Introduction: Combined allergic rhinitis and asthma syndrome (CARAS) is a concurrent allergic symptom of diseases of allergic rhinitis and asthma. However, the mechanism of CARAS remains unclear. The study aimed to investigate the impact of microRNA-21 (miR-21) on CARAS via targeting poly (ADP-ribose) polymerase-1 (PARP-1) and phosphoinositide 3-kinase (PI3K)/AKT pathways. Methods: The levels of miR-21-5p and PARP-1 in CARAS patients were detected by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). An ovalbumin-sensitized mouse model of CARAS was established. And knock down of miR-21-5p was constructed by intranasally administering with miR-21-5p shRNA-encoding adeno-associated virus vector. Airway resistance and airway inflammatory response were detected. ELISA was used to evaluate IL-4/IL-5/IL-13 levels in bronchoalveolar lavage fluid (BALF). Expression levels of E-cadherin, fibronectin, and α-SMA were determined using Western blotting. The levels of PARP-1 and the activation of PI3K/AKT were assayed. Results: Downregulation of miR-21-5p relieved pathophysiological symptoms of asthma including airway hyperreactivity and inflammatory cell infiltration. Downregulation of miR-21-5p significantly reduced the levels of IL4, IL-5, and IL-13 in BALF. Additionally, downregulation of miR-21-5p inhibited the epithelial-mesenchymal transition (EMT) process in CARAS mice. Furthermore, miR-21-5p regulated PARP-1 and was involved in PI3K/AKT activation in CARAS mice. Conclusion: Downregulation of miR-21-5p ameliorated CARAS-associated lung injury by alleviating airway inflammation, inhibiting the EMT process, and regulating PARP-1/PI3K/AKT in a mouse model of CARAS.

Combined allergic rhinitis and asthma syndrome (CARAS) is a concurrent allergic symptom of diseases of the upper (allergic rhinitis [AR]) and the lower respiratory tracts (asthma) [1]. AR is a chronic inflammatory disease of the nasal mucosa caused by allergens [2]. Asthma is a chronic respiratory disease characterized by chronic inflammation of the airways and reversible airway obstruction [3]. The prevalence of asthma is 4–20 times higher among AR patients than in the normal population (2–5%) [1]. Rhinitis and asthma have similar causes and pathogenesis [4]. Combined treatment of AR and asthma may improve the diagnostic accuracy of the disease and reduce the reuse of medications [5]. Therefore, it is necessary to explore the pathogenesis of AR developing into CARAS for the prevention and treatment of asthma.

In the development of airway inflammation in AR and asthma, a Th1/Th2 cell imbalance predominates, with the involvement of a variety of immune cells, including eosinophils, mast cells, and T lymphocytes, which secrete inflammatory factors leading to airway hyperresponsiveness and chronic airway inflammation [6, 7]. However, repeated release of inflammatory mediators in asthma leads to airway epithelial damage and undergoes epithelial-mesenchymal transition (EMT), which promotes airway fibrosis and extracellular matrix deposition, ultimately leading to the development of airway remodeling [8‒10].

There are more studies on AR and asthma, but effective markers for CARAS are lacking. Previous studies have shown that many microRNAs (miRNAs), such as miR-21, miR-223, and miR-146a, are crucial in causing airway inflammation in asthma [11]. miR-21 is a widely studied miRNA and has a significant association with asthma, which could regulate the inflammatory response via several pathways [12, 13]. In macrophage-derived exosomes, miR-21-5p decreases Smad7 expression and promotes EMT in airway epithelial cells by the TGFβ7/Smad signaling pathway [14]. Poly (ADP-ribose) polymerase-1 (PARP-1), a member of a protein family comprising 18 members, is involved in DNA repair and inflammatory response [15]. The levels and activities of PARP-1 are increased in a mouse model of airway inflammation, indicating a correlation with inflammation [16]. Our previous research using sequencing and bioinformatics revealed an association between CARAS and miR-21/PARP-1 [17]. Additionally, PARP-1 is directly targeted by miR-21. They regulate migration and EMT of 16HBE cells via the phosphoinositide 3-kinase (PI3K)/AKT pathway [18]. However, there is a lack of in vivo experimental evidence on the regulation of miR-21/PARP-1 in asthma and CARAS. The present study aimed to investigate the effect of miR-21 in CARAS airway inflammation and EMT and to explore the relationship of miR-21 with PARP-1 and PI3K/AKT pathways using an animal model of CARAS.

Patient Samples and Protocols

Approval for this study was granted by the Ethics Committee of Changzhou Second People’s Hospital (2020KY213-01), and written informed consent was obtained from every participant. Clinical information for each participant is shown in Table 1. The protein level of PARP-1 in plasma was measured using the Human PARP-1 ELISA Kit (Shanghai Kexing, China) according to the manufacturer’s protocol. Plasma samples were obtained from 44 patients with CARAS, 31 patients with AR, and 42 healthy controls. Blood samples used to perform quantitative reverse transcription polymerase chain reaction (RT-qPCR) were obtained from 38 patients with CARAS, 25 patients with AR, and 42 healthy controls. These participants were the same individuals from whom plasma samples were collected. Transcript levels of miR-21-5p and PARP-1 were detected in blood samples using RT-qPCR. Total RNA was extracted using the RNAliquid RNA Blood Kit (Aidlab, China). The primer sequences are shown in Table 2. The diagnostic criteria and severity classification of AR were referred to the Chinese guidelines for the diagnosis and treatment of AR [19], and the diagnosis was made by considering the patient’s allergy history, clinical symptoms, and allergen testing results. Asthma diagnosis was based on China guidelines for bronchial asthma prevention and management (2020 version) [20], and CARAS was diagnosed by meeting the diagnostic criteria for both AR and asthma. CARAS patients were primary patients who were not treated with medications. Normal controls were individuals without a history of AR and asthma and free of other allergic, pulmonary, and immune system disorders. Participants were excluded if they had bronchiectasis, tuberculosis, chronic obstructive pulmonary disease, hematological disorders, malignancy, or other confounding factors. During the clinical visit, height, weight, lung function, and hematology were measured, and blood and plasma were collected.

Table 1.

Clinical characteristics of subjects

VariableHealthy (n = 42)AR (n = 31)CARAS (n = 44)p value
Sex (M/F) 19/23 17/14 21/23 0.732 
Age 37.38±1.56 41.45±2.13 43.68±2.13 0.0582 
Allergic history (Y/N) 0/42 8/23 15/29 <0.001 
Smoking history (Y/N) 6/36 5/26 9/35 0.747 
FEV1%, pre 104.08±3.99 97.10±4.88 81.10±2.82 <0.001*,+ 
FEV1/FVC 81.54±1.79 78.95±2.23 70.78±1.71 0.003*,+ 
EOS 0.13±0.07 0.21±0.03 0.48±0.36 <0.001#,+ 
FeNO (ppb) 27.04±19.89 25.26±2.66 59.83±7.23 <0.001*,+ 
FnNO (ppb) 423.30±27.77 520.89±37.38 522.14±52.48 0.1401 
VariableHealthy (n = 42)AR (n = 31)CARAS (n = 44)p value
Sex (M/F) 19/23 17/14 21/23 0.732 
Age 37.38±1.56 41.45±2.13 43.68±2.13 0.0582 
Allergic history (Y/N) 0/42 8/23 15/29 <0.001 
Smoking history (Y/N) 6/36 5/26 9/35 0.747 
FEV1%, pre 104.08±3.99 97.10±4.88 81.10±2.82 <0.001*,+ 
FEV1/FVC 81.54±1.79 78.95±2.23 70.78±1.71 0.003*,+ 
EOS 0.13±0.07 0.21±0.03 0.48±0.36 <0.001#,+ 
FeNO (ppb) 27.04±19.89 25.26±2.66 59.83±7.23 <0.001*,+ 
FnNO (ppb) 423.30±27.77 520.89±37.38 522.14±52.48 0.1401 

#Healthy versus AR: p < 0.05.

+Healthy versus CARAS: p < 0.05.

*AR versus CARAS: p < 0.05.

Table 2.

Primer sequences used for the RT-qPCR

Primer namePrimer sequences (5′ to 3′)
Human GAPDH-F (internal reference) CTG​GGC​TAC​ACT​GAG​CAC​C 
Human GAPDH-R (internal reference) AAG​TGG​TCG​TTG​AGG​GCA​ATG 
Human ACTB-F (internal reference) TCC​GCA​AAG​ACC​TGT​ACG​C 
Human ACTB-R (internal reference) CTG​GAA​GGT​GGA​CAG​CGA​G 
Human PARP-1-F CGG​AGT​CTT​CGG​ATA​AGC​TCT 
Human PARP-1-R TTT​CCA​TCA​AAC​ATG​GGC​GAC 
Human hsa-miR-21-5p TAG​CTT​ATC​AGA​CTG​ATG​TTG​A 
Mouse β-actin-F (internal reference) CTC​CTG​AGC​GCA​AGT​ACT​CT 
Mouse β-actin-R (internal reference) TAC​TCC​TGC​TTG​CTG​ATC​CAC 
Mouse PARP-1-F GCT​TTA​TCG​AGT​GGA​GTA​CGC 
Mouse PARP-1-R GGA​GGG​AGT​CCT​TGG​GAA​TAC 
Mouse U6-F (internal reference) CTC​GCT​TCG​GCA​GCA​CAT​ATA​CT 
Mouse U6-R (internal reference) ACG​CTT​CAC​GAA​TTT​GCG​TGT​C 
Mouse miR-21-5p-F ACA​CTC​CAG​CTG​GGT​AGC​TTA​TCA​GAC​TGA 
Mouse miR-21-5p-R GTG​TCG​TGG​AGT​CGG​CAA​TTC 
Primer namePrimer sequences (5′ to 3′)
Human GAPDH-F (internal reference) CTG​GGC​TAC​ACT​GAG​CAC​C 
Human GAPDH-R (internal reference) AAG​TGG​TCG​TTG​AGG​GCA​ATG 
Human ACTB-F (internal reference) TCC​GCA​AAG​ACC​TGT​ACG​C 
Human ACTB-R (internal reference) CTG​GAA​GGT​GGA​CAG​CGA​G 
Human PARP-1-F CGG​AGT​CTT​CGG​ATA​AGC​TCT 
Human PARP-1-R TTT​CCA​TCA​AAC​ATG​GGC​GAC 
Human hsa-miR-21-5p TAG​CTT​ATC​AGA​CTG​ATG​TTG​A 
Mouse β-actin-F (internal reference) CTC​CTG​AGC​GCA​AGT​ACT​CT 
Mouse β-actin-R (internal reference) TAC​TCC​TGC​TTG​CTG​ATC​CAC 
Mouse PARP-1-F GCT​TTA​TCG​AGT​GGA​GTA​CGC 
Mouse PARP-1-R GGA​GGG​AGT​CCT​TGG​GAA​TAC 
Mouse U6-F (internal reference) CTC​GCT​TCG​GCA​GCA​CAT​ATA​CT 
Mouse U6-R (internal reference) ACG​CTT​CAC​GAA​TTT​GCG​TGT​C 
Mouse miR-21-5p-F ACA​CTC​CAG​CTG​GGT​AGC​TTA​TCA​GAC​TGA 
Mouse miR-21-5p-R GTG​TCG​TGG​AGT​CGG​CAA​TTC 

Experimental Animal

Twelve female BALB/c mice, weighing 16–18 g and aged 6 weeks each, were purchased from the Beijing HFK Bioscience Co., Ltd (Beijing, China). The mice were specific-pathogen-free and were kept in conditions that included a temperature of 20–24°C, a 12-h light-dark cycle, and a humidity of 40–70%. The animals were fed in separate cages at least 7 days before experiments. The animal license was No. SCXK 2019-0008 (Beijing, China). The quality test of animals was performed by the Institute of Laboratory Animals Sciences, CAMS (Beijing, China).

Construction of Animal Models

Specific-pathogen-free female BALB/c mice were randomly divided equally into four groups (3 mice per group): control, CARAS, CARAS+AAV (adeno-associated virus), CARAS+AAV-miR-21-5p inhibitor. The mice were sensitized and challenged with ovalbumin (OVA) to establish a mouse model of CARAS. In the CARAS+AAV-miR-21-5p inhibitor group, AAV carrying miR-21-5p was administered nasally into CARAS mice to knock down miR-21-5p. The CARAS+AAV group served as the control, receiving an empty AAV. On day 0, the AAV group of mice received 30 μL of AAV (1E + 13 GC/mL) (AAV9, Azenta) via nasal drip. And the AAV-miR-21-5p inhibitor group of mice received 0.3 mL of the AAV (miR-21-5p) virus (1E + 12 GC/mL), inhibiting the miR-21-5p gene. Mice in other groups were maintained without viral treatment. The mice were sensitized by intraperitoneal injection of 0.1 mL OVA emulsified in AL(OH)3 on days 7 and 14. The mice were challenged by a 10-μL droplet of OVA (1 mg/mL) into each nostril using a micropipette, 3 times a week for 3 consecutive weeks from day 28. Twenty-four h after the last intranasal drip, the mice were challenged with 5 mL of 2% OVA using an ultrasonic atomizer (REF, China) once a day for 30 min on 5 consecutive days. Airway resistance was measured, and samples were harvested on day 51 (48 h after the last challenge). The CARAS mouse model was constructed according to the method of reference [21].

Measurement of Airway Resistance

Mice were weighed and anesthetized with 60 mg/kg pentobarbital sodium intraperitoneally [22]. An 18 G tube (Buxco, USA) was inserted into the pharynx via an incision. One end of the cannula was connected to an experimental precision injection pump with a flow rate of 1 mL/s and an injection/retraction of 10 mL. The other end of the cannula was connected to a bio-signal collector (Zhongshi Technology, China) that detected the pressure value. Airway resistance was acquired through the ratio of pressure difference to the flow rate minus intubation resistance. After measuring the baseline resistance, a series of increasing doses (with concentration doubling) of Ach (6.25, 12.5, 25, and 50 mg/mL) was given to oral cavity through a nebulizer. Meanwhile, the injection pump was withdrawn, and a barrier was set up to prevent the aerosol from entering the lower airway. The change of the resistance was recorded after 3 min.

Enzyme-Linked Immunosorbent Assay

The levels of IL-4 (Shanghai Kexing, China), IL-5 (Shanghai Kexing, China), and IL-13 (Shanghai Kexing, China) in bronchoalveolar lavage fluid were evaluated by enzyme-linked immunosorbent assay according to the manufacturer’s instructions.

Lung Pathology

Lung tissues from mice were extracted and preserved in 4% paraformaldehyde for 24 h. Then, the tissues were embedded and sectioned. The inflammatory cell infiltration was evaluated with hematoxylin and eosin staining. Following dewaxing and rehydration, the sections were stained in hematoxylin staining solution for 5–10 min and treated with differentiation solution for 30 s. After washing, the sections were placed in eosin staining solution for 30 s to 2 min and immersed in running water for 2–5 min. Finally, the sections were dehydrated, sealed, and photographed.

The lung tissues were quantified with periodic acid-Schiff staining, respectively. The sections were immersed in periodate alcohol solution for 10 min and placed in reducing solution for 1 min. The sections were stained with a saline magenta solution for 1–1.5 h. After washing, the nuclei were incubated with Mayer’s Hematoxylin Solution for 5 min and then differentiated with 1% hydrochloric acid alcohol. Finally, the sections were dehydrated, sealed, and photographed.

Immunohistochemistry

Lung tissue sections were permeabilized and blocked for 30 min (preheat 40 mL of PBS with 120 μL of TritonX-100 for a few minutes and add 400 μL of 30% H2O2 before use). The antigen was then repaired by sodium citrate buffer (pH = 6.0) (ZSGB-BIO, China). After blocking, the sections were incubated with an anti-PARP-1 antibody (1:1,000, Bioss, China) overnight at 4°C in wet environments and incubated with a secondary antibody for 30 min at 37°C, followed by immersing in SP for 30 min at 37°C and staining with DAB for 3–10 min. Sections were washed 3 times for 5 min with PBS between each step. Nuclear, plasma, and membrane proteins were stained, respectively. The expression of PARP-1 was quantified by the brown area/total tissue area using ImageJ (https://imagej.net/ij/).

Immunofluorescence

Lung tissue sections were permeabilized and then blocked with serum for 1 h. The sections were incubated with anti-fibronectin (1:200, Bioss, China) antibodies at 4°C overnight and washed five times for 3 min with PBS. Then they were labeled with FITC-conjugated antibodies (1:200, Bioss, China) for 60 min at 37°C, washed, and preceded with DAPI (Beyotime, China) staining. After washing, the sections were sealed with an Anti-Fluorescence Quenching Agent (MDL, China). Finally, the fluorescence intensity was quantified by the red area/total tissue area using ImageJ.

Quantitative Reverse Transcription PCR

TRIzol® solution (Invitrogen, USA) was used to extract total RNA from lung tissues. The concentration and purity of extracted total RNA were determined by a nucleic acid concentration tester. RNA concentrations were transferred to cDNA with the SuperScript III Reverse Transcriptase Kit (Invitrogen, USA). mRNA expression levels of PARP-1 and miR-21-5p were detected with RT-qPCR using the SYBR Premix ExTaq (Takara Bio, Inc., Otsu, Japan), which used β-actin and U6 as internal reference genes, respectively. The reaction system with primers, cDNA, and mix was programmed as follows: incubation at 95°C for 5 min, followed by 40 cycles for 10 s at 95°C, 20 s at 58°C, and 20 s at 72°C. The relative expression was determined using the 2−ΔΔct method, considering p < 0.05 as statistically significant. The primer sequences are shown in Table 2.

Western Blotting Analysis

Fresh lung tissues were collected and homogenized in liquid nitrogen. Then, the samples were added to 1 mL PBS, centrifuged, and lysed in lysis buffer. Subsequently, ultrasonication was performed to the samples in ice for 10 s, repeated 3 times, and centrifuged for 15 min to collect supernatant. Therefore, total protein was obtained. The BCA protein assay kit (MDL, China) was employed to quantify protein concentrations. Equal amounts of protein were transferred to the PVDF membrane by transfer gel electrophoresis. The membranes were incubated with anti-PARP-1 (1:2,000), fibronectin (1:2,000), α-SMA (1:2,000, Bioss, China), E-cadherin (1:100), PI3K (1:2,000), AKT (1:2,000, ImmunoWay, USA), phosphorylated AKT (p-AKT, 1:5,000, Proteintech, China), and β-actin (1:1,000, Affinity, China) antibodies for overnight at 4°C, followed by incubation with secondary antibody for 1 h at room temperature. The intensity of each band was quantitatively determined using Gel-Pro Analyzer Software (CLINX, China). The bands were analyzed by ImageJ (https://imagej.nih.gov/ij/). The values of the target genes were normalized by the internal reference genes.

Statistical Analysis

Data were shown as the means ± standard deviation. A t test was used between two samples. One-way ANOVA followed by Dunnett’s multiple-comparison test was performed on more than two samples using GraphPad Prism version 8.3.0 (USA, www.graphpad.com). p values <0.05 were considered statistically significant.

miR-21-5p Expression Is Upregulated in CARAS Patients

We analyzed miR-21-5p and PARP-1 expression levels in blood samples from patients suffering from AR and CARAS. miR-21-5p was significantly upregulated in CARAS patients compared with controls, while there was no significant difference in miR-21-5p expression levels between AR and CARAS patients (Fig. 1a). There is a tendency for transcript levels of PARP-1 to decrease in CARAS patients (Fig. 1a). The protein levels of PARP-1 were significantly downregulated in serum samples from patients with CARAS (Fig. 1b). The raw expression data for miR-21-5p and PARP-1 are presented in online supplementary File 1 (for all online suppl. material, see https://doi.org/10.1159/000538252).

Fig. 1.

miR-21-5p expression is upregulated in CARAS patients. Detection of miR-21-5p and PARP-1 levels in blood (a) by RT-qPCR from healthy subjects (n = 42) and AR (n = 25) and CARAS (n = 38) patients and in plasma (b) by ELISA from healthy subjects (n = 42) and AR (n = 31) and CARAS (n = 44) patients. *p < 0.05, **p < 0.01. ELISA, enzyme-linked immunosorbent assay.

Fig. 1.

miR-21-5p expression is upregulated in CARAS patients. Detection of miR-21-5p and PARP-1 levels in blood (a) by RT-qPCR from healthy subjects (n = 42) and AR (n = 25) and CARAS (n = 38) patients and in plasma (b) by ELISA from healthy subjects (n = 42) and AR (n = 31) and CARAS (n = 44) patients. *p < 0.05, **p < 0.01. ELISA, enzyme-linked immunosorbent assay.

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Downregulation of miR-21-5p Attenuates Pathophysiological Symptoms in the CARAS Mouse Model

As shown in Figure 2a, b, miR-21-5p was successfully knocked down in BALB/c mice using the AAV vector (Fig. 2a, b). We administered different concentrations of Ach to four groups to evaluate miR-21-5p effects on airway resistance. The baseline resistance showed no significant differences among the four groups. At doses of Ach ranging from 12.5 to 50 mg/mL, the airway resistance of mice increased substantially in the CARAS group compared with the control group. As miR-21-5p levels decreased, the CARAS mice had significantly lower airway resistance than controls (Fig. 2c).

Fig. 2.

Downregulation of miR-21-5p alleviates pathological features in the CARAS mice models. a Mice models of CARAS were established, and AAV-miR-21-5p was administered before the nasal challenge. b Levels of miR-21-5p were detected by RT-qPCR. c Airway resistance detected in mice exposed to Ach. #p < 0.05 compared with the control group, *p < 0.05 compared with the CARAS+AAV group. d ELISA for the detection of IL-4, IL-5, and IL-13 in BALF. e Detection of airway inflammation using HE and PAS staining. Inflammatory substances (black arrow). Connective tissue (yellow arrow). Intercellular edema with tissue congestion (green arrow) (original magnification, ×400, scale bar, 200 μm). Data were presented as mean ± SD, n = 3. ***p < 0.001, ****p < 0.0001. ELISA, enzyme-linked immunosorbent assay; BALF, bronchoalveolar lavage fluid; HE, hematoxylin and eosin; PAS, periodic acid-Schiff; SD, standard deviation.

Fig. 2.

Downregulation of miR-21-5p alleviates pathological features in the CARAS mice models. a Mice models of CARAS were established, and AAV-miR-21-5p was administered before the nasal challenge. b Levels of miR-21-5p were detected by RT-qPCR. c Airway resistance detected in mice exposed to Ach. #p < 0.05 compared with the control group, *p < 0.05 compared with the CARAS+AAV group. d ELISA for the detection of IL-4, IL-5, and IL-13 in BALF. e Detection of airway inflammation using HE and PAS staining. Inflammatory substances (black arrow). Connective tissue (yellow arrow). Intercellular edema with tissue congestion (green arrow) (original magnification, ×400, scale bar, 200 μm). Data were presented as mean ± SD, n = 3. ***p < 0.001, ****p < 0.0001. ELISA, enzyme-linked immunosorbent assay; BALF, bronchoalveolar lavage fluid; HE, hematoxylin and eosin; PAS, periodic acid-Schiff; SD, standard deviation.

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We analyzed the cytokine levels of IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid to confirm the impact of miR-21-5p on pulmonary function. Inhibition of miR-21-5p expression in CARAS mice significantly reduced the levels of IL-4, IL-5, and IL-13 (p < 0.05) (Fig. 2d).

We also investigated the histological morphology and inflammatory cell infiltration of the lungs in the mouse model of CARAS by examining lung tissue sections (Fig. 2e). The results showed diffuse exudation of inflammatory substances, deformation of the trachea, formation of a significant amount of connective tissue around the trachea, and intercellular edema with tissue congestion. A low level of miR-21-5p showed typical histological lung morphology and a decrease in inflammatory cell infiltration, including a decrease in monocytes, macrophages, neutrophils, and lymphocytes (Fig. 2e). Periodic acid-Schiff staining showed a significant decrease in positive reactions along with decreased miR-21-5p levels (Fig. 2e; online suppl. File 2).

Downregulation of miR-21-5p Alleviates EMT in the CARAS Mouse Model

In both asthma mice models and human asthmatics, the presence of EMT in airway epithelial cells was investigated [23, 24]. To investigate the impact of miR-21-5p on CARAS mice, we analyzed the expression of E-cadherin, a marker for epithelial cells, and fibronectin and α-SMA, which are markers for mesenchymal cells. Downregulation of E-cadherin and upregulation of fibronectin and α-SMA were observed in CARAS mice compared with controls (Fig. 3a). Immunofluorescence results also showed that the expression levels of fibronectin were increased in CARAS mice (Fig. 3b). These results indicated that EMT took place. Knockdown of miR-21-5p reversed these results (Fig. 3; online suppl. File 3).

Fig. 3.

Downregulation of miR-21-5p alleviates EMT in the CARAS mice models. a Western blotting analysis evaluated the expression of EMT-associated makers in mice lung tissues, including E-cadherin, fibronectin, and α-SMA. ****p < 0.0001. b Levels of fibronectin were assessed in mice lung tissues by immunofluorescence (original magnification, ×400, scale bar, 30 μm) (left) and scored (right). Data were presented as mean ± SD, n = 3. SD, standard deviation.

Fig. 3.

Downregulation of miR-21-5p alleviates EMT in the CARAS mice models. a Western blotting analysis evaluated the expression of EMT-associated makers in mice lung tissues, including E-cadherin, fibronectin, and α-SMA. ****p < 0.0001. b Levels of fibronectin were assessed in mice lung tissues by immunofluorescence (original magnification, ×400, scale bar, 30 μm) (left) and scored (right). Data were presented as mean ± SD, n = 3. SD, standard deviation.

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Downregulation of miR-21-5p Increases PARP-1 Levels in the CARAS Mouse Model

In our previous study, we demonstrated that PARP-1 is a direct target of miR-21 by in vitro experiments [25]. Then, we examined the changes in PARP-1 levels in CARAS mice with downregulated miR-21-5p expression to determine whether miR-21-5p affects CARAS in mice by targeting PARP-1. The results showed that PARP-1 levels were significantly increased in CARAS mice compared with controls. Downregulation of miR-21-5p increased PARP-1 mRNA (Fig. 4a) and protein (Fig. 4b) levels in CARAS mice. Immunohistochemistry also showed consistent results (Fig. 4c) (online suppl. File 4).

Fig. 4.

Downregulation of miR-21-5p increases PARP-1 levels in the CARAS mice models. RT-qPCR (a), Western blotting (b), and immunohistochemical assessment (c) of PARP-1 levels in mice lung tissues. Representative images (original magnification, ×400) (left) and scores (right). Data were presented as mean ± SD, n = 3. *p < 0.05, ***p < 0.001, ****p < 0.0001. SD, standard deviation.

Fig. 4.

Downregulation of miR-21-5p increases PARP-1 levels in the CARAS mice models. RT-qPCR (a), Western blotting (b), and immunohistochemical assessment (c) of PARP-1 levels in mice lung tissues. Representative images (original magnification, ×400) (left) and scores (right). Data were presented as mean ± SD, n = 3. *p < 0.05, ***p < 0.001, ****p < 0.0001. SD, standard deviation.

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PI3K/AKT May Be a Downstream Signaling Pathway of miR-21-5p

We measured the protein levels of PI3K, AKT, and p-AKT to investigate how miR-21-5p affects CARAS. PI3K, AKT, and p-AKT levels to be significantly increased in CARAS mice compared to the control group. In CARAS mice, downregulation of miR-21-5p was followed by a decrease in PI3K and AKT/p-AKT protein levels (Fig. 5a, b; online suppl. File 5). These results suggested that miR-21-5p may regulate CARAS by affecting the PI3K/AKT signaling pathway. All original images of the Western blots are shown in online supplementary File 6.

Fig. 5.

PI3K/AKT may be a downstream signaling pathway of miR-21-5p. The protein levels of p-AKT (a) and the protein levels of PI3K and AKT (b) were evaluated using Western blotting analysis in lung tissue. Data were presented as mean ± SD, n = 3. ***p < 0.001, ****p < 0.0001. SD, standard deviation.

Fig. 5.

PI3K/AKT may be a downstream signaling pathway of miR-21-5p. The protein levels of p-AKT (a) and the protein levels of PI3K and AKT (b) were evaluated using Western blotting analysis in lung tissue. Data were presented as mean ± SD, n = 3. ***p < 0.001, ****p < 0.0001. SD, standard deviation.

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AR and asthma have similar pathogenesis, and CARAS refers to patients with both [1]. Many studies reported the higher incidence and deaths in asthma [26]. AR is a risk factor for the development of asthma [27]. It is important to identify key biomarkers for AR developing into CARAS to prevent and treat asthma. In the present study, we first demonstrated that downregulation of miR-21-5p attenuated the airway inflammation in CARAS mice, and PARP-1/PI3K/AKT might be involved in the molecular mechanism.

Several studies have shown the significant impact of miR-21 on the development of asthma. miR-21 levels are increased in individuals with asthma and can be used as a biomarker for allergic inflammatory diseases in humans [28, 29]. Meanwhile, a comprehensive analysis of microRNAs has shown that miR-21 may be a biomarker for diagnosing asthma [30]. In a mice model of allergic airway disease, high levels of miR-21 allow for the activation of PI3K/AKT and the inhibition of histone deacetylase 2, resulting in severe steroid insensitivity [31]. miR-21 inhibitor reduces the proliferation and migration of human airway smooth muscle cells by regulating PTEN/PI3K [32]. Moreover, miR-21 can exacerbate asthma symptoms by increasing airway inflammation and oxidative stress [33]. Previous studies indicated that inhibition of miR-21 can improve lung function by reducing airway inflammation, hyperreactivity, and remodeling, thereby treating acute lung injury [34, 35]. According to our research, we found that miR-21-5p was significantly elevated in both CARAS patients and CARAS mice. In addition, the knockdown of miR-21-5p in CARAS mice resulted in a decrease in airway hyperresponsiveness and airway inflammation. These results suggested the importance of miR-21 in the treatment of CARAS.

In recent years, repairing damage to the airway epithelium has become a major focus of research. Airway remodeling has been reported to be a primary pathological characteristic of asthma [18]. The pathological process of asthma can cause airway remodeling due to the release of inflammatory mediators [8, 36]. It is widely acknowledged that EMT plays a crucial role in promoting airway remodeling in asthma [37]. It has been reported that EMT-mediated airway remodeling can be attenuated by DTA-64 through TGF-β1/Smad, MAPK, and PI3K pathway in asthma [24]. miR-21 has also been shown to be associated with the EMT process. There is increasing evidence that miR-21 regulates cancer progression by inhibiting EMT through multiple signaling pathways [38, 39]. In the present study, we discovered that the knockdown of miR-21-5p expression prevented the EMT process in CARAS mice. Therefore, miR-21-5p may be a potential target for regulating airway remodeling in CARAS mice.

The PI3K/AKT signaling pathway is an important signaling pathway in cells. Inhibition of the PI3K/AKT signaling pathway with targeted molecules has been shown to attenuate pathological changes in asthma and play an important role in airway protection [40]. Inhibition of the PI3K/AKT pathway reduces inflammatory cell infiltration and airway remodeling and improves lung function in in vivo studies [41, 42]. Moreover, miR-21 can be a target to inhibit the PI3K/AKT signaling pathway to enhance corticosteroid sensitivity in severe asthma [43]. Several studies have shown that miR-21 is associated with the PI3K/AKT signaling pathway and EMT in regulating biological processes [44‒46].

PARP-1 is involved in various cellular processes such as DNA repair, recombination, proliferation, apoptosis, transcription, and inflammation [47‒49]. Numerous studies have indicated that PARP-1 plays a role in regulating the inflammatory response of the airways and airway remodeling is associated with asthma [50, 51]. In addition, PARP-1 has been shown to regulate the EMT process in multiple cell types [52, 53]. In this study, consistent with bioinformatics analyses and cellular experiments [17, 18], we demonstrated that miR-21-5p can modulate PARP-1 and PI3K/AKT. Our previous study demonstrated that PARP-1 is a direct target of miR-21 in 16HBE cells and human airway smooth muscle cells [18, 25]. Taken together, miR-21-5p may regulate CARAS-related symptoms by targeting PARP-1 and activating PI3K/AKT in CARAS mice. However, it may be necessary to conduct further studies to investigate the mechanisms of PARP-1 to regulate asthma as previous studies have produced contradictory results.

In conclusion, our study indicated that miR-21-5p/PARP-1 may be a potential target for the treatment of CARAS. Knockdown of miR-21-5p resulted in increased PARP-1 levels, decreased PI3K/AKT levels, alleviated airway hyperresponsiveness and inflammation, and inhibited the progress of EMT. miR-21-5p may be involved in the activation of PI3K/AKT signaling by targeting PARP-1, which in turn regulates inflammatory and EMT processes, thus affecting the development of AR to CARAS. However, further studies are necessary to investigate the relationship between miR-21, PARP-1, PI3K/AKT, and EMT using the CARAS animal model.

We thank all the members for their generous participation.

The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Changzhou Second People’s Hospital, Jiangsu Province (2020KY213-01), for the human participants, and written informed consent was obtained from every participant. The study was approved by the Ethics Committee of Changzhou Second People’s Hospital, Jiangsu Province (2020KY213-01), for the animals used. All methods were conducted in accordance with the ARRIVE guidelines (https://arriveguidelines.org).

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflicts with the subject matter or materials discussed in the manuscript apart from those disclosed.

This study was supported by grants from the Jiangsu Province Social Development Project (BE2020651 to Qian Zhang) and in part from the Changzhou High-Level Medical Talents Training Project (2022CZLJ013 to Qian Zhang) and the Nanjing Medical University Changzhou Medical Center Research Project (CMCB202214 to Qian Zhang).

Z.M.: conceptualization, data curation, investigation, methodology, validation, and writing – original draft. Z.D.: conceptualization, data curation, investigation, methodology, visualization, and writing – original draft. Z.L. and Y.S.: methodology, software, validation, and formal analysis. Q.Z.: conceptualization, supervision, project administration, resources, and writing – review and editing. All the authors read and approved the final manuscript.

Additional Information

Zhengdao Mao and Ziqi Ding contributed equally to this work.Edited by: H.-U. Simon, Bern.

All data generated or analyzed during this study are included in this article and its online supplementary files. Further inquiries can be directed to the corresponding author.

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