Male and female Sprague-Dawley (SD) rats (Charles River, Beijing, China), weighing 200–220 g, received ad libitum food and water, and were housed in a temperature-controlled room (25 ± 3 °C) under a 12-h light/dark cycle. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academies Press) and was approved by ethical committee of Chengdu University of Traditional Chinese Medicine (no. 2014–03).
Recombinant virus plasmid and the carriers of auxiliary packaging original plasmids were purchased from Shanghai Genechem Co., Ltd. (Shanghai, China). Adora1, adora2a, and adora2b mRNA in situ hybrization kits were purchased from Boster Biological Technology Co., Ltd. (CA, USA). 293 T cell was purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences (Shanghai, China).
After 1-week acclimatization, 140 rats were randomly divided equally into 14 groups, namely normal control (NC) group, sham operation (SO) group, model (M) group, Neiguan (N) group, Hegu (H) group, sham acupoint (SA) group, negative lentivirus infected (NLI) group, A1AR lentivirus-infected (A1LI) group, A2aAR lentivirus-infected (A2aLI) group, A2bAR lentivirus-infected (A2bLI) group, negative lentivirus-infected Neiguan (NLIN) group, A1AR lentivirus-infected Neiguan (A1LIN) group, A2aAR lentivirus-infected Neiguan (A2aLIN) group, and A2bAR lentivirus-infected Neiguan (A2bLIN) group. In NC group, rats received no operation; in SO group, rats were only threaded, but not ligated left anterior descending coronary artery (LADCA); in M group, rats received ligation of LADCA; in N group, rats received ligation of LADCA and electroacupuncture (EA) treatment at bilateral PC6; in H group, rats received ligation of LADCA and EA treatment at bilateral LI4; in SA group, rats received ligation of LADCA and EA treatment at bilateral sham acupoint; in NLI, A1LI, A2aLI, and A2bLI groups, rats received corresponding lentivirus injection and ligation of LADCA; in NLIN, A1LIN, A2aLIN, and A2bLIN groups, rats received corresponding lentivirus injection, ligation of LADCA, and EA treatment at bilateral PC6.
The rats were anesthetized with 10% chloral hydrate (0.4 ml/100 g IP) and were positioned on an operating table for small animals (Fig. 1). After a thoracotomy performed in the fourth intercostal space, the LADCA was ligated 2 to 3 mm from its origin between the pulmonary artery conus and the left atrium with 6–0 silk. Occlusion of LADCA was verified by regional color change in the myocardial surface local and distal to the ligation, and change in electrocardiography (ECG). For M group, the LADCA of rats were only threaded with 6–0 silk.
The rats were anesthetized by intraperitoneal injection of chloral hydrate and received above thoracotomy. After exposure of heart, 50 μl corresponding lentivirus solution (1 × 107 TU/100 μl) was injected into left ventricle anterior wall at four different points. Finally, the chest was closed.
The rats received EA treatment a day after MI operation. Each EA treatment lasted 20 min; the rats received 5 times of treatment once every day for 5 consecutive days. Once-off stainless steel acupuncture needle (Suzhou Hualun Medical Appliance Co., Ltd., Jiangsu, China) was inserted to a depth of 1–2 mm. The acupuncture needle was connected to the HANS Acupuncture Point Nerve Stimulator (Nanjing Jisheng Medical Technology Co., Ltd., Jiangsu, China). The frequency was set at 2/100 Hz, and the intensity of stimulation was 0.1–1 mA. The procedures of all EA groups (N, H, SA, NLIN, A1LIN, A2aLIN, A2bLIN groups) were the same. The location of acupoints, which refers to previous literatures [23, 24], is as following: (1) PC6: on the anteromedial aspect of the forelimb, between the ulna and the radius, 3 mm proximal to the wrist joints; (2) LI6: on forelimb, between the first metacarpal bone and the second metacarpal bone; and (3) sham acupoint: on the dorsal aspect of the forelimb, the spatia between the third metatarsal bone and the fourth metatarsal bone.
Determination of rate pressure product and ST segment
Left ventricular developed pressure (LVDP) and heart rate (HR) obtained by Data Acquisition System (iWorx, IL, USA) were used to calculate rate pressure product (RPP), according to the previously described method : RPP = LVDP × HR. Data Acquisition System was also adopted to record ECG. All rats were calculated RPP and were recorded ECG before taking sample.
High-performance liquid chromatography
HPLC was used to determine the production of ATP, AMP, ADP, and ADO in myocardial tissue. The samples were determined by 2 mol/l HClO4 (20 μl/mg). After centrifugation at 4 °C and 20,000×g for 10 min, aliquots of the supernatant were collected. To collect supernatant to regulate PH value to 6.5–7.0, 1 mol/l K2CO3 was added. After blending, the mixture was stood on ice for 5 min. After centrifugation at 4 °C and 20,000×g for 10 min, the supernatant was collected for HPLC determination of ATP, AMP, ADP, and ADO concentrations. The chromatographic conditions were as follows: (1) chromatographic column: hypersil C18, 5 μm, 4.6 × 200 mm; (2) mobile phase: 0.2 mol/l phosphate potassium salt buffer (pH 6.0); and (3) flow rate 0.9 ml/min. Ultraviolet-visible detector (Dalian Elite Analytical instrument Co., Ltd., Liaoning, China) was adopted to carry out chromatographic analysis.
In situ hybrization
Cardiac tissue was fixed in 4% paraformaldehyde overnight and was soaked in phosphate buffer solution (PBS) 4–5 times for 5 min. Cardiac tissue was put into 30% sucrose/0.1 M PBS. Two days later, cardiac slice was soaked in 0.1 M PBS (once for 5 min), 0.1 M glycine/0.1 M PBS (once for 5 min), 0.3% Triton X-100/0.1 M PBS (once for 10 min), and 0.1 M PBS (three times for 5 min). Fifty microliter proteinase was added to incubate at 37 °C for 30 min. Cardiac slice was soaked in 4% paraformaldehyde for 5 min and was washed by 0.1 M PBS twice for 5 min. Cardiac slice was put into 0.25% acetic anhydride/0.1 M triethanolamine for 10 min. Cardiac slice was pre-hybridized by pre-hybridization solution at 42 °C for 30 min and was hybridized by hybridization solution at 42 °C overnight. Four times SSC, 2× SSC, 1× SSC, 0.5× SSC, 0.2× SSC, 0.2× SSC/0.1 M PBS, 0.05 M PBS were, respectively, used to wash cardiac slice. Then, cardiac slice was coated by 3% BSA/0.05 M PBS at 37 °C for 30 min. Anti-digoxin/antiserum alkaline phosphatase was added to cardiac slice, and cardiac slice was, respectively, washed by 0.05 M PBS, TSM1, and TSM2. ImagePro Plus 6.0 software (Media Cybernetics, Inc., MD, the USA) was adopted to analyze results.
Design and construction of lentivirus RNA interference vector
Short hairpin RNA (shRNA) interference sequences were designed for A1AR, A2aAR, and A2bAR (accession no. NM_017155, NM_053294, and NM_017161) to construct recombinant shuttle plasmids and packaging plasmids (pFU-GW-RNAi). The target sequences for A1AR, A2aAR, A2bAR, and negative control are as follows: 5′-CTTCTTTGCGTTCGTGTTA-3′, 5′-GATTTGGAATGACCACTTC-3′, 5′-GTGTCTCTTTGAGAACGTA-3′, 5′-TTCTCCGAACGTGTCACGT-3′. 293 T cells were transfected by sequenced plasmids and transfection reagent RNA-mate. The supernatants with lentivirus particles were concentrated and purified.
Statistical analysis was made using SPSS 26.0 software (IBM, NY, USA). All data were expressed as mean ± SD and were analyzed using one-way ANOVA with post hoc Tukey tests or Games-Howell tests. Differences were considered statistically significant when P values were less than 0.05.