Experimental animals
Sixty healthy, clean, male, Sprague Dawley (SD) rats aged 8 weeks (weighing 200 ± 20 g) were purchased from the Experimental Animal Center of Jiangxi University of Traditional Chinese Medicine, of Jiangxi Province, China (certificate no. JZLLSC (jiangxi) 2018-0033). The rats were fed with standard fodder and with food and water freely available in a controlled environment with a constant temperature of 20 ~ 25℃ and a 12/12-hr light/dark cycle. All procedures were conducted in accordance with guidelines reviewed and approved by the Institutional Animal Care and Use Committee of Jiangxi University of Traditional Chinese Medicine, China.
Modeling
Sixty rats were randomly divided into the normal group (norm, n = 12) and the model-building group (n = 48). Referring to literature [19] and making improvements, each rat in the model-building group was given intramuscular injection of hydrocortisone(Huazhong pharmaceutical co., LTD., national drug approval no. H420201507) at a dose of 0.03 mg/g·bw in left and right hind limbs of the rat alternately, and the modeling was carried out continuously for 14 days. Each rat in the normal group was given an intramuscular injection of 0.9% normal saline at a dose of 0.03 mg/g·bw for 14 days. During this period, natural light and free eating and drinking will be adopted. Criteria for model success [20[: decreases in body weight, withered clothing hair, hunched back, aversion cold, tendency to cluster, slowed reaction, weakness, decreased activity, scrotal shrinkage, and significantly increased urine output.
Grouping and treatment
The 48 rats successfully modeled were then randomly subdivided into model group (model, n = 12), carbenoxolone intraperitoneal injection group (CBX, n = 12), moxibustion group (moxi, n = 12) and moxibustion + carbenoxolone intraperitoneal injection group (moxi + CBX, n = 12).
In the moxibustion treatment group, guanyuan (CV4) and shenshu(BL25) (double laterals) were selected, and acupoints locating according to the experimental acupuncture [21]. Moxibustion treatment began on the 1st day after modeling finished. Before the treatment, the hair on the acupoints was cut off to expose the skin in advance. guanyuan (CV4) and shenshu (BL25) points were heated by suspended moxibustion using a moxa-stick produced from mugwort (custom-made for use with animals, length 12 cm, diameter 0.6 cm, made in the Affiliated Hospital of Jiangxi University of Traditional Chinese Medicine, Nanchang, China) at a height of approximately 3 cm over a hairless area of the skin for 20 min, once a day for 14 days.
Rats in the CBX group were given intraperitoneal injection of carbenoxolone at a dose of 10 mg /( kg·d) (11) once a day for 14 days. Fixation was performed as in the moxibustion group for a total of 14 times.
Rats in the moxi + CBX group were received moxibustion as in the moxibustion group. After moxibustion treatment, the rats were given intraperitoneal injection of carbenoxolone as in the CBX group.
Rats in the normal group and model group were fixed in the same way as the moxibustion group did, and were given intraperitoneal injection of normal saline (10 mg /( kg·d) once a day for 14 days.
Sampling
After 14 days of treatment, rats were anesthetized with an intraperitoneal injection of sodium pentobarbital 3% (weight/volume) at a dose of 40 mg/kg. Arterial blood is extracted and centrifuged to extract the supernatant, and stored in the − 20℃ refrigerator for later use. Tissues such as amygdala, hypothalamus and pituitary were extracted and stored in the − 80℃ refrigerator for later use.
Enzyme linked immunosorbent assay (ELISA) assessment
The levels of CORT, ACTH and CRH were determined using an ELISA kit purchased from Shanghai bogu biotechnology co., LTD (Shanghai, China). The ELISA-based method was conducted according to protocol provided by manufacturer.
Reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR was performed as described previously with minor modifications [22]. Total RNA was isolated from amygdala, hypothalamus and pituitary of rats in each group using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). The mRNA expression levels of MR, GR, 11β-HSD1,CRH and ACTH were measured using an RT-qPCR system with SYBR Green (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was amplified by PCR using primers for each target gene. cDNA was synthesized from each RNA using a random primer and RevertAid™ M-MuLV reverse transcriptase (Fermentas), according to the manufacturer's instructions. PCR was performed in a final volume of 25 µL, containing 12.5 µL 2 × PCR master mix, 2 µL cDNA, 1 µL forward primer, 1 µL reverse primer, and 8.5 µL sterilized DEPC water. RT-qPCR conditions were as follows: 94 °C for 5 minutes, followed by 40 cycles of 95 °C for 15 seconds, 60 °C for 45 seconds and 72 °C for 30 seconds. The fluorescence signal was detected at 60 °C, and the samples were finally extended at 72 °C for 7 minutes. The amplification efficiency was compared between the target and reference control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the delta-delta Ct (∆∆Ct) method [23]. The primers employed are listed in Table 1.
Table 1
Primer sequences for real-time quantitative polymerase chain reaction
Gene
|
Full name
|
Sequences (5–3′)
|
Product size (bp)
|
11β-HSD1
MR
|
11 beta- hydroxysteroid dehydrogenase type 1
mineralocorticoid receptor
|
Sense primer: AAA ATA CCT CCT CCC CGT CC
antisense primer: AGG CAG CGA GAC ACC ACC
Sense primer: AGA AGC TGG GGA AGT TAA AAG G
antisense primer: TCG GAG CGA TGT ATG TGG TC
|
219
102
|
GR
|
glucocorticoid receptor
|
Sense primer: CAT TAC CAC AGC TCA CCC CTA C
antisense primer: GCA ATC ACT TGA CGC CCA C
|
148
|
CRH
ACTH
GAPDH
|
corticotropin-releasing hormone
adrenocorticotrophic hormone
glyceraldehyde 3-phosphate dehydrogenase
|
Sense primer: TGG CTC TGT CGC CCT GTC
antisense primer: CAG CGG GAC TTC TGT TGA GG
Sense primer: CTC CTG CTT CAG ACC TCC ATA G
antisense primer: GGC TGT TCA TCT CCG TTG C
Sense primer: GGA GTC TAC TGG CGT CTT CAC
antisense primer: ATG AGC CCT TCC ACG ATG C
|
186
161
237
|
Western blot assay
Western blot analysis was performed as described previously with minor modifications [24]. All amygdala and hypothalamus tissues obtained in each group were homogenized in lysis buffer (JRDUN Biotechnology, Shanghai, China). The sample was centrifuged at 12,000 r/min for 20 minutes at 4 °C, and an aliquot of the supernatant was taken for protein concentration estimation using the BCA assay. Equal amounts of proteins were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), and then the resolved proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C. Monoclonal antibodies used for Western blotting included a rat monoclonal anti-MR antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The antibodies used in this study were rat monoclonal antibodies, 1:1000 and were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), except for those specifically indicated. The other antibodies included anti-GR, anti-11β-HSD1 and anti-GAPDH. The membranes were washed 5 minutes with Tris-buffered saline Tween 3 times and incubated with goat anti-rabbit IgG-HRP (1:1000; Beyotime Biotechnology, Shanghai, China) and goat anti-mouse IgG-HRP PS1 (C-20) (1:1000; Beyotime Biotechnology, Shanghai, China) antibodies at 37 °C for 1 hour. The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Beyotime Biotechnology, Shanghai, China). The grayscale values of bands were quantified using Image J software (Fujifilm, Tokyo, Japan). The relative expression of protein was calculated based on the ratio of target grayscale values to loading control grayscale values.
in situ hybridization
in situ hybridization was performed as described previously with minor modifications [25]. Six paraffin blocks were selected randomly and placed into a refrigerator at -20℃ for at least 30 minutes. Then, the blocks were cut into 4-7-µm-thick serial sections. After being dewaxed and dehydrated, each section was incubated in 50 µl hybridization buffer containing 10 µM oligonucleotide probe at 95℃ for 5 minutes and 37–40℃ for 12 hours, washed three times with 5×, 1 × and 0.2 × saline sodium citrate, and treated with blocking buffer at 37℃ for 15 minutes. Then, the blocking buffer was blotted with paper. Each section was then incubated in 30 µl biotinylated anti-digoxin antibody (1:50) at 37℃ for 1 hour, washed four times with 0.5 M PBS, incubated in streptavidin-biotin-peroxidase complex at 37℃ for 30 minutes, and rinsed four times in 0.5 M PBS. Subsequently, the sections were developed in 3, 3, diaminobenzidine, counterstained with hematoxylin, dehydrated in alcohol, permeabilized in xylene, mounted with proof quench mounting agent, and photographed with a fluorescence microscope [26]. The upper, middle, lower, left and right visual fields were randomly selected for each section and the image-pro Plus 6.0 software was used to detect the integrated optical density (IOD) of positive cells, the average value was used for statistical analysis. The negative control was incubated in 0.01M PBS without primary antibody. The primers employed are shown in Table 2.
Table 2
Primer sequences for Hybridization in situ
Gene
|
Full name
|
Sequences (5–3′)
|
11β-HSD1
CRF
MR
|
11 beta- hydroxysteroid dehydrogenase type 1
corticotropin-releasing factor
mineralocorticoid receptor
|
CAGAUGCCAGGUUUGUGCUCAAGUAAAUCCAAAAUGGAUAGCCUUACUCA
CAAUACAAAUAACGCUGUUUUGUUACUACAAAGAAACACACUUUGUGCA
GGAUGGAGAGGAUAGCAAUCCCGGCAGUCGCCCUACUGACGGUGGG
|
GR
|
glucocorticoid receptor
|
UGCUUGUGGAGCCUUUCGAGAAAUCAAGGAGAAUCCUCUGCUGCUU
|
Statistical analyses
All data are presented as the mean ± SD. Data were analyzed by one-way analysis of variance followed by a post hoc Student-Newman-Keuls (SNK) test using SPSS 19.0 software (SPSS, Chicago, IL, USA). A value of P < 0.05 was considered statistically significant.