathione peroxidase 4 (GPX4), and cystine glutamate reverse transporter (xCT) were examined by western blot. N = 6. **P < 0.01 vs. Control; ##P < 0.01 vs. OVA + LV-shNC

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SMAD4 silencing alleviated cell injury and inflammation in IL-13-induced BEAS-2B model

Subsequently, we further explored the role of SMAD4 in the IL-13-induced BEAS-2B cells model. qRT-PCR results showed that the expression level of SMAD4 was increased in IL-13-induced BEAS-2B cells compared to the untreated control group, further validating the high expression of SMAD4 in asthma. Then, SMAD4 expression was effectively knocked down by transfection of si-SMAD4-1, si-SMAD4-2, and si-SMAD4-3, and si-SMAD4-2 was selected for subsequent experiments (Fig. 3A). CCK-8 was utilized to assess BEAS-2B cell viability. The results showed that IL-13 induction inhibited cell viability, which was significantly restored after SMAD4 silencing (Fig. 3B). Similarly, EdU results demonstrated that SMAD4 knockdown notably reversed the reduction in cell proliferation induced by IL-13 (Fig. 3C). Flow cytometry revealed that IL-13 induction promoted apoptosis, whereas silencing of SMAD4 inhibited cell apoptosis (Fig. 3D). Cell inflammatory factor levels were assessed using ELISA, revealing that SMAD4 knockdown considerably counteracted the increased inflammatory factor levels caused by IL-13 induction (Fig. 3E).

Fig. 3
figure 3

Silencing of SMAD4 ameliorated IL-13-induced BEAS-2B cell injury and inflammatory factor levels. A. qRT-PCR was utilized to assess the expression level of SMAD4 and transfection efficiency for si-SMAD4-1, si-SMAD4-2, and si-SMAD4-3. B. IL-13 induced BEAS-2B cell viability was assessed by cell counting kit-8 (CCK-8) assay. C. The proliferation capacity in IL-13-induced BEAS-2B cells was evaluated with the the 5-ethynyl-2-deoxyuridine (EdU) assay. D. Flow cytometry was used to detect apoptosis. E. Cell inflammatory factor levels (TNF-α, IL-4, and IFN-γ) were examined by ELISA assay. **P < 0.01 vs. Control; ##P < 0.01 vs. IL-13 + si-NC

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SMAD4 silencing suppressed ferroptosis in IL-13-induced BEAS-2B model

The previous our data indicated that knocking down SMAD4 inhibited ferroptosis in OVA-induced asthmatic mice. Then, we further verified it in the IL-13-induced BEAS-2B cell model. Induction of BEAS-2B cells with IL-13 resulted in higher Fe2+, MDA, and ROS levels, and lower GSH level compared to the control group. This trend was significantly reversed when SMAD4 was knocked down (Fig. 4A). The DCFH-DA assay was also employed to assess intracellular ROS level. The findings revealed that the level of ROS in IL-13-induced BEAS-2B cells was higher than that in untreated controls. Meanwhile, SMAD4 silencing reduced the ROS level (Fig. 4B). The elevated levels of ACSL4 protein expression, and reduced protein expression levels of GPX4 and xCT in the IL-13-induced BEAS-2B cell model were obviously reversed after SMAD4 knockdown (Fig. 4C). Furthermore, IL-13 treatment upregulated the expression of the autophagy-associated protein Beclin-1 and the pro-apoptotic protein BAX, while simultaneously downregulating the expression of the anti-apoptotic protein Bcl-2. These trends were significantly reversed by SMAD4 silencing (Fig. 4D), and the extent of this reversal was less pronounced than that of the regulation of ferroptosis-related proteins. This suggests that ferroptosis may be the primary regulatory mechanism through which SMAD4 influences asthma.

Fig. 4
figure 4

Silencing SMAD4 suppressed ferroptosis in IL-13-induced BEAS-2B cells. A. Fe2+, MDA, GSH, and ROS levels were assessed by ELISA in IL-13-induced BEAS-2B cells after SMAD4 silencing. B. The DCFH-DA method was employed to assess the level of ROS. C. Protein levels of ACSL4, GPX4, and xCTs, which were associated with ferroptosis, were examined by western blot in IL-13-induced BEAS-2B cells. D. Western blot was used to detect autophagy-associated protein Beclin-1, pro-apoptotic protein BAX, and anti-apoptotic protein Bcl-2 levels in IL-13-induced BEAS-2B cells after SMAD4 silencing.**P < 0.01 vs. Control; #P < 0.05; ##P < 0.01 vs. IL-13 + si-NC

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Transcriptome sequencing and functional enrichment analysis of DEGs

Three lung tissue samples from mice in the OVA + LV-shNC group and OVA + LV-shSMAD4 group were subjected to transcriptome sequencing to explore the molecular mechanism of SMAD4 in asthma. Sequencing results showed that there were 2,121 DEGs between the two groups, of which 1,496 genes were highly expressed and 625 genes were lowly expressed. The top 10 DEGs that were up-regulated and down-regulated are shown in Supplementary Table 2. In addition, DEGs were visualized by volcano and heatmap (Fig. 5A and B). To investigate the potential biological roles of DEGs, we performed GO and KEGG analyses. DEGs between OVA + LV-shNC group and OVA + LV-shSMAD4 group were primarily associated with the biological process (BP) of macromolecule metabolic process, gene expression, and nitrogen compound metabolic process. For cell component (CC), DEGs were primarily related to membrane-bounded organelle, intracellular, and nucleus. For molecular function (MF), DEGs were mostly related to DNA-binding transcription factor activity, RNA polymerase II transcription regulatory region, and double-stranded DNA binding (Fig. 5C). In addition, KEGG pathway analysis demonstrated that DEGs were mostly involved in the IL-17 pathway, p53 pathway, NF-kappa B pathway, and TNF signaling pathway (Fig. 5D).

Fig. 5
figure 5

Transcriptome sequencing and functional enrichment analysis. A. Volcano plots of differentially expressed genes (DEGs) obtained by transcriptome sequencing of mouse lung tissues from OVA + LV-shNC group and OVA + LV-shSMAD4 group. Red represents up-regulated genes, blue represents down-regulated genes, and gray represents genes with no differential expression. DEGs were screened based on |log2FoldChange| >1 and P < 0.05. B. Heatmap of DEGs, with red indicating up-regulated genes and green indicating down-regulated genes. C. The top 10 terms of Gene Ontology (GO) annotation, including molecular functions (MF), biological processes (BP), and cellular components (CC) were displayed in bar charts. D. The top 20 Kyoto Encyclopedia of Genomes (KEGG) pathways were shown in bubble plots

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To verify the reliability of the transcriptome sequencing results, the expression level of the top 2 candidate genes for up- and down-regulation was measured by qRT-PCR at the tissue level. The analysis revealed that in contrast to the OVA + LV-shNC group, KDM5D and DDX3Y expression levels were substantially increased in the OVA + LV-shSMAD4 group, and the expression levels of MARCO and PAX1 were significantly diminished, which was in accordance with the transcriptome sequencing results (Supplementary Fig. 1).

SMAD4 silencing alleviated IL-13-induced BEAS-2B cell injury and inflammation by blocking the IL-17 A signaling pathway

Due to the critical role of the IL-17 signaling pathway in asthma and the important correlation with ferroptosis [35, 36], we then explored the relationship between SMAD4 and IL-17 pathways. In KEGG enrichment analysis, 12 up-regulated DEGs and 4 down-regulated DEGs were enriched in the IL-17 A pathway after SMAD4 knockdown (Supplementary Table 3). We found that the protein levels of IL-17RA, IL-17 A, and Act1 were elevated in the IL-13-induced cell model, and knockdown of SMAD4 reduced their protein levels (Fig. 6A). To assess whether SMAD4 silencing affected asthma progression by regulating the IL-17 signaling pathway, rhIL-17 A was added to activate IL-17 signaling in the IL-13-induced BEAS-2B model with SMAD4 silencing for reversal feedback. According to the CCK-8 assay, cell viability inhibited by IL-13 was restored upon SMAD4 silencing. However, when rhIL-17 A was added, the improvement in cell viability of SMAD4 knockdown was significantly reduced (Fig. 6B). EdU results also demonstrated that rhIL-17 A significantly suppressed the elevation of IL-13-induced BEAS-2B cell viability caused by silencing SMAD4 (Fig. 6C). Flow cytometry data showed that SMAD4 silencing inhibited apoptosis in the asthma cell model, an effect that was substantially reduced by rhIL-17 A treatment (Fig. 6D). Levels of inflammatory factors, including TNF-α, IL-4, and TNF-γ, were reduced in IL-13-induced BEAS-2B cells after silencing of SMAD4, which was substantially counteracted by rhIL-17 A treatment (Fig. 6E).

Fig. 6
figure 6

Silencing SMAD4 alleviated IL-13-induced BEAS-2B cell injury and inflammation by blocking the interleukin 17 A (IL-17 A) signaling pathway. A. The expression levels of IL-17 signaling pathway-related proteins in the IL-13-induced BEAS-2B cells of different groups were measured using western blot analysis. B. IL-13-induced BEAS-2B cell viability was assessed by CCK-8 after treatment with the IL-17 signaling pathway activator rhIL-17 A. C. The EdU assay was used to evaluate cell proliferation after treatment with rhIL-17 A. D. After treatment with the rhIL-17 A, cell apoptosis was assessed using the flow cytometry assay. E. ELISA assay was performed to assess the effect of rhIL-17 A treatment on the levels of inflammatory factors. **P < 0.01 vs. Control; ##P < 0.01 vs. IL-13 + si-NC; $$P < 0.01 vs. IL-13 + si-SMAD4

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SMAD4 silencing suppressed IL-13-induced BEAS-2B cell ferroptosis by blocking the IL-17 A signaling pathway

We then explored the effects on ferroptosis after knocking down SMAD4 to block the IL-17 signaling pathway. ELISA was employed to detect Fe2+, MDA, ROS, and GSH levels, and the results indicated that rhIL-17 A treatment promoted Fe2+ and MDA levels and decreased GSH level compared with the IL-13 + si-SMAD4 group (Fig. 7A). Intracellular ROS level was reduced by SMAD4, which was significantly reversed by rhIL-17 A addition (Fig. 7B). Additionally, the level of the ferroptosis-associated protein ACSL4 was increased, whereas and the protein levels of GPX4 and xCT were reduced after rhIL-17 A treatment in SMAD4-silenced asthma cell model (Fig. 7C).

Fig. 7
figure 7

SMAD4 silencing suppressed IL-13-induced BEAS-2B cell ferroptosis by blocking the IL-17 A signaling pathway. A. After treatment with rhIL-17 A, ELISA was employed to assess the levels of Fe2+, MDA, GSH, and ROS in IL-13-induced BEAS-2B cells. (B) DCFH-DA was conducted to evaluate ROS level following treatment with rhIL-17 A. (C) The protein levels of ACSL4, GPX4, and xCT, which were related to ferroptosis, were determined by western blot analysis. **P < 0.01 vs. Control; ##P < 0.01 vs. IL-13 + si-NC; $$P < 0.01 vs. IL-13 + si-SMAD4

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SMAD4 silencing inhibited ferroptosis in OVA-induced asthmatic mice by blocking the IL-17 A signaling pathway

Furthermore, IL-17RA, IL-17 A, and Act1 protein levels in in lung tissue of mice exhibited the same trend of change as those observed in IL-13-induced BEAS-2B cells. Compared with controls, protein levels of IL-17RA, IL-17 A, and Act1 were elevated in lung tissues, suggesting that the IL-17 A pathway was activated in the asthma mice model. Additionally, we also observed that SMAD4 silencing inhibited the IL-17 signaling pathway in OVA-induced asthmatic mice, accompanied by a decrease in IL-17RA, IL-17 A, and Act1 protein levels (Fig. 8A). Subsequently, rescue experiments were performed using the IL-17 signaling pathway activator rhIL-17 A, to investigate how SMAD4 regulated the IL-17 signaling pathway and its effects on asthma progression in vivo. HE staining revealed that SMAD4 knockdown notably reduced the injury and inflammatory cell infiltration in alveolar tissues of mice. However, the addition of rhIL-17 A significantly reversed these beneficial effects (Fig. 8B). Immunohistochemistry indicated that SMAD4 silencing decreased Caspase-3 expression, which was significantly reversed by rhIL-17 A treatment (Fig. 8C). In addition, lgG in the serum of asthmatic mice and inflammatory factors TNF-α, IL-4, and IFN-γ in the lung tissues were elevated in the OVA + LV-shSMAD4 + rhIL-17 A group in comparison with the OVA + LV-shSMAD4 group (Fig. 8D and E). SMAD4 silencing also reduced the levels of ferroptosis-related factors, including Fe2+, MDA, and ROS and promoted GSH. This trend was significantly attenuated by the rhIL-17 A treatment (Fig. 8F). Moreover, the administration of rhIL-17 A counteracted the suppression of ACSL4 protein expression and the promotion of GPX4 and xCT protein expressions induced by SMAD4 knockdown (Fig. 8G).

Fig. 8
figure 8

SMAD4 silencing suppressed ferroptosis in OVA-induced asthmatic mice by obstructing the IL-17 A signaling pathway. A. After SMAD4 silencing, western blot was used to assess protein levels of IL17RA, IL-17 A, and Act1. B. HE staining was used to assess lung histopathologic changes after SMAD4 silencing and IL-17 pathway activator rhIL-17 A treatment. C. Caspase-3 expression levels was evaluated using immunohistochemistry after SMAD4 silencing and rhIL-17 A treatment. D-E. lgG level in mouse serum and TNF-α, IL-4, and IFN-γ levels in lung tissues were measured by ELISA assay. F. ELISA was used to detect levels of Fe2+, MDA, ROS, and GSH in lung tissues. G. Western blot was performed to assess the expression levels of ferroptosis-related proteins, including ACSL4, GPX4, and xCT. Scale: 100 μm, Magnification: 200x. N = 6. **P < 0.01 vs. Control; ##P < 0.01 vs. OVA + LV-shNC; $$P < 0.01 vs. OVA + LV-shSMAD4

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Discussion

Asthma is a complicated, chronic, and heterogeneous lung disease. Despite notable improvements in asthma morbidity and mortality in the past 15 years, the clinical management of asthma treatment still poses challenges [37]. Hence, further investigation into the molecular mechanisms and potential treatments for asthma is crucial. The TGF-β/SMAD pathway is a key regulator of lung remodeling in asthma, significantly contributing to epithelial changes, subepithelial fibrosis, goblet cell hyperplasia, and smooth muscle proliferation [38]. Multiple studies have demonstrated that targeting the TGF-β/SMAD pathway may be an effective therapy to asthma [39, 40]. For instance, quercetin mitigates asthma-induced airway inflammation and remodeling by reducing periostin through the TGF-β1/SMAD pathway inhibition [41]. Similarly, cordyceps polysaccharide prevents the TGF-β1/SMAD pathway activation, thereby suppressing OVA-induced airway hyperresponsiveness in asthmatic mice [42]. SMAD4, a crucial mediator in the TGF-β signaling pathway, has been associated with various cellular activities, such as inflammation and cell death [43,44,45]. SMAD4 is implicated in Th2 cytokine production, Treg and Th17 differentiation, and the pro-allergic cytokine IL-9 [46, 47]. It is reported that the SMAD4 gene may be a target for age-related genetic risk variants for the first appearance of allergic disease symptoms [21]. Additionally, cryptotanshinone has been shown to attenuate airway remodeling partly by inhibiting SMAD4 expression [48]. Our results corroborate these findings, showing that SMAD4 was highly expressed in the OVA-induced asthma mouse model and IL-13-induced BEAS-2B cells and silencing SMAD4 alleviated inflammation in asthma. Importantly, we found that silencing SMAD4 inhibited inflammation and ferroptosis in asthma by blocking the IL-17 A signaling pathway, which further elucidated the mechanism of the TGF-β/SMAD pathway in asthma as well as provides a new potential target for asthma therapy.

Ferroptosis is a newly identified iron-dependent programmed cell death type, with underlying mechanisms mainly associated with intracellular iron accumulation, GSH depletion, GPX4 inactivation, and lipid peroxidation [49]. Research has demonstrated that ferroptosis in asthmatic epithelial cells is facilitated by IL-13 via SOCS1-mediated ubiquitination and degradation of SLC7A11 [50]. Furthermore, in asthma, excessive iron levels trigger airway inflammation and a hyperoxidative environment, potentially causing ferroptosis [51]. Similarly, in the current research, we found that ferroptosis was enhanced in an OVA-induced asthma animal model and an IL-13-induced cell model, as evidenced by increased levels of Fe2+, MDA, and ROS, as well as decreased level of GSH. Ferroptosis can promote asthma progression. House dust mite exposure disrupts iron homeostasis, increases lipid peroxidation, and activates ferritin phagocytosis in asthma, leading to ferroptosis in airway epithelial cells and promoting inflammation [52]. Ferroptosis plays a role in the disruption of E-cadherin in airway epithelial cells in a mixed granulocytic asthma mouse model [53]. Furthermore, ferroptosis is involved in the exacerbation of OVA-allergic asthma mice induced by dibutyl phthalate through increased levels of ROS and lipid peroxidation [54]. Therefore, inhibiting ferroptosis may be a promising therapeutic strategy for asthma [55]. In IL-13-induced BEAS-2B cells and lung tissues of asthmatic mice, Ferrostatin-1 and 3-methyladenine treatment reduced iron deposition, along with suppression of inflammatory factors (IL-1β, IL-6, and TNF-α) and oxidative stress markers (ROS and MDA), and increased SOD levels, ultimately alleviating asthma [29]. By inhibiting the ferroptosis, Liproxstatin-1 mitigates LPS/IL-13-induced injury to bronchial epithelial cells and neutrophilic asthma in mice [56]. In addition, the study has shown that the effectiveness of acupuncture in treating asthma was associated with the modulation of ferroptosis and the ACSL4-15LO1 pathway [57]. These studies provide the molecular mechanism and theoretical basis for targeting ferroptosis to treat asthma. In this investigation, SMAD4 knockdown inhibited ferroptosis in asthma. Earlier studies have revealed that SMAD4 is involved in iron metabolism by positively modulating hepcidin expression [58]. SMAD4 can regulate the expression of antioxidant enzymes, such as GPX4, to influence the cellular response to oxidative stress and ferroptosis [59]. Our study found that silencing of SMAD4 inhibited ferroptosis in asthma, which has rarely been reported before. In addition, this study further demonstrated that SMAD4 regulated ferroptosis by affecting Fe2+, MDA, ROS, and GSH levels, as well as the protein expressions of ACSL4, GPX4, and xCT. Additionally, programmed cell death, including autophagy, ferroptosis, and necrotic apoptosis contributes to the pathogenesis of asthma. It has been reported that ferroptosis is predominantly associated with airway inflammation, whereas autophagy contributes to airway remodeling, and necroptosis is engaged in the immune response to viral infections in asthma [19, 60]. In the present study, we also found that silencing SMAD4 inhibited autophagy and apoptosis in the IL-13-induced BEAS-2B cells.

We then explored the downstream targets of SMAD4. By transcriptome sequencing of untreated OVA-induced mice and SMAD4-silenced OVA-induced mice, there were 2121 DEGs were screened, which were primarily associated with IL-17 pathway, p53 pathway, NF-kappa B pathway, and TNF pathway. Previous studies have shown that these signaling pathways play an important role in asthma [61,62,63], and SMAD4 may regulate asthma by modulating these pathways. We subsequently chose the IL-17 pathway for an in-depth exploration. Patients with severe asthma typically show a higher frequency of dual-positive Th2/Th17 cells in bronchoalveolar lavage [64]. Furthermore, individuals with IL-17-high asthma exhibit frequent exacerbations, airway neutrophilia, diminished lung microbiota diversity, and urinary biomarker evidence of thromboxane B2 pathway activation [65]. Therefore, defective or blocked IL-17 A signaling may be a promising target for asthma therapy. For example, anti-IL-17 monoclonal antibody attenuates asthma-chronic obstructive pulmonary disease overlap responses [66]. By suppressing the TGF-β1/SMAD2 and IL-17 A signaling pathways, stigmasterol mitigates airway inflammation in OVA-induced asthmatic mice [67]. However, another clinical trial based on 302 asthmatic subjects demonstrates that anti-IL-17 receptor monoclonal antibody does not elicit a therapeutic response in moderate to severe asthma [68]. Therefore, this may require anti-IL-17 receptor monoclonal antibody in combination with other strategies for the treatment of moderate to severe asthma. A study has shown that acupuncture diminishes the level of IL-17 A in BALF by modulating ferroptosis and oxidative stress [69], which points to a notable connection between the IL-17 A signaling pathway and ferroptosis. In addition, another study suggests that reduced IL-17 A levels protect airway epithelial cells via the xCT-GSH-GPX4 antioxidant system and TNF signaling, resulting in the inhibition of OVA-induced ferroptosis and airway inflammation in allergic asthma mice [36]. In house dust mite-induced asthma, Bcl11b influences IL-17 signaling through the TGF-β/SMAD pathway [70]. The present findings further revealed that silencing SMAD4 suppressed inflammation and ferroptosis through the IL-17 A signaling pathway inhibition in an OVA-induced asthma mice model and an IL-13-induced cell model. Therefore, we hypothesize that targeting the TGF-β/SMAD pathway and IL-17 A signaling pathway, in combination with ferroptosis inhibitors may be a promising approach for the treatment of asthma.

Conclusion

In this research, we explore the functions of silencing SMAD4 in an OVA-induced asthma mouse model and an IL-13-induced cellular model, specifically focusing on its impact on ferroptosis and the IL-17 A pathway. Our findings suggest that targeting SMAD4 can mitigate asthma symptoms by inhibiting ferroptosis through the IL-17 A pathway inhibition. This study offers a novel insight into the molecular mechanisms underlying asthma and presents a new avenue for treatment strategies.

Data availability

All data in the manuscript is available through the responsible corresponding author.

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Funding

1. The Natural Science Foundation of Jiangxi Province (no. 20224BAB206009); 2.Science and Technology Research Project of Jiangxi Provincial Department of Education (no. GJJ211528); 3.Ganzhou Guiding Science and Technology Plan Project (no. GZ2023ZSF114); 4. Science and Technology Plan of Jiangxi Provincial Administration of Traditional Chinese Medicine (no. 2023A0189); 5. Science and Technology Plan of Jiangxi Provincial Health and Wellness Committee (no. 202410364); 6. Ganzhou science and technology plan project (no. 2023LNS37749); 7. Key Support Projects of Gannan Medical College (no. ZD201832).

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Authors and Affiliations

  1. Department of Pediatrics, First Affiliated Hospital of Gannan Medical University, No.128, Jinling Road, Zhanggong District, Ganzhou, 341000, China

    Xingyu Rao, Hong Luo & Kaiyuan Luo

  2. Department of Surgery I, The Third Affiliated Hospital of Gannan Medical University/Affiliated Stomatological Hospital, No. 46, Jingjiu Road, Zhanggong District, Ganzhou, Jiangxi Province, 341000, China

    Chaohua Hu

Authors
  1. Xingyu Rao
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  2. Hong Luo
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  3. Kaiyuan Luo
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  4. Chaohua Hu
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Contributions

Xingyu Rao: Substantial contributions to conception and design, data acquisition, drafting the article; Hong Luo and Kaiyuan Luo: data acquisition, drafting the article; Chaohua Hu: data acquisition; reviewing the article; All the authors took part in the experiment.All the authors read and approved the manuscript.

Corresponding author

Correspondence to Chaohua Hu.

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Ethics approval and consent to participate

All methods are reported in accordance with ARRIVE guidelines for the reporting of animal experiments. The animal experiments conformed to the Guide for the Care and Use of Laboratory Animals. Animal study has been approved by the Animal Ethics Committee of First Affiliated Hospital of Gannan Medical University (22SC-2023-290). All methods were performed in accordance with Declaration of Helsinki.

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Not applicable.

Competing interests

The authors declare no competing interests.

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Rao, X., Luo, H., Luo, K. et al. Silencing SMAD4 inhibits inflammation and ferroptosis in asthma by blocking the IL-17A signaling pathway. Respir Res 25, 429 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-024-03052-1

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-024-03052-1

Keywords

Respiratory Research

ISSN: 1465-993X

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