Thirty 6-week-old female BABL/c mice with SPF condition, weighing 20 $\pm$ 2 g, were obtained from the Beijing Baiaosike Biomedical Technology Co., Ltd (Beijing, China). The RSV Long strain was acquired from the Institute of Virology of Jiangsu Provincial Center for Disease Control and Prevention (Nanjing, China), and grown in Hep-2 cells, which are derived from human epithelial carcinoma. Additionally, the viral concentration was determined using the Reed-Muench technique, and the resulting titers of the samples fell within the range of 10⁸ TCID50 (median tissue culture infective dose)/mL in this investigation. All experimental protocols of this study were approved by ethics committee (NO.DWYJ-2022-001).

The research found that ribavirin + budesonide has therapeutic efficacy for asthma [5]. The BABL/c mice were randomly assigned to six groups, each consisting of five mice: the asthma model (M) group, the normal control (NC) group, the ribavirin + budesonide (PC) group serving as the positive control, and three groups receiving different doses of IC (GN10278, GLPBIO, Montclair, CA, USA): low (IC-L), medium (IC-M), and high (IC-H) dose. The asthma model in these mice was created based on previously described methods with some modifications [18]. With the exception of the NC group, all mice in the remaining groups were sensitized on day 1 and day 14 by intraperitoneal injection of 100 µg ovalbumin (OVA, S7951, Sigma, Saint Louis, MO, USA) and 2 mg aluminum hydroxide (239186, Merck, Germany) dissolved in 200 µL of physiological saline. During days 15–49, the mice were exposed every other day with a 20-minute exposure to aerosolized 0.99% OVA in saline. Additionally, on days 21, 35, and 49, the mice were intranasally given 50 µL RSV (1 $\times$ 10⁶ TCID50/mL). Furthermore, from day 15 to day 49, the IC-L, IC-M, and IC-H groups received intragastric doses of IC at 10 mg/kg/d, 20 mg/kg/d, and 40 mg/kg/d, respectively. The mice in the NC and M groups received an equivalent quantity of normal saline. In contrast the Rbv + Bud group was given ribavirin (60269ES03, Yeasen Biotechnology Co. Ltd., Shanghai, China) at 90 mg/kg/day orally and aerosolized budesonide at 0.2 mg/kg/day (H20140087, AstraZeneca AB, Sweden) from days 15 to 49. Apart from the Rbv + Bud group, all other mice received 30-minute daily treatments of aerosolized normal saline. On the 50th day, after the last OVA/RSV challenge, the mice were observed for sneezing, nose-scratching, and the severity of asthma, were assessed over a 15-minute interval. Following this, anesthesia was administered by intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg) (20090512, SINOPHARM, Beijing, China). Cervical dislocation method: the cervical vertebrae of the mouse are quickly twisted by hand, causing spinal cord injury, resulting in the mouse losing consciousness and stopping breathing immediately. Finally, blood, bronchoalveolar lavage fluid (BALF) and lung tissues were extracted for subsequent analysis.

Following euthanasia, blood samples from the abdominal aorta. The blood samples were spun in a centrifuge at a speed of 2700 g for 15 minutes at a temperature of 4 °C to separate and analyze the inflammatory factors present in the serum. Ultimately, the concentrations of OVA-specific IgE, malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) were measured using ELISA kits (OVA-specific IgE, ml037618, Enzyme-linked Biotechnology Co., Ltd. Shanghai, China; MDA, SP30131; SOD, SP14390; CAT, SP14946; GSH, SP14856, Saipei Biotechnology Co., Ltd., Wuhan, China). The concentrations of IL-13, IL-4, and IL-5 were assessed using Beyotime provided kits (IL-13, PI539; IL-4, PI612; IL-5, PI620, Shanghai, China).

Lung tissues from each mouse were excised and weighed. The left lung tissues were then fixed in a 4% paraformaldehyde solution (41678, Acros Organics, Brussels, Belgium) for 24 hours before being processed in paraffin for further processing. Subsequently, the preserved lung tissues were sliced into sections that were 3 micrometers thick. These sections were then stained using hematoxylin and eosin (HE, GF1010, Shanghai G$\cdot$fan Biological Co. Ltd., Shanghai, China) as well as TUNEL staining (C1088, Beyotime, Shanghai, China), to evaluate inflammation in the lung airways. Images of the stained tissue sections were taken with a microscope at a magnification of 200 times. To determine the severity of inflammatory cell infiltration, a blind cell count of peribronchial cells was conducted using a 5-point scoring system described by Duan et al. [19], with specific scoring criteria: 0, a score of 0 meant there were no inflammatory cells; 1, a score of 1 indicated a small number of inflammatory cells; 2, a score of 2 suggested a thin layer of one or two inflammatory cells around the bronchi or blood vessels; 3, a score of 3 showed a noticeable layer of three to five inflammatory cells around the bronchi or vessels; 4, a score of 4 indicated more than five layers of inflammatory cells surrounding these structures [18, 19, 20]. The thickness of the airway wall was assessed using Image-Pro® Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) from Media Cybernetics. Five random, non-overlapping areas were selected from each lung tissue sample to count the number of apoptotic cells.

After euthanizing the mice, their lungs were rinsed with cold PBS. The resulting BALF was then spun at 2700 g for 10 minutes at 4 °C. The liquid part was separated and kept at –80 °C for later analysis. To count the cells, the samples were stained with Wright-Giemsa dye from Nanjing Jiancheng Bioengineering Institute (D010-1-1, Nanjing, China). The quantity of inflammatory cells, such as neutrophils, eosinophils, and lymphocytes, was then determined using a light microscope (XSP-17C, Leica, Germany).

Both the PD-1-specific siRNA and its corresponding control siRNA were designed and synthesized by HUAGENE (Shanghai, China). The sequences for si-PD-1 was 5^′-CATTCACTTGGGCTGTGCTGCAGTT-3^′. Lipofectamine 3000 (L3000015, Invitrogen, Mountain View, CA, USA) served as the transfection reagent, while si-NC acted as the control. The sequence of si-NC was 5^′-CTACTTACTGCGTGGTTCGGCTTGA-3^′. The si-NC and si-PD-1 were injected via the tail vein separately for 48 hours. The injection frequency was once every two days, and after three repetitions of siRNA treatment, PD-1 knockdown was assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot.

To extract total protein from 100 mg of right lung tissue, the tissue was treated with cold-RIPA lysis buffer. The amount of protein was then measured using a bicinchoninic acid (BCA) reagent kit (P0010S, Beyotime, Shanghai, China). Following this, protein samples with uniform concentrations were separated through 10~15% SDS-PAGE (P0012A, Beyotime, Shanghai, China). Upon transfer to the PVDF membranes (FFP19, Beyotime, Shanghai, China) provided by Millipore in the USA, the membranes were blotted and subjected to overnight incubation with the primary antibodies (Bax, ab53154, Abcam, 1:1000; Bcl-2, ab194583, Abcam, 1:2000; Cleaved-Caspase 3, ab2302, Abcam, 1:2000; Nrf2, AF0639, Affinity, 1:2000; HO-1, AF5393, Affinity, 1:1000; PD-1, ab89828, Abcam, 1:1000; Lamin B1, ab65986, Abcam, 1:5000; $\beta$-actin, ab230169, Abcam, 1:4000) at 4 °C. Subsequently, the blots underwent incubation with the appropriate goat anti-rabbit or goat anti-mouse secondary antibody (1:4000, Cell Signaling Technology Inc, Boston, MA, USA) at 37 °C for one hour. Finally, enhanced chemiluminescence (ECL) reagents (PE0010, Solarbio, Beijing, China) were used to visualize the protein bands. The relative amounts of these proteins were calculated using Image J software (https://imagej.nih.gov/ij/, National Institutes of Health, Bethesda, MD, USA) from the National Institutes of Health.

Following the manufacturer’s guidelines, the reaction system was generated using TB Green® Premix Ex TaqTM Kit (RR071A, BIOSCIENCE, Boston, MA, USA). The prepared mixture was reacted in the Real-Time PCR System (Roche, Shanghai, China) with the reaction program consisting of an initial denaturation at 97 °C for 4 minutes, followed by 38 cycles of denaturation at 97 °C for 4 seconds and annealing/extension at 60 °C for 30 seconds. All measurements were performed in triplicate to eliminate operational errors. The primers sequences listed in Table 1. The relative expression levels of the genes were calculated using the 2^{-Δ⁢Δ⁢Ct} method.

The data were presented as mean $\pm$ standard deviation (SD), and the statistical analysis was performed using SPSS 22.0 software (IBM, Armonk, NY, USA). For comparisons involving more than two groups, one-way analysis of variance with Tukey’s post hoc test was employed. A p-value of less than 0.05 was used to determine statistical significance. Each experiment was repeated biologically thrice.

The mice belonging to the M group exhibited elevated instances of sneezing and nose-catching associated with asthma compared to mice in the NC group (Fig. 1A,B) (p 0.001). Conversely, the mice treated with PC or IC showed a dose-dependent decrease in the frequency of sneezing and nose-catching when compared to the M group (p 0.001). Additionally, it was observed that M resulted in a significant increase in OVA-specific IgE levels in comparison to the NC group (Fig. 1C) (p 0.01). In the asthma model group (M), the frequency of sneezing, nose scratching, and head/face scratching gradually increased, accompanied by slowed breathing and nodding respiration, leading to the gradual exacerbation of asthma symptoms. These manifestations indicate the successful establishment of the asthma model. Following this, mice were administered PC and varying concentrations of IC, which resulted in substantially lower OVA-specific IgE levels in the PC, IC-M and IC-H group compared to the M group (Fig. 1C) (p 0.01). These results indicated that IC effectively reduced the frequency of sneezing and catching nose in RSV-induced asthmatic mice and decreased OVA specific IgE levels.

Fig. 1.

Effect of the icariin on the overall behavior of mice with asthma induced by respiratory syncytial virus (RSV) was examined. The number of sneezing (A), and catching nose (B) of the mice. (C) The contents of ovalbumin (OVA)-specific IgE in serum were detected by Enzyme-Linked Immunosorbent Assay (ELISA). n = 5. **p 0.01, ***p 0.001 vs NC group;^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 vs M group. NC, normal control; M, asthma model; PC, ribavirin + budesonide; IC, icariin.

A predominant clinical characteristic of asthma is the excessive accumulation of inflammatory cells in lung tissue. Subsequently, lung tissue from asthmatic mice was gathered for HE staining. HE staining showed a noticeable increase in inflammatory cells around the alveolar walls compared to the NC group (p 0.001). Treatment with IC or PC significantly reduced the presence of inflammatory cells surrounding the airways (Fig. 2A). The analysis of inflammatory cell infiltration in lung sections was conducted according to previously described methods, and the results are shown as the inflammation score in Fig. 2A [19]. Additionally, BALF was collected and the count of inflammatory cells was conducted. Examination of the neutrophils, eosinophils, and macrophages in BALF revealed a notable rise in the M group. Conversely, the RSV-infected asthmatic mice treated with IC at 20 mg/kg/d and 40 mg/kg/d exhibited a marked decrease in the count of inflammatory cells compared to the M group (Fig. 2B) (p 0.001). Besides, ELISA results demonstrated a notable rise in the levels of cytokines IL-13, IL-5, and IL-4 in the BALF of RSV-infected asthmatic mice in the M group when compared to the NC group (p 0.001). Remarkably, the levels of these cytokines were reduced by IC-M and IC-H treatment (Fig. 2C). Overall, these findings indicate that IC treatment played a role in alleviating airway inflammation in RSV-infected asthmatic mice.

Fig. 2.

Effect of the icariin (IC) on inflammatory cells in RSV-infected asthmatic mice. (A) Histopathological changes were assessed using HE staining of mouse lung tissue. Bar = 200 µm. (B) The cell numbers of neutrophils, eosinophils, and macrophages in bronchoalveolar lavage fluid (BALF) of mice in each group. (C) Inflammatory cytokines IL-4, IL-13, and IL-5 in BALF were measured using ELISA. n = 5. ***p 0.001 vs NC group; ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 vs M group. IL-4, interleukin-4; IL-5, interleukin-5; IL-13, interleukin-13; NC, normal control; M, asthma model.

To further analyze the effect of IC on oxidative stress in RSV-induced asthma mice, we examined the levels of MDA, GSH, SOD and CAT in lung homogenates. The results from the ELISA analysis revealed that MDA level of RSV-induced asthma mice was markedly elevated, and GSH, SOD and CAT was markedly reduced compared with that in NC group (Fig. 3A) (p 0.001). In contrast with the M group, the level of MDA in tissue reduced remarkably after PC and IC treatment (p 0.001), while the level of GSH, CAT and SOD were markedly increased (Fig. 3A) (p 0.001). In addition, the nuclear factor-erythropoietin 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins in RSV-induced asthma mice were estimated via western blot and reverse transcription quantitative polymerase chain reaction (RT-qPCR). Versus the NC group, the protein and messenger ribonucleic acid (mRNA) expression of Nrf2 and HO-1 in the M group reduced remarkably (p 0.001). When IC intervention, Nrf2 and HO-1 protein and mRNA expression was markedly upregulated (Fig. 3B–E) (p 0.001). Hence, the results demonstrated that IC ameliorated oxidative stress in asthmatic mice induced by RSV.

Fig. 3.

Effect of the IC on oxidative stress in asthmatic mice induced by RSV. (A) The levels of MDA, SOD, CAT and GSH in lung tissue homogenates were detected by ELISA. (B,C) Western blot was conducted to investigate the expression levels of Nrf2 and HO-1. (D,E) RT-qPCR was used to assess the mRNA levels of Nrf2 and HO-1. n = 5. ***p 0.001 vs NC group; ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 vs M group. MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GSH, glutathione; Nrf2, nuclear factor-erythropoietin 2-related factor 2; HO-1, heme oxygenase-1; RT-qPCR, reverse transcription quantitative polymerase chain reaction.

In the control group of NC mice, a limited number of TUNEL-positive cells were detected. Contrastingly, a notable rise in TUNEL-positive cells was observed in the M group, which was substantially reduced by the treatment with IC or PC (Fig. 4A) (p 0.001). Additionally, western blot and RT-qPCR analysis showed that IC decreased the amounts of apoptosis markers Cleaved caspase-3 and Bax in the lung tissues of asthmatic mice, while increasing the levels of the anti-apoptotic markers Bcl-2 (Fig. 4B,D). The results illustrate that IC treatment reduced apoptosis in RSV-infected asthmatic mice.

Fig. 4.

Effect of the IC on apoptosis and the expression of PD-1 in asthmatic mice infected with RSV. (A) The number of apoptotic cells in lung tissue by TUNEL assay. Bar = 200 µm. (B) The expression of Bax, Bcl-2 and cleaved caspase-3 was detected by Western blot. (C) The expression of PD-1 in lung tissue was detected by Western blot. (D,E) RT-qPCR was used to determine the mRNA levels of Bax, Bcl-2, cleaved caspase-3 and PD-1. n = 5. ***p 0.001 vs NC group; ^#p 0.05, ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 vs M group. Bax, Bcl-2 associated X Protein; Bcl-2, B-cell lymphoma-2; PD-1, programmed cell death protein-1.

PD-1 plays an important role in asthma, and its activation can reprogram T cells to alleviate asthma. To this end, we further analyzed whether the improvement of RSV-induced asthma by IC is related to PD-1 [13]. Compared with NC group, the protein and mRNA expression of PD-1 was markedly reduced in the M group (p 0.001). In contrast, the PD-1 expression of IC- and PC mice were significantly higher than in model mice (Fig. 4C,E). The results showed that the expression of PD-1 increased, indicating that IC may reduce apoptosis and improve asthma in mice by regulating the expression of PD-1. In addition, based on the therapeutic effects of the above three concentrations of IC it was found that a medium dose of IC not only achieves the experimental results, but also reduces some of the damages caused by high doses of the drug in rats. Therefore, we used the medium dose of Icariin (IC-M; 20 mg/Kg) for the next experiment.

To further determine whether IC ameliorates RSV-induced asthma in mice by modulating PD-1 expression. RT-qPCR results showed that the expression of si-PD-1 was significantly lower than that of si-NC, indicating that the si-PD-1 construct was successful (Fig. 5A) (p 0.001). ELISA detected the levels of Inflammatory cytokines IL-4, IL-13, and IL-5 in BALF. The results showed compared with NC group, levels of IL-4, IL-13 and IL-5 were notably higher in M group (p 0.001). Compared with M group, levels of IL-4, IL-13 and IL-5 were markedly lower after IC treatment, but si-PD-1 significantly reversed IC inhibition of IL-4, IL-13 and IL-5 (Fig. 5B) (p 0.01).

Fig. 5.

IC affects RSV-induced asthma in mice by modulating PD-1 expression. (A) Transfection efficiency of PD-1 knockdown was verified using RT-qPCR. (B) Inflammatory cytokines IL-4, IL-5, and IL-13 in BALF were determined by ELISA. (C) The expression of Bax, Bcl-2 and cleaved caspase-3 was detected by Western blot. (D) Western blot detected the expression of PD-1 in lung tissue. (E,F) The mRNA levels of Bax, Bcl-2, cleaved caspase-3 and PD-1 was detected using RT-qPCR. n = 5. ***p 0.001 vs NC group; ^#⁢#p 0.01, ^#⁢#⁢#p 0.001 vs M group; ^▲⁢▲p 0.01, ^{▲⁢▲⁢▲}p 0.001 vs IC+si-NC group. Bax, Bcl-2 associated X Protein; Bcl-2, B-cell lymphoma-2; PD-1, programmed cell death protein-1; IL-4, interleukin-4; IL-5, interleukin-5; IL-13, interleukin-13.

In addition, the mRNA and protein expression of cleaved caspase-3 and Bax was markedly reduced and Bcl-2 expression was markedly increased after IC treatment (Fig. 5C,E). Knockdown of PD-1 reversed the inhibitory effect of IC on Bax and cleaved caspase-3, as well as the promotional effect on Bcl-2 (Fig. 5C,E). Besides, the results showed that asthmatic mice infected with RSV had an increased in PD-1 after IC treatment. Conversely, si-PD-1 treatment reversed PD-1 mRNA and protein levels in asthmatic mice (Fig. 5D,F) (p 0.001). In summary, our findings suggested that that IC ameliorated RSV-induced asthma mice by regulating PD-1 expression.