Chemicals
ALA was purchased from Yuanye Biotechnology (Shanghai, China). Rabbit polyclonal anti-NOX4 antibody was obtained from Novus Biologicals (Littleton, USA). Anti-NOX2 and goat anti-rabbit IgG/HRP were purchased from Bioss Biotechnology (Beijing, China). The reactive oxygen species (ROS) detection kits were purchased from Beyotime Biotechnology (Shanghai, China). The malondialdehyde (MDA) detection kits and superoxide dismutase (SOD) activity assay kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ELISA kits for the protein Nox4 were purchased from Westang Biotechnology (Shanghai, China).
ALA solution preparation
ALA (80 mg/mL) was dissolved in 10% ethanol and sterilized by filtering. ALA solutions were prepared immediately before use.
Animals
In this study, 30 healthy New Zealand white rabbits weighing 2.9 ± 0.2 kg (2.7–3.1 kg) were selected regardless of sex. All rabbits were randomly assigned to the SHAM group (n = 10), the RAP group (n = 10), or the ALA + RAP group (n = 10). All of them were fed adaptively in the Laboratory Animal Center for 7 d before experiments. In the ALA + RAP group, ALA (30 mg/kg) was administered intraperitoneally daily (i.p.) to the rabbits for 3 days [27]. In the remaining groups, equal volume of normal saline was injected intraperitoneally. All rabbits were obtained from Xuzhou Medical University Animal Center. This study was approved by the Animal Ethics Committee of Xuzhou Medical University Animal Center.
Construction of the RAP model
First, 1% pentobarbital sodium solvent was injected slowly through the ear vein at 2 ml/kg. After successful anesthesia induction, the anesthesia was maintained through an indwelling needle in the ear vein and a constant speed micropump. An incision was made in the middle of the neck, the trachea was isolated, tracheal intubation was conducted, and an animal ventilator was connected to assist in ventilation. The right internal jugular vein was isolated, and distal shaping of the 10-pole coronary sinus electrode was conducted, which was sent to the right atrium through the 6F sheathing canal, and the chest lead V1 was connected to the distal end of the coronary sinus buttcock line.
The BL-420 s biological function experimental system was connected, and continuous stimuli were released at a frequency 10–20% higher than the sinus rhythm. Microadjustment of the coronary sinus electrode direction was performed so that the stimulating electrode adhered to the right atrium. Then, 1:1 atrial and ventricular conduction observed simultaneously in the intracavity lead and the body surface lead electrocardiograms revealed complete atrial pacing. The position of the coronary sinus electrode was maintained, and 12 h continuous stimuli were carried out by pacing at a rate of 600 times/min at a pressure twice that of the pacing threshold.
After pacing, the rabbits were euthanized by intravenous injection of 100 mg/kg pentobarbital sodium.
AERP determination
The 2-fold diastolic pacing threshold was treated as the output voltage, and the pulse width was set at 0.5 ms to measure AERP under the basic condition. Programmed premature extrastimulation (S1S2) was adopted at a stimulation frequency of 8:1. Decreasing scanning was carried out at S1S2 interphase at a step length of 10 ms and a scanning interval of 30 s. The refractory period was defined as the longest S1S2 interphase that S2 did not induce atrial activation in the time of S1S2 stimulation. AERP was measured 3 times, and the mean was treated as the baseline AERP value. AERP200 and AERP150 at the S1S1 of 200 ms and 150 ms were measured, respectively. The frequency adaptability is expressed as AERP200 − AERPl50.
Tissue processing [30]
The hearts were excised and washed immediately with buffered saline (pH 7.4) to remove any red blood cells and clots. The tissues were then weighed and homogenized in normal saline (0.9%) for 2 min. All homogenate samples were centrifuged for 20 min at 3,000 rpm at 4 °C in a refrigerated centrifuge to remove debris.
Detection of NOX4 and oxidative stress-related parameters
The levels of malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen species (ROS) were detected using the respective assay kits. The levels of the NOX4 protein were determined by ELISA.
Histopathological analysis [30]
Samples of heart tissues were washed immediately with saline (0.9%) and fixed in 10% phosphate buffered formalin solution for 24 h. The tissues were then dehydrated in ascending grades of ethyl alcohol, cleared with xylene, and embedded in paraffin. Paraffin blocks were cut by a microtone to prepare 5 µm thick sections. Sections were stained with hematoxylin-eosin (H&E) to stain the nucleus and cytoplasm and with Masson’s trichome to stain collagen fibers for the detection of fibrosis.
Ultrastructural changes
After precooling the relevant instruments at 4 °C, the samples of heart tissues were washed with saline (0.9%) then cut into 1 × 1 × 1 mm3 pieces and placed immediately in 4% glutaraldehyde at 4 °C for 4 hours. Afterward, the samples were sent to the Neuroelectrometer Experimental Center of Xuzhou Medical University, sliced and stained, and observed by transmission electron microscopy.
Immunohistochemistry [30]
For the detection of p47 phox, paraffin sections were deparaffinized, rehydrated and washed in 0.05 M Tris-buffered saline (TBS) pH 7.6. Immunostaining was performed using a rabbit polyclonal antibody (p47-phox-H-195/sc-14015) at a dilution of 1:250 for 1 h at room temperature. Negative controls were performed by omission of the primary antibody. Then, the sections were incubated with NovoLink Polymer Detection System (product No. RE 7280-K; Leica Biosystems, Newcastle, UK) for 30 min to detect any tissue-bound primary antibody. Sections were further incubated with the substrate/chromogen, 3,3-diaminobenzidine (DAB), prepared from Novocastra DAB chromogen (50 µl) and 1 ml of NovoLink Substrate Buffer (Polymer). Washing with TBS (0.05 M, pH 7.6) twice for 5 min was performed following each step of incubation. Immunostained sections were then counterstained with Mayer’s hematoxylin. Finally, sections were dehydrated rapidly in ascending grades of ethanol, cleared in xylene, and mounted in DPX mounting medium. The stained sections were evaluated and photographed using an Olympus BX51 bright field microscope equipped with an Olympus DP72 camera (Olympus Corporation, Japan).
Western blot
The whole-cell lysates of the cortical samples (N = 3 for each group) were obtained using tissue protein extraction kits supplemented with protease inhibitors (Roche, IN, USA). The total protein concentration was determined using BCA kits. An equal amount of protein was separated by electrophoresis in 10% sodium dodecyl sulfate polyacrylamide gels and then transferred to nitrocellulose membranes. After washing with TBST, the blots were then incubated with the respective secondary antibodies for 2 h and developed by chemiluminescence. Relative optical densities of the blots were quantified by densitometry.
Statistical analysis
SPSS 19.0 statistical software was adopted for statistical analysis. Measurement data were expressed as mean ± standard deviation. Data among multiple groups at all time points were compared using one-way analysis of variance. Differences in continuous repeated measuring data were compared by the repeated data analysis of variance. Further pairwise comparison was carried out using a Dunnett t-test. Differences were considered significant when P < 0.05.