Laboratory animals and grouping
Sixty C57BL/6J mice, male, seven weeks old, were purchased from Beijing Huafukang Laboratory Animal Technology Co., Ltd. Mice were breed in 24-26℃，40-70% humidity in the laboratory animal center of Jilin University. Mice were randomly divided into five groups(n=15): sham operation group (SH), HF model group (HF), HF + NBP (HN), HF + NBP+ CaMKⅡ (KN93) (HNKC), HF + NBP + Nrf2 inhibitor (HNM). This whole animal experiment was approval by Laboratory Animal Welfare and Ethics Committee of Jilin University (IACUC No.2019156).
Mice HF model preparation
The preparation of mice HF model was followed as this protocol: Except for mice in the SH group, mice in other group were performed with the method of constriction of abdominal aorta to prepare HF mouse models 21. Firstly, mice were anesthetized by 1% pentobarbital sodium (45 mg/Kg) with intraperitoneal injection. Place mice in supine position on a surgery platform with a heating pad to maintain the body temperature. The abdominal region of the mice with an animal hair clipper and hair remover lotion were shaved for avoiding surgical contaminations. Open the abdominal cavity at the center of the abdomen for 2~3 cm, bluntly strip the muscles, expose the abdominal aorta, place a 7-gauge surgical needle parallel to the abdominal aorta, ligate the aorta and the needle with 0-gauge silk thread, Then remove the surgical needle to confirm the aorta is unobstructed and the abdomen is closed. In the sham operation group, only the abdominal aorta was isolated, not ligated. Mice were injected intraperitoneally with 50,000 U penicillin daily for 3 days after operation for infection prevention.
The mice of sham group were given the same amount of penicillin daily antibiotics after operation for 3 days. After surgery, mice in HN group were given NBP (60mg/kg) daily by intragastric administration by tail vein injection. Besides, mice in the HNK were co-treated by the same amount (60mg/kg) of NBP and CaMKII antagonist (KN93) with injection via tail vein post-surgery for 15 days. Moreover, mice of HNM group were treated with Nrf2 inhibitor (ML385, MCE, USA) before intragastric administration of NBP for 15 days.
Mice heart function detection
Mice heart function was detected by Cardiac ultrasound. M-mode echocardiography detection was performed for examination. And measurement of thickness of left ventricular anterior wall (LVAW) , left ventricular posterior wall (LVPW) and diameters of left ventricular end-diastolic (LVEDD) and left ventricular end-systolic (LVEDs), left ventricular ejection fraction (LVEF) and left ventricular short-axis shortening rate (LVFS) were made. These results were measured for three cardiac cycles, and the average value was calculated as the final test result.
Haematoxylin-eosin (H&E) staining
The formalin-fixed cardiac tissues were fixed in formalin solution. Then the tissues were gradually dehydrated in gradient ethanol, transparent in xylene, and embedded in paraffin. Then tissue samples were cut into 4μm sections. Following that, the slices were immersed into haematoxylin for 10 min, rinsed with 1*PBS, then differentiated in 1% hydrochloric acid alcohol. Eosin-stained solution was stained for 30 s. After that, slices were dehydrated, transparented and sealed. Finally, the slices were observed under microscope.
The slices containing heart tissue were deparaffinised and rehydrated with ethanol, and then stained in Regaud haematoxylin solution. Then slices were stained in Masson ponceau-acid solution for 15min, then differentiated in 1% molybdenum phosphate acid. The sections were directly transferred to aniline blue solution and stained, afterwards, sections were differentiated in 0.2% glacial acetic acid. Following with that, slices were dehydrated in 100% ethanol, rinsed in xylene and mounted in neutral resins. The slices were examined using a light microscope (Olympus, Tokyo, Japan).
TUNEL assay kit (Roche Diagnostics, Mannheim, Germany) was used for cell apoptosis detection . The protocol followed with detail instructions in kit. The sample were observed and detected with fluorescence microscope, then the apoptosis rate was calculated.
Immunofluorescence assay (IFA)
Post-deparaffinised slices were submerged in hydrogen peroxide solution, rinsed in PBS and put into 0.1M sodium citrate solution. After incubating with goat serum solution for 30 min, the slices was added with the probe of ROS and Nrf2 antibody, following with incubation overnight at 4℃. After washing in PBC, fluorescent dye-labelled secondary antibody was added to the sections and incubated at 37℃ for 30 min in darkness. DAPI dye was use to stain cell nuclear. After slices mounted in mounting solution, the targeting proteins were observed using the fluorescence microscope.
ELISA Kit (ab108816, Abcam) was used to detect the level of BNP and the ELISA Kit (CEA485Ra, USCN, China) was also performed to examine the level of NT-proBNP in serum. In addition, the ELISA kits of superoxide dismutases (SOD) (ab65354, Abcam), malondialdehyde (MDA) (ab238537, Abcam) and catalase (CAT) (ab83464, Abcam) were performed according to the manufacture’s protocols.
Heart tissue of mice were homogenized with RIPA buffer (CST, USA) at 15,000 rpm for 30 seconds and centrifugated for 15 min at 15,000g at 4 ℃. The protein concentration in the supernatant were determined by BCA assay. 30μg protein were loaded and seperated in 10-12% SDS-PAGE gels under electrophoresis and transferred to PVDF membranes. After blocked in 5% skim milk, membranes were seperately incubated overnight at 4℃ with primary antibodies against Bcl-2 (3498, CST, USA), Bax (5023, CST, USA), CaMKII (13-7300, Abcam, USA), phospho-CaMKII (12716, CST, USA), cAMP response element-binding (CREB) (4820, CST, USA), p-CREB (9198, CST, USA), SERCA2a (4388, CST, USA), dihydropyridine receptor (ab232983, Abcam, USA), Cav1.2 (ab84814, Abcam, USA), Caspase-12 (2202, CST, USA), Nrf2 (12721, CST, USA), HO-1 (43966, Abcam, USA), thioredoxin (Trx-1) (2298, CST, USA), GRP78 (ab21685, Abcam, USA) and GAPDH (51742, CST, USA) as the internal control. Then, PVDF membrane was added with HRP-conjugated goat anti-rabbit secondary antibody (1:1000,5597, CST, USA) for 1 h, the intensity of protein bands was visualized with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (CAT#3480, Thermo Fisher Scientific, USA)
Results were analyzed by Graphpad Prism 8.0 and represented as mean ± SD. To determine the statistic significant differences, un-paired Student’s t-test was used for two group comparison, One-way analysis of variance (ANOVA) was performed followed by multiply comparisons. P value less than 0.05 was considered to be statistically significant different.