Involvement of alpha-7 nicotinic receptor (α7nAChR) in respiratory syncytial virus infection-induced asthma in rats

This study aimed to investigate the role of alpha−7 nicotinic receptor (α7nAChR) in rats with asthma caused by respiratory syncytial virus (RSV) infection. An asthmatic rat model was established by RSV infection and conrmed based on pathological changes. sh-α7nAChR lentivirus was constructed to reduce α7nAChR expression in rats. The animals were divided into a control group, a model group, a sh-α7nAChR lentivirus group, a nerve growth factor (NGF) antibody group, a dexamethasone (DXMS) group, a sh-α7nAChR lentivirus + NGF antibody group, and a sh-α7nAChR lentivirus + DXMS group. The expression of α7nAChR was detected by quantitative real-time PCR and western blotting. Serum levels of adrenaline and norepinephrine were detected by ELISA. Transmission electron microscopy determined the morphological change of chroman cells and synaptic vesicles. Immunouorescence was used to detect the expression of synaptophysin (SYN) and nuclear factor-kappa B (NF-κB). Compared with the control group, the model group showed signicantly increased expression of α7nAChR, SYN, and NF-κB. Compared with the model group, the sh-α7nAChR lentivirus, NGF antibody, DXMS, sh-α7nAChR lentivirus + NGF antibody, and sh-α7nAChR lentivirus + DXMS groups showed signicantly decreased expression of α7nAChR (P < 0.05). After repeated infection with RSV, the number of chroman cells and synaptic vesicles increased, which were signicantly reduced after silencing α7nAChR by NGF antibody or DXMS treatment. The model group showed signicantly lower serum adrenaline than the control group (P < 0.05). Similarly, the serum adrenaline in the sh-α7nAChR lentivirus, sh-α7nAChR lentivirus + NGF antibody, and sh-α7nAChR lentivirus + DXMS groups, but not the NGF antibody and DXMS groups, was signicantly lower than in the model group. Together, upregulation of α7nAChR is involved in RSV infection-induced asthma in rats. Silencing of α7nAChR reduces the numbers of chroman granules and synaptic vesicles and adrenaline release.


Introduction
Bronchial asthma is a common and frequently occurring respiratory disease, with paroxysmal wheezing, shortness of breath, chest tightness, and cough being the main symptoms. Asthma is characterized by chronic airway in ammatory reaction, which is associated with airway hyperresponsiveness (1). Infants with recurrent respiratory syncytial virus (RSV) infection have a high risk of bronchial asthma in adolescence (2,3), but the pathogenesis is not yet fully known. Presently, bronchial asthma is treated using glucocorticoid and β-2-adrenergic receptor agonists (4). As a representative β-2-adrenergic receptor agonist, adrenaline is still the most effective treatment (5) and acts by binding with adrenergic receptors in the airway smooth muscle (5). Insu cient adrenaline secretion in the asthmatic state leads to unbinding of adrenaline with the receptors, which inhibits alleviation of bronchospasm during an asthmatic attack (6,7).
Adrenaline is synthesized by adrenomedullary chroma n cells and released into the circulatory system; the chroma n cells originate from the neural crest and possess the phenotype and function of endocrine cells (8). Their differentiation is regulated by nerve growth factor (NGF) and glucocorticoids (9,10). After continuous injection of NGF into pregnant rats, chroma n cells from the offspring rats can transform into sympathetic neurons (11). Similarly, primary cultured chroma n cells gradually lose their endocrine phenotypes and are transformed into sympathetic neurons after NGF treatment; meanwhile, glucocorticoids could also block NGF-induced phenotypic transformation of chroma n cells (12).
Seventeen subunits of nicotinic acetylcholine receptors (nAChR) have been identi ed based on molecular cloning technique: 10 α-subunits; four β-subunits; and one each of γ, δ, and epsilon subunits. These subunits assist the nACh receptor subtypes to regulate different physiological functions (13).
Redistribution of nAChR subunits, primarily the α7nAChR, occurs in the adrenal medulla of asthmatic mice (14,15). A previous study reported that α7nAChR was a potential therapeutic target for the treatment of type-2 innate lymphoid cells (ILC2s)-dependent asthma (16). However, to our knowledge, the function of α7nAChR in RSV infection-induced asthma has not yet been veri ed. In this study, we aimed to clarify the function of α7nAChR in RSV infection-induced asthma by silencing α7nAChR or by the application of NGF antibody or dexamethasone (DXMS).

Materials And Methods
Construction of α7nAChR shRNA lentivirus Three pairs of interference sequences were designed and synthesized as per gene sequences. The conjugated products were transformed into susceptible bacteria and cultured overnight in a 24-well plate. Complete colonies were randomly selected from the plate. The positive clones were detected by PCR, sequenced, and validated. The bacteria with correct sequence of extraction and plasmid detection were ampli ed. The interfering lentiviruses were extracted by Axygen Plasmid Extraction Kit (Cat. APGX250, Axygen). Plasmid vector was detected by optical density (OD) values (data not shown). The sequences of shRNA for α7nAChR are listed in Table 1. Brie y, 293T cells were digested with 0.05-0.25% trypsin and suspended as single cells in complete Dulbecco's Modi ed Eagle Medium (DMEM) (Hyclone/Thermo SH30023.01B). The cells were counted and inoculated into the dish (106 cells per 10-cm dish) and cultured overnight at 37˚C in a 5% CO 2 incubator. Before transfection, the medium was removed and replaced with 5 mL Opti-MEM medium (31985, GIBCO). 9 µg Packaging Mix and 3 µg lentiviral expression plasmids were added into 1.5 mL Opti-MEM (preheated at 37˚C). 36 µL lipofectamine 2000 was added to 1.5 mL Opti-MEM and kept at room temperature for 5 min. Lipofectamine 2000 solution, kept at room temperature for 20 min, was added to the dish, gently mixed, and incubated at 37˚C in a 5% CO 2 incubator for 6 h. Then, the medium was replaced with complete medium (DMEM+10% fetal bovine serum [FBS]). After 48 h, the virus was collected and the titer tested.

Animal model
Experimental protocols were approved and supervised by the Animal Care and Use Committee of People's Hospital of Nanchang University (No. 2019093). In all, 42 male Sprague-Dawley rats (weight: 180±5 g) were purchased from Vital River Laboratories Co., Ltd. (Beijing, China) and maintained in speci c pathogen-free conditions at a temperature of 23±2˚C, relative humidity of 45-65%, and a controlled 12/12 h light/dark cycle. The asthmatic rat model was established by repeated RSV infection as previously described (17). 12 weeks after infection, the rats were anesthetized by inhalation of iso urane (5%) and decapitated. The health and behaviors were monitored every day and there was no death of the animals during the experiments.
RSV virus was obtained from the Institute of Microbiology, Xiangya Medical College, and rats were infected by RSV inhalation once a day (105 PFU/infection) for one week. Goat anti-rabbit NGF antibody (11050-MM06, Sino Biological) was injected intraperitoneally every week before the virus infection, and the negative control group was injected with virus-free medium. The source of infection was immediately isolated, and cages were replaced every three days and disinfected. The rats were sacri ced 12 weeks after infection. The animals were divided into seven groups (n=6 in each group): a control group; a model group (RSV-infected rats, 105 PFU/infected for one week); a sh-α7nAChR lentivirus group (200 µl, tail vein injection); a NGF antibody treatment group (anti-NGF, goat anti-rabbit β-NGF antibody was injected intraperitoneally every week before virus infection at a dose of 4 mL/kg/day for one week before infection); a dexamethasone (DXMS) treatment group (i.p. 2 mg/kg/day for one week before infection); a sh-α7nAChR lentivirus+NGF antibody group, and a sh-α7nAChR lentivirus+DXMS group. After 12 weeks' treatment, the rats were anesthetized by iso urane and decapitated. Pathological changes were observed in modeled rats by hematoxylin-eosin (HE) staining. Adrenal tissues were collected for subsequent experiments.

Transmission electron microscopy
The ultrastructure of rat chroma n cells was detected by transmission electron microscopy. The dust and impurities on the surface of the samples were washed repeatedly with phosphate buffer, xed for 2 h with 3% glutaraldehyde at room temperature. The tissues were washed three times with phosphate buffer for 10 min each time; dehydrated with 50%, 70%, 80%, and 90% ethanol for 15 min; and removed with 100% ethanol. The tissues were sectioned into 70-nm slices, which were then stained by 3% uranyl acetate and lead citrate and imaged with transmission electron microscopy (80 kV) (JEOL JEM-1230).

Immuno uorescence
The tissues were xed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS, and permeated with 0.5% Triton X-100 (PBS) at room temperature for 20 min. 5% BSA was used to block the unspeci c staining (30 min at 37℃). The tissues were incubated with the antibodies against synaptophysin (1:200; ab32127, Abcam) and NF-κB (1:200; ab32536, Abcam) overnight at 4℃. After washing, Alexa Fluor 593 goat anti-mouse IgG (1:100; catalog no. CW0159S, CW BiotechCWBIO, Beijing, China) was incubated with the slides for 30 min at room temperature. The images were taken using uorescent microscopy. At least four elds in each image were analyzed. The uorescence intensity was analyzed by ImageProPlus software 6.0 (National Institutes of Health, Bethesda, MD, USA).
The ampli cation reactions were performed with an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scienti c, Inc., Waltham, MA, USA), with initial denaturation at 95˚C for 3 min, followed by 40 cycles of a two-step PCR at 95˚C for 10 s, 53˚C for 30 s, and 72˚C for 30 s. The 2-ΔΔCt method was used to determine the amount of target, normalized to the endogenous reference, βactin, as previously described (18).
Western blotting. Proteins were extracted from adrenal tissues as previously described (19) using a protein isolation kit (ReadyPrep; GE Healthcare Life Sciences). Protein concentration was determined using a bicinchoninic assay kit (Thermo Fisher Scienti c, Inc.). A total of 20-μg protein was loaded into each lane and separated via SDS-PAGE on a 12% gel and transferred onto nitrocellulose membranes. Subsequently, membranes were blocked in 5% skim milk for 2 h in room temperature and incubated with the following primary antibodies overnight at 4˚C: α7nAChR (1:1,000; Proentech, USA) and anti-β-actin (1:1,000; cat. no. 4970; Cell Signaling Technology, Inc., Danvers, USA). The nitrocellulose membranes were washed three times and incubated with HRP-labeled goat anti-rabbit IgG secondary antibody (1:10,000, cat. no. A16104SAMPLE; Thermo Fisher Scienti c, Inc.) at 4˚C for 2 h. Protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scienti c, Inc.), and the blots were scanned using a ChemiDoc XRS (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein expression was normalized to β-actin, and densitometric analysis was performed by ImageJ Software version 7.0 (National Institutes of Health, Bethesda, MD, USA).

ELISA
Serum adrenaline and norepinephrine (NE) were detected using ELISA, according to the manufacturer's instructions (Adrenaline: abx514232, ABCAM; NE: KA3836, Abnova). The absorbance (OD value) of each well was measured at 450 nm. The measurements were carried out within 15 min.

Statistical analysis
Data were presented as the mean ± standard error of the mean and analyzed using SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA). Signi cant differences were determined using one-way analysis of variance followed by the Bonferonni's test. P<0.05 was considered to indicate a statistically signi cant difference.

Results
Sh-α7nAChR lentivirus was established As shown in Figure 1, expression of α7nAChR was signi cantly down-regulated in 293T cells by all three interference sequences compared with the control group, and the effect of sh-α7nAChR1 was the most obvious. Therefore, sh-α7nAChR1 was packaged into lentiviruses. Brie y, 1-mL virus solution was diluted to a nal titer of 1.0*108 TU/mL. α7nAChR expression was promoted in the adrenal tissues after RSV infection, but down-regulated by sh-α7nAChR lentivirus and/or DXMS Initially, HE staining was only carried out in normal control and model groups to ensure the success of the modeling. In the later experiments, we did not do the experiment in other groups. Compared with the control group, rat adrenal tissues with RSV infection showed obvious in ltration ( Fig. 2A). As shown in Fig. 2B, the expression of α7nAChR at the mRNA level in the model group was higher than in the control group (P<0.05). The expression of α7nAChR in the sh-α7nAChR lentivirus, NGF antibody, DXMS, sh-α7nAChR lentivirus+NGF antibody, and sh-α7nAChR lentivirus+DXMS groups was signi cantly lower than that in the model group (P<0.05). Consistent with the mRNA expression, the protein expression of α7nAChR was also reduced in the sh-α7nAChR lentivirus, NGF antibody, DXMS, sh-α7nAChR lentivirus+NGF antibody, and sh-α7nAChR lentivirus+DXMS groups compared with the model group (P<0.05) (Fig. 2C). Importantly, α7nAChR expression at both mRNA and protein levels were signi cantly reduced in the sh-α7nAChR lentivirus+NGF antibody and sh-α7nAChR lentivirus+DXMS groups compared with the sh-α7nAChR lentivirus group.

Silencing of α7nAChR reducedchroma n granules and synaptic vesicles
In the model group, cell membranes were shrunken and showed clubbed and villous processes; chroma n granules were uniformly distributed; the contents of granules were fewer, and more vesicles were observed (Figure.3). By contrast, the cell membranes were also shrunken in the sh-α7nAChR group, while chroma n granules were obviously reduced. The number of chroma n granules and synaptic vesicles decreased in the anti-NGF treatment group. The distribution of chroma n granules was slightly sparse in the DXMS treatment group. In the sh-α7nAChR+DXMS group, the nuclear membrane was shrunken. In addition, the mitochondria shrank slightly; the number of chroma n granules and synaptic vesicles obviously decreased.
Silencing of α7nAChR reduced the expression of synaptophysin and NF-κB As shown in Figs. 4 and 5, the expression of synaptophysin and NF-κB in the model group was signi cantly higher than that in the control group (P<0.05), while the expression of synaptophysin and NF-κB was signi cantly lower in the sh-α7nAChR lentivirus, NGF antibody, DXMS, sh-α7nAChR lentivirus+NGF antibody, and sh-α7nAChR lentivirus+DXMS groups than that in the model group (P<0.05). Importantly, the expression of synaptophysin and NF-κB was signi cantly reduced in the sh-α7nAChR lentivirus+NGF antibody and sh-α7nAChR lentivirus+DXMS groups compared with the sh-α7nAChR lentivirus group.
Silencing of α7nAChR reduced serum adrenaline levels Compared with the control group, the model group showed decreased levels of serum adrenaline (P<0.05). Compared with the model group, the sh-α7nAChR lentivirus, sh-α7nAChR lentivirus+NGF antibody, and sh-α7nAChR lentivirus+DXMS groups showed signi cantly decreased serum adrenaline level (P<0.05). By contrast, single treatment with anti-NGF or DEX did not alter the adrenaline levels (vs. model, P>0.05, Fig. 6A). As shown in Fig. 6B, serum norepinephrine levels were not signi cantly altered in any group (P>0.05).

Discussion
In this study, we demonstrated that α7nAChR expression was elevated in asthmatic rats. The expression of α7nAChR was reduced by sh-α7nAChR virus, NGF antibody, and DXMS. Moreover, the chroma n granules and synaptic vesicles were accordingly reduced by α7nAChR silencing. Moreover, the effects of α7nAChR silencing were promoted by NGF antibody or DXMS. nAChR subunits, primarily the α7nAChR has been reported to redistribute in asthmatic mice (14,15). Especially, α7nAChR was a potential therapeutic target for the treatment of ILC2s-dependent asthma (16). Nevertheless, our present study would further support the function of α-7nAChR in asthma using a different model. Especially, both pharmacological and genetic methods have been utilized in this study.
First, the rat asthmatic model was established upon RSV infection in our study. The expression of α7nAChR increased in asthmatic rats and decreased after treatment with sh-α7nAChR viruses, anti-NGF antibody, and DXMS. These results further supported that NGF antibody suppressed the expression of α7nAChR in chroma n cells caused by RSV infection (20). Second, the results of transmission electron microscopy further revealed that the numbers of chroma n cells and synaptic vesicles increased after repeated RSV infection, while these numbers decreased after silencing of α7nAChR and anti-NGF antibody and DXMS treatment. The present study also implicated that occurrence of asthma was related to the increase of chroma n cells and synaptic vesicles.
In ammation or stimulation may elicit nerve terminals in the airway to release tachykinin, causing neurogenic in ammation and increased susceptibility to afferent nerves, respiratory changes, and cough (21). Our data also showed that the expression of synaptophysin after RSV infection increased signi cantly. However, silencing of α7nAChR and treatment with anti-NGF and DXMS decreased the expression. As synaptophysin is an important structural protein of sensory neurons (22), the increase of synaptophysin indicates changes to plasticity in nerve terminals.
The release of adrenaline from chroma n cells depends on the activation of calcium channels to produce su cient concentration of [Ca2+], whereas [Ca2+] mainly depends on the regulation of Ca2+ channels by subtypes of nAChR on chroma n cell membranes (15). Previous studies have con rmed that the Ca2+ permeability of different subtypes of nAChR is different (23)(24)(25). The nAChR subtype composed of alpha 7-9 subunits has a higher Ca2+ permeability, while the α7nAChR subtype has the highest Ca2+ permeability (26). The α7nAChR subtypes of chroma n cells are regulated by NGF and glucocorticoids (27). To maintain the endocrine phenotype of chroma n cells, appropriate concentration of glucocorticoids in the physiological state can inhibit or reduce the unnecessary release of adrenaline by inhibiting the activation of Ca2+ channels (28).
Acetylcholine is released when sympathetic nerves projecting into the adrenal medulla are stimulated (29). Thereafter, acetylcholine binds to the receptor and activates nAChR in chroma n cells (30). Activated calcium channels promote calcium in ux, resulting in a signi cant increase of intracellular calcium concentration, which triggers the release of catecholamines such as adrenaline, dopamine, and norepinephrine (31). In this study, we also detected serum levels of adrenaline and norepinephrine.
Adrenaline, but not norepinephrine, level was reduced after RSV infection. Compensatory increase of adrenaline release is required in the body. Over time, the compensatory release of adrenaline by chroma n cells gradually weakens, resulting in decreased adrenaline release; on the other hand, reduced adrenaline levels after silencing of α7nAChR is due to disorder of the hypothalamus pituitary adrenal (HPA) axis in asthma (32).
During the course of asthma after RSV infection, the synthesis of adrenaline and the release process of chroma n cells were abnormal (33). After repeated infection with RSV, increase of NGF concentration and decrease of glucocorticoid concentration induced the transformation of chroma n cells from endocrine phenotype to neuronal phenotype, resulting in the decrease of adrenaline synthesis (34). It may be through a negative feedback mechanism that NGF induced the increase of α7nAChR expression in chroma n cells. The decrease of cell threshold and abnormal release of adrenaline result in insu cient secretion of adrenaline, provoking asthma.
Our study has some limitations. First, although 293 cells were an approved cell tool to evaluate the e ciency of gene silencing, the more speci c respiratory cell lines would strengthen our conclusion. Moreover, the mechanisms could also be deeply investigated using the cultured respiratory cell lines.
Second, pathological changes of adrenal tissues (shown by HE staining) were only con rmed in the model group. Whether α7nAChR is involved in the pathological changes remains an important question. In our future study, we will detect the pathological changes of all groups using HE staining. Third, it might be also interesting to verify whether sh-α7nAChR could repair the airway resistance. Fourth, synaptophysin and NF-κB expression will be further checked using western blotting. Moreover, its clinical application also warrants more pharmacological and toxicological studies. Conclusion α7nAChR was elevated in rats after asthmatic modeling with RSV infection. The expression of α7nAChR was eliminated by sh-α7nAChR lentivirus, NGF antibody, and DXMS. Importantly, the silencing of α7nAChR reduces the number of chroma n granules and synaptic vesicles and adrenaline release.

Declarations
Tables Table 1 Sequences of sh-α7nAChR.

Figure 5
The silencing of α7nAChR reduced expression of synaptophysin. The expression of synaptophysin in the model group was signi cantly higher than that in the control group, and the expression of synaptophysin and NF-κB was signi cantly lower in the sh-α7nAChR lenti-virus, NGF antibody, DXMS, sh-α7nAChR lenti-virus+NGF antibody, and sh-α7nAChR lenti-virus+DXMS groups. *P<0.05 compared with control; #P<0.05 compared with model group (Bonferonni's test). Scale bar: 100 μm.