The research was performed in Tangshan Workers’ Hospital from December, 2022 to January, 2024.

Mesoporous BG microspheres were prepared to adopt the sol-gel methodology [13]. A mixture of tri-block copolymer P123 (4.0 g), triethyl phosphate (0.73 g), 0.5 mol/L hydrochloric acid (1.0 g), ethanol (60 g), tetraethyl orthosilicate (6.7 g) and calcium nitrate (1.4 g) was stirred continuously at 25 °C for 24 hrs. They were calcined at 700 °C for 5 hrs to obtain mesoporous BG. A 1 g/L suspension of the mesoporous BG was prepared and mixed with a 1 g/L aqueous solution of SVST. The mixture was shaken horizontally at 24 °C for 24 hrs in the dark, then centrifuged at 3000 rpm for 5 min to remove supernatant. Precipitate was rinsed thrice with deionized water, dried at 37 °C using a WH-A-5 electric hot air-drying oven (WHA5, Nanjing Wohuan Technology Co., Ltd., Nanjing, China) and ground to obtain the mesoporous BG microspheres loaded with SVST (SVST/MNBG).

The microscopic morphology and particle size of SVST/MNBG were examined using a ZEM20 scanning electron microscope (ZEM 20, Anhui ZEPTOOLS Technology Co., Ltd., Tongling, China). Nitrogen adsorption/desorption experiments were conducted at liquid nitrogen temperature using an ASAP2010 analyzer (Micromeritics Instrument Corp., Norcross, GA, USA). Pore distribution was computed using Barrett-Joyner-Halenda (BJH) methodology [13].

The drug release profile of SVST/MNBG was assessed using a UV5 Nano UV-Vis spectrophotometer (Mettler Toledo, Columbus, OH, USA). Fifty milligrams of SVST/MNBG particles were placed in a dialysis bag with a molecular weight cutoff of 3500 and diluted with 5 mL of phosphate-buffered saline (PBS). The dialysis bag was immersed in PBS (45 mL, pH = 7.4), sealed and incubated at 37 °C with shaking. Samples were collected at 3, 6, 12, 24, 72, 168, 336 and 504 hrs. Absorbance was measured at 350 nm to determine the content of sacubitril/valsartan sodium, using a standard curve for calculation. After each sampling, an equal volume of PBS was applied to maintain the consistency of liquid volume in the system.

Sixty specific pathogen-free Sprague Dawley rats (male, 200–250 g; Chengdu Dashuo Experimental Animal Co., Ltd., Chengdu, China) were selected for this experiment. Rats were kept at 23 $\pm$ 2 °C, relative humidity of 50 $\pm$ 5%, low light with a 12 hrs light/dark cycle, well-ventilated and noise levels below 80 dB for one week before modeling. Fasting lasted for 8 hrs before the procedure and rats can have water.

Sixty rats were randomly allocated into four groups: Ctrl group, CHF group, SVST group and SVST/MNBG group (each with 15 rats). The Ctrl group received no treatment. The CHF, SVST and SVST/MNBG groups first underwent CHF modeling. For this, rats were anesthetized with a 40 mg/kg intraperitoneal injection of sodium pentobarbital (Suicheng Pharmaceutical Co., Ltd., Zhengzhou, China) and a midline abdominal incision 2.5 cm below the xiphoid process was made. The abdominal cavity was exposed and the abdominal aorta was isolated and ligated to achieve a 50% circumferential constriction. After 4 weeks of modeling, a cardiac ultrasound was performed, with a left ventricular ejection fraction (LVEF) of 45% considered as the criterion for successful modeling [14]. The SVST group received a continuous gavage of 10 mg/kg/day SVST dissolved in water for 6 weeks post-surgery. The SVST/MNBG group received a continuous gavage of 10 mg/kg/day MNBG suspension for 6 weeks post-surgery.

All stages of the study were carried out according to ethical principles and accepted by the Ethics Committee for Animal Experiments.

Four weeks after cessation of treatment, rats were anesthetized by intraperitoneal injection of sodium pentobarbital (40 mg/kg). Upon achieving anesthesia, they were secured in a supine position for cardiac function assessment. Cardiac function parameters, including LV end-diastolic dimension (LVEDD), LVE-systolic dimension (LVESD), LVEF, LV fractional shortening (LVFS), LV posterior wall end-diastolic thickness (LVPWD) and LV posterior wall end-systolic thickness (LVPWS), were measured via DW-T5 color Doppler ultrasound diagnostic system (Dawei Medical Co., Ltd., Shenzhen, China). Each parameter was recorded in triplicate for statistical analysis.

Following the completion of ultrasound examinations, all rats were euthanized via intraperitoneal injection of sodium pentobarbital (150 mg/kg) in accordance with experimental animal ethics guidelines and the chemical euthanasia standards recommended by the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020 Edition) [15]. After confirming deep anesthesia and complete cessation of vital signs, the hearts were excised. One-third of the myocardial tissue was homogenized using a high-throughput vibration grinder (Model HG100, Zhiheng Scientific Instruments Co., Ltd., Shanghai, China). The homogenized tissue was centrifuged at 2500 rpm for 10 min and the supernatant was collected. The expression levels of Reactive Oxygen Species (ROS), nitric oxide (NO), Creatine Kinase-MB isoenzyme (CK-MB) and NT-proBNP in the myocardial tissue were measured via kits (ROS kit, Cat# E004-1-1; NO kit, Cat# A012-1-2; CK-MB kit, Cat# H197; NT-proBNP kit, Cat# H255, Shanghai Westang Biotechnology Co., Ltd., Shanghai, China).

One-third of the myocardial tissue was processed for routine dehydration and sectioned into 4 µm thick paraffin slices. The slices were dewaxed by baking at 60 °C for 20 min, followed by immersion in Xylene I and Xylene II for 5 min each and sequentially rinsed with graded alcohol solutions from high to low concentrations. According to the instructions of the H&E staining kit (Cat# G1120, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), the tissue sections were stained with H&E for 10 min each, rinsed with tap water for 10 min and then rinsed with distilled water before dehydration with 95% ethanol. After clearing with xylene, sections were mounted with neutral gum and examined under a BX53 optical microscope (BX53, Olympus Corp., Tokyo, Japan) to visualize pathological changes.

One-third of the myocardial tissue was taken and 100 µL of Radio-immunoprecipitation assay (Cat# R0278, Sigma-Aldrich, St. Louis, MO, USA) buffer was applied to thoroughly lyse the tissue and extract the total protein. The extract was then heated to 100 °C in a DK-S12 water bath (Model DK-S12, Shanghai Jingqi Instrument Co., Ltd., Shanghai, China) to denature the proteins. The bicinchoninic acid kit (Cat# BCA1, Sigma-Aldrich, St. Louis, MO, USA) determined protein concentrations. Protein (30 µg) was loaded for electrophoresis, separated and transferred to a membrane, which was subsequently blocked at 25 °C for 2 hrs. It was incubated overnight at 4 °C with primary antibodies against Bcl-2, Bax, Atg5, Beclin1, p62, LC3-I, LC3-II and GAPDH (all diluted 1:2000; Cat# ab182858, ab32503, ab109490, ab62557, ab109012, ab192890, ab232940, ab8245; Abcam, Cambridge, MA, USA). After the membranes were washed, the secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG-HRP (diluted 1:5000; Cat# ab6721, Abcam, Cambridge, MA, USA), was added and incubated at 25 °C for 2 h. Following additional washes, an enhanced chemiluminescence reagent (Cat# E2529, Sigma-Aldrich, St. Louis, MO, USA) was utilized for detection and imaging was conducted with a WD-9413A gel imaging system (Model WD-9413A, Beijing Liuyi Biotechnology Co., Ltd., Beijing, China). The GAPDH was utilized as a loading control and the relative optical density of the target proteins was calculated employing Image-Pro Plus 6.0 (Media Cybernetics Inc., Rockville, MD, USA) [13].

Data were processed using SPSS 26.0 (IBM, Armonk, NY, USA). Quantitative data (Mean $\pm$ Standard Deviation ($\overline{\chi }$$\pm$ s)) were compared among groups utilizing One-way ANOVA, with between-group comparisons conducted via t-test. Categorical data (frequencies or rates) were compared between groups via $\chi$² test. The p 0.05 implied statistical significance.

First, nitrogen adsorption/desorption isotherm analysis were conducted for MNBG and SVST/MNBG (Fig. 1a). Both samples exhibited type IV isotherms, indicative of typical mesoporous solid adsorption. Next, pore size distribution analysis was performed for MNBG and SVST/MNBG (Fig. 1b), revealing a single characteristic peak for both samples, located at 4.1 and 6.0 nm, respectively. Finally, the in vitro drug release profile of SVST/MNBG was analyzed (Fig. 1c). It was observed that a burst release occurred within the first 0–12 hrs, followed by a stable drug release concentration of approximately 45 mg/L from 24 to 504 hrs, with a drug loading capacity of 33%.

Fig. 1.

Characterization of MNBG. (a) Nitrogen adsorption/desorption isotherms, (b) Pore size distribution curve and (c) Drug release profile over time. SVST, Sacubitril/valsartan tablets; MNBG, Mesoporous nano-bioactive glass.

Comparisons were made of cardiac function parameters among the different groups, including LVEDD, LVESD, LVEF, LVFS, LVPWD and LVPWS (Fig. 2a–f). The CHF group exhibited drastic increases in LVEDD, LVESD, LVPWD and LVPWS versus Ctrl group, along with great declines in LVEF and LVFS (p 0.05). In comparison to the CHF group, SVST and SVST/MNBG groups showed prominent reductions in LVEDD, LVESD, LVPWD and LVPWS and substantial increases in LVEF and LVFS (p 0.05). Furthermore, relative to the SVST group, the SVST/MNBG group demonstrated notably lower LVEDD, LVESD, LVPWD and LVPWS and higher LVEF and LVFS (p 0.05).

Fig. 2.

Cardiac function indicators of rats in each group. (a) LVEDD, (b) LVESD, (c) LVEF, (d) LVFS, (e) LVPWD and (f) LVPWS. LVEDD, Cardiac function parameters; including LV end-diastolic dimension; LVESD, LVE-systolic dimension; LVEF, Left Ventricular Ejection Fraction; LVFS, LV fractional shortening; LVPWD, LV posterior wall enddiastolic thickness; LVPWS, LV posterior wall end-systolic thickness, *p 0.05 vs Ctrl group, ^#p 0.05 vs CHF group and ^{^}p 0.05 vs SVST group.

Comparisons were made of ROS (Fig. 3a), NO (Fig. 3b), CK-MB (Fig. 3c) and NT-proBNP (Fig. 3d) in myocardial tissue across various groups. The CHF group exhibited greatly increased ROS, CK-MB and NT-proBNP and a markedly decreased NO versus Ctrl group (p 0.05). The SVST and SVST/MNBG groups showed notable reductions in ROS, CK-MB and NT-proBNP and increased NO versus the CHF group (p 0.05). Additionally, relative to the SVST group, SVST/MNBG group demonstrated notably lower ROS, CK-MB and NT-proBNP and a considerably higher NO (p 0.05).

Fig. 3.

Cytokine levels in rat myocardial tissue. (a) ROS, (b) NO, (c) CKMB and (d) NT-proBNP. ROS, Reactive Oxygen Species; NO, Nitric oxide; CK-MB, Creatine Kinase-MB isoenzyme; NT-proBNP, N-terminal pro-B-type natriuretic peptide, *p 0.05 vs Ctrl group, ^#p 0.05 vs CHF group and ^{^}p 0.05 vs SVST group.

In Fig. 4a–d, Ctrl group exhibited clear myocardial tissue structure with orderly fiber arrangement and well-defined, uniform-sized nuclei. In contrast, the CHF group showed blurred myocardial tissue structure with disorganized fiber arrangement, including fragmentation and nuclei appeared fragmented and incomplete. Myocardial cell hypertrophy and swelling were evident. Relative to the CHF group, SVST and SVST/MNBG groups displayed relatively clearer myocardial tissue structures and uniform nuclei sizes. Nevertheless, a small amount of fiber fragmentation and cytoplasmic vacuolation in myocardial cells was still observed.

Fig. 4.

Pathological morphology of myocardial tissue in each group of rats ($\mathrm{\times }$200, $\mathrm{\times }$400). (a) Ctrl group, (b) CHF group, (c) SVST group and (d) SVST/MNBG group. CHF, Chronic heart failure; SVST, Sacubitril/valsartan tablets; MNBG, Mesoporous nano-bioactive glass. Scale bar = 20 µm, 50 µm.

Comparisons were made of the relative expression levels of Bcl-2 and Bax (Fig. 5a–c) in myocardial tissue across different groups. The CHF group exhibited a notable decrease in the relative expression level of Bcl-2 and a marked elevation in the relative expression level of Bax (p 0.05). The SVST and SVST/MNBG groups showed markedly higher relative expression levels of Bcl-2 and greatly lower expression levels of Bax (p 0.05). Additionally, relative to the SVST group, the SVST/MNBG group demonstrated a substantially greater increase in Bcl-2 expression level and a more pronounced decrease in Bax expression level (p 0.05).

Fig. 5.

Apoptosisrelated protein expression level in rat myocardial tissue. (a) Western blot detection result, (b) Relative expression level of Bcl-2 (Bcell lymphoma 2) and (c) Relative expression level of Bax. *p 0.05 vs Ctrl group, ^#p 0.05 vs CHF group and ^{^}p 0.05 vs SVST group.

Comparisons were made of the relative expression levels of autophagy-related proteins Atg5, Beclin1, p62 and LC3 (Fig. 6a–e) in myocardial tissue across various groups. Relative to Ctrl group, CHF group showed notably reduced relative expression levels of Atg5, Beclin1 and p62, along with a marked increase in the LC3II/I relative expression level (p 0.05). The SVST and SVST/MNBG groups exhibited markedly higher relative expression levels of Atg5, Beclin1 and p62 and a greatly lower LC3II/I ratio (p 0.05). Additionally, SVST/MNBG group demonstrated notably higher relative expression levels of Atg5, Beclin1 and p62 and a greatly lower LC3II/I relative expression level (p 0.05).

Fig. 6.

Autophagyrelated protein expression level in rat myocardial tissue. (a) Western blot detection result, (b) Relative expression level of Atg5, (c) Relative expression level of Beclin1, (d) Relative expression level of p62 and (e) Relative expression level of LC3II/I. *p 0.05 vs Ctrl group, ^#p 0.05 vs CHF group and ^{^}p 0.05 vs SVST group.