Effect of typhaneoside on ventricular remodeling and regulation of PI3K/Akt/mTOR pathway

A total of 150 specific-pathogen-free (SPF) male Sprague–Dawley rats, 8 weeks old, weighing 200 ± 20 g, were obtained. After fasting for 12 h before surgery, the rats were provided with free drinking water. Using a noninvasive inhalation anesthesia system, with the ventilation rate set to 1.5 l/min, the rats inhaled 2% isoflurane. An electrocardiogram (II lead) was then recorded. The left anterior descending coronary artery was ligated. The rats were placed on the test bench; at 2 cm to the intercostal space between the fourth and fifth left sternum, a 2-cm longitudinal incision was made with ophthalmic scissors, and the pectoralis major and the chest were bluntly separated. A broken purse-string suture was made around the incision, and the suture was temporarily not tightened.

With the help of a hemostat, the pleura and pericardium were penetrated at the fourth intercostal space, the chest was opened and the left chest wall was pressed clockwise. The apex was then opened toward the chest wall, and the heart was squeezed out of the chest with the index finger and thumb of the left hand. At 2 cm to the lower edge of the left atrial appendage, the left coronary artery was ligated with a small round needle and a 6/0 nonabsorbable line. The apex instantly turned white, and the heart was quickly placed in the chest cavity; the purse-string suture was tightened, the chest wall was squeezed to remove thoracic gas, and the chest cavity was closed.

In the sham group, 20 rats were treated with the same surgical procedure, but the anterior descending artery was only threaded without ligation. Serum troponin I levels were measured 24 h after surgery to determine whether the myocardial infarction model was successfully established. Postoperatively, 200,000 u/kg penicillin via an intraperitoneal injection was routinely administered for 7 days as anti-inflammatory treatment. The animals received routine feeding for 4 weeks. After the myocardial infarction model was established, the rats were included in the experiments.

The 16 live rats in the sham group (group A) were given the same dose of normal saline. The 80 successfully modeled rats were randomly divided into four groups: the model group (group B) was given the same dose of normal saline; the typhaneoside low-dose group (group C) was given 10 mg/kg of typhaneoside; he typhaneoside middle-dose group (group D) was given 20 mg/kg of typhaneoside; the typhaneoside high-dose group (group E) was given 40 mg/kg of typhaneoside. The typhaneoside infusion was administered once a day for 4 weeks.

After 4 weeks of treatment, the left ventricular short axis shortening rate (LVFS), stroke volume (SV), heart rate (HR), cardiac output (CO), left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), and left ventricular ejection fraction (LVEF) were all measured.

After 4 weeks of treatment, mean arterial pressure (MAP), left ventricular diastolic pressure (LVDP), left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), left ventricular maximum ascending rate (+dp/dtmax), and the maximum rate of decrease in left ventricular pressure (−dp/dtmax) were all determined.

Enzyme-linked immunosorbent assay (ELISA) was used to detect the expressions of serum N‑terminal pro-brain natriuretic peptide (NT-proBNP), matrix lysin 2 (ST2), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), matrix metalloproteinase 2 (MMP-2), and MMP-9. The aluminum foil bag with slats was equilibrated at room temperature for 20 min, and the required strips were removed. Standard holes, sample holes, and blank holes were set on the strips; the blank holes were not filled with any liquid, while the standard and sample holes were filled with different concentrations of 50-μl samples. A total of 10 ml sample was added to the holes for testing, and then a 40-ml sample dilution was added. Subsequently, 50 ml horseradish peroxidase-labeled detection antibody was added to the standard and the sample holes, which were sealed with a film and placed in an incubator at 37 °C. The incubation time was set to 60 min. The liquid was discarded and the required strips patted dry on the absorbent paper. Washing liquid was injected into the hole. After 1 min, the washing liquid was removed and the strips then placed on the absorbent paper to dry; this was repeated five times. Substrate A and B at 50 ml were added to each hole, respectively, and then placed in an incubator at 37 °C in darkness. Stop solution 50 μl was added to each hole, and the optical density (OD) value at a wavelength of 450 nm was measured within 15 min.

The skin of the chest wall of the suckling rat was disinfected, the chest cavity was cut open along the left edge of the xiphoid process of the sternum, the heart was fully exposed, and the apex was clamped with ophthalmology scissors. The heart was then bluntly separated and placed in a Petri dish containing pre-cooled phosphate buffered saline (PBS). The heart was rinsed thoroughly three times. The pericardium, large blood vessels, and atrial tissue around the heart were removed. The ventricular muscles were cut into 1–3-mm tissue pieces and then placed in a serum bottle. An equal volume of type I collagenase (0.08%) and trypsin (0.08%) was placed in a serum bottle, then gently pipetted and mixed at 37 °C for 5‑min digestion in a magnetic stirrer (200 rpm). The first aspirated supernatant was discarded, and the digestion was continued for 5 min. Following this, the aspirated supernatant was added to the serum-containing medium to terminate the digestion, and the digested product was filtered through a 200-mesh filter until the tissue block disappeared or the supernatant was clear. The filtrate was centrifuged on a horizontal centrifuge (1000 rpm) for 5 min, the supernatant was removed, and the pellet was fully resuspended in the complete medium. The cells were suspended in a Petri dish and placed in a cell culture incubator with 5% CO₂ at 37 °C, and then subjected to differential adherence. Following this, the unattached cardiomyocyte suspension was aspirated by pipetting the bottom of the culture dish, and then placed in a complete medium containing 20% fetal bovine serum, after which the cells were counted with a cell-counting plate. The cells were inoculated into different cell culture plates at a density of 5 × l0⁵/ml. The cells were added to 0.1 mol/l Brdu and placed in a cell culture incubator with 5% CO₂ at 37 °C. Pulsation was observed for a small portion of cells after 24 h, and the cells were basically adherent after 48 h. The medium was changed every 2–3 days according to the metabolic state of the cells.

We randomly divided 60 male Sprague-Dawley rats into two groups: a drug-administered group and a control group, with 30 rats in each group. The drug-administered group was given 40 mg/kg of typhaneoside, while the control group was given an equal volume of saline. Rats were routinely fed for 4 days, with fasting on the fourth day after the last gavage. Saline or typhaneoside was administered to the rats twice on the fifth day, with a 1-h gap between administration, and blood samples were obtained 1 h after the last gavage. After anesthetizing the rats with 3% pentobarbital, blood was obtained from the abdominal aorta of the rats with a negative-pressure blood collection needle, and the group number was indicated. The blood sample was centrifuged at 3000 rpm at room temperature for 15 min. After pipetting the supernatant, the blood was placed in a water bath for 30 min and the temperature was set to 56 °C. After deactivation, the cells were sterilized by filtration using a 0.22-μm microporous membrane. Following this, the blood was stored in 2‑ml sterile EP tubes.

After pretreatment of primary cardiomyocytes with drug-containing serum, the medium was removed, the cells were washed once with PBS, then PBS was added again, and the cells were placed in an oxygen-deficient box. Nitrogen was replaced with oxygen in the hypoxia box, and the box was sealed. The box was placed in an incubator at 37 °C, with hypoxia conditions for 12 h. Following this, the cells were removed from the hypoxia box, the PBS was removed and replaced with the complete medium, and the cells were cultured in a constant temperature box at 37 °C, with reoxygenation for 4 h. The experimental grouping was as follows:

The A, B, and C groups were prepared for electron microscopy and immunofluorescence, and the A, B, C, D, E, F, and G groups were prepared for Western blot analysis.

Glutaraldehyde (2.5%) was prepared with phosphate buffer. The tissue samples were fixed and rinsed with 0.1 M phosphate for 15 min, three times, and then fixed with 2% citrate fixative for 1 h. The tissue samples were rinsed with 0.1 M phosphate solution for 15 min, three times. Following this, the samples were placed in different concentrations of ethanol (50%, 70%, and 90%) and acetone (90%) for 15 min, then placed in 100% acetone at room temperature three times, for 15–20 min each time. The tissue samples were then placed in a solution with a 2:1 ratio of acetone to embedding solution for 3–4 h. Following this, the tissue samples were placed in a solution with a 1:2 ratio of acetone to embedding solution and left overnight. The tissue samples were placed in 37 °C pure embedding solution for 2–3 h. After the tissue samples were embedded, they were placed in an oven at 37 °C for 24 h, then in an oven at 45 °C for 12 h, and successively in an oven at 60 °C for 24 h. The tissue was sliced into 50–70-mm slices on a microtome. The slices were double-stained with uranyl acetate-lead citrate (3%) and prepared for transmission electron microscopy for observation.

The paraffin slices were dewaxed in xylene I for 10 min, and then in xylene II for 10 min. The slices were successively rinsed in 100% xylene I, xylene II, and 80% alcohol for 10 min, respectively. The slices were then placed on a shaker and rinsed thoroughly with PBS for 5 min. After the antigen was repaired, the slices were naturally cooled at room temperature for 20–30 min, then placed on a shaker and rinsed thoroughly with PBS buffer for 3 min three times. The slices were thoroughly rinsed with PBS for 3 min three times, and 50 μl of primary antibody (LC3B) was added dropwise . The mixture was then incubated for 60 min; the incubator temperature was set to 37 °C. Subsequently, the slices were thoroughly rinsed with PBS for 3 min three times. The secondary antibody was added dropwise and the slices were incubated at room temperature for 60 min. The slices were thoroughly rinsed with PBS for 3 min three times, and then stained with DAPI for about 2 min. Following this, the slices were thoroughly rinsed with PBS for 3 min and observed under a fluorescence microscope.