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 amplified. 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.
Briefly, 293T cells were digested with 0.05–0.25% trypsin and suspended as single cells in complete Dulbecco’s Modified 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% CO2 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% CO2 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 specific 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 isoflurane (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 sacrificed 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 isoflurane 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 chromaffin cells was detected by transmission electron microscopy. The dust and impurities on the surface of the samples were washed repeatedly with phosphate buffer, fixed 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).
Immunofluorescence
The tissues were fixed 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 unspecific 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 fluorescent microscopy. At least four fields in each image were analyzed. The fluorescence intensity was analyzed by ImageProPlus software 6.0 (National Institutes of Health, Bethesda, MD, USA).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR).
Total RNA was extracted from the adrenal tissues using a TRIzol kit (Thermo Fisher Scientific, Inc.). RNA concentrations were determined spectrophotometrically, and 1 μg total RNA was reverse transcribed into cDNA using an Avian Myeloblastosis Virus Reverse-Transcriptase kit (Promega Corporation, Madison, WI, USA). RT-qPCR was performed using the TB GreenTM Fast qPCR Mix (Takara Biotechnology Co., Ltd., Dalian, China). The following PCR primer sequences were used (5’–3’):
α7nAChR-F: ACAACATCTGCCAACACG,
α7nAChR-R: TTTCCCAACCTTTCTCCC;
β-actin-F: ATCGTCCACCGTAAATGC,
β-actin-R: TGAAGTGGTAGTCGGGTG.
The amplification reactions were performed with an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, 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, β-actin, 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 Scientific, 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 Scientific, Inc.) at 4˚C for 2 h. Protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific, 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). Significant differences were determined using one-way analysis of variance followed by the Bonferonni’s test. P<0.05 was considered to indicate a statistically significant difference.