Female healthy Sprague-Dawley (SD) rats weighing 220–250 g, which were purchased from Beijing Vital River Company (Beijing, China), were used for this study. The animals were maintained in a 25°C atmosphere, with a cycle of 12 h light/12 h dark and ad libitum access to food and water. The animal model of endotoxin shock was established by injecting LPS (10 mg/kg) into the peritoneal cavity of the rats and the control group was injected with normal saline. Blood was collected at 6, 12, and 24 h after LPS injection, rats were anesthetized by injection with 3% (v/v) pentobarbital sodium solution (40 mg/kg) into the abdominal cavity, and blood was collected from the inferior vena cava. The serum was separated by centrifugation at 1,000 × g for 10 min at 4°C and preserved at −80°C for subsequent experimentation. The cardiac troponin T (CTnT) levels in the serum were assessed by ELISA. In addition, heart specimens were taken 6, 12 and 24 h after LPS injection. After anesthesia, rats were sacrificed using a continuous flow of CO₂ using a flow meter unit for 3–5 min at the flow rate of 2 l/min (displacement rate, 20% chamber volume/min). The right region of the heart was homogenized for ELISA detection of TNF-α and IL-6 levels, as well as colorimetric analysis for malondialdehyde (MDA). The left region of the heart was placed into paraformaldehyde fixative for 24 h, and used for paraffin sections (4-µm thick) for hematoxylin and eosin (H&E) staining. All procedures for animal care were approved by the Animal Management Committee of The First Affiliated Hospital of Soochow University.

H9c2 rat embryo cardiomyocytes were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and used for cell transfection and LPS injury. Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) were used for cell culture. Sestrin2 siRNA was purchased from MISSION predesigned siRNA (Sigma-Aldrich; Merck KGaA). The sequences of si-Sestrin2 were as follows: Sense, 5′-CAGAGUAUUGUAACAU-3′ and antisense, 5′-AUAGUGUUACAAUACUCUG-3′. Sestrin2 siRNA (50 nM, siSestrin2 group) and Sestrin 2 overexpression (Sestrin2 group) recombinant plasmids (Sigma-Aldrich; Merck KGaA), as well as the empty vector (NC group) were respectively transfected into H9c2 cells using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells without any treatment were used for control. Then, 50 µg/ml LPS was applied in all the above groups for 48 h for most experiments of cell functions, namely, LPS, LPS+NC, LPS+Sestrin2, LPS+siSestrin2 groups. After a 48-h incubation, the cells were collected and used for subsequent experiments.

The paraffin sections of the hearts excised at 6, 12 and 24 h after LPS injection were first dewaxed with xylene I and II for 15 min, and then dewaxed in gradient ethanol at different concentrations (100, 95, 85 and 70%, respectively) for 3 min each, and subsequently rinsed with steaming water for 5 min. Next, the sections were stained with hematoxylin for 10 min and then stained with eosin for 5 min. The sections were again placed into gradient concentrations of ethanol (70, 85, 95 and 100%, respectively) for 3 min each for dehydration. After that, the slices were clarified with xylene I, II for 5 min each time and finally sealed with neutral gum.

The cell viability of H9c2 cells treated with LPS (0, 5, 10, 50 µg/ml) for 0, 24 and 48 h was measured using a Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology, Nantong, China) first. In addition, cell viability of the cells in the different groups (control, NC, Sestrin2, siSestrin2, LPS, LPS+NC, LPS+Sestrin2, LPS+siSestrin2 groups) was also determined. The processes were mainly performed as follows: cells were cultured in 96-well plates (5×10³ cells/well) for 0, 24 and 48 h and stained with 20 µl CCK-8 stain for 1 h. The optical density values (OD) at 450 nm as detected by a microplate reader (BioTek, Winooski, VT, USA) were used for cell viability analysis.

The quantities of CTnT in rat blood, and TNF-α and IL-6 in rat hearts collected at 6, 12, and 24 h after LPS injection, as well as CTnT, TNF-α and IL-6 in the different cell groups (control, NC, Sestrin2, siSestrin2, LPS, LPS+NC, LPS+Sestrin2, LPS+siSestrin2 groups) were determined by ELISA kits (Cusabio, Wuhan, China), following the manufacturer's instructions. The samples and standard substances were added into 96-well plate and incubated for 90 min at 37°C, cultured with biotinylated antibodies for 60 min, and later seeded with avidin peroxidase complex for 30 min before tetramethylbenzidine (TMB) coloration. Finally, OD values at 450 nm as read by a microplate reader (BioTek) were used for quantity calculation.

The levels of lipid peroxidation product MDA in rat heart collected at 6, 12, and 24 h after LPS injection, as well as the levels in the different cell groups (control, NC, Sestrin2, siSestrin2, LPS, LPS+NC, LPS+Sestrin2, LPS+siSestrin2 groups) were determined with the Lipid Peroxidation MDA Assay kit (Beyotime Institute of Biotechnology). According to the manufacturer's instructions, MDA reacts with thiobarbituric acid (TBA) and the red MDA-TBA products were observed at 535 nm using a colorimetric method.

The protein levels of ribosomal protein S6 kinase (p-S6K), phosphorylated AMP-activated protein kinase (p-AMPK) and Sestrin2 in the heart tissues of rats treated with LPS (10 mg/kg) for 6, 12 and 24 h, as well as these proteins in the different cell groups (control, NC, Sestrin2, siSestrin2, LPS, LPS+NC, LPS+Sestrin2, LPS+siSestrin2 groups) were detected by western blot analysis. First, the total proteins were extracted using RIPA lysis buffer (Boster Biological Technology, China), and quantified by BCA protein assay reagent kit (Beyotime Institute of Biotechnology). Then, 10 µg protein was loaded in each lane and were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Amersham; GE Healthcare, UK). The PVDF membranes were blocked with Tris-buffered saline (TBS) containing 5% skim milk with 0.1% Tween 20 at 37°C for 1 h, and then the membranes were incubated overnight at 4°C with the following primary antibodies: p-AMPK antibody (cat. no. 4186; dilution, 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), AMPK antibody (cat. no. 12063; dilution, 1:1,000; Cell Signaling Technology, Inc.), p-S6K antibody (cat. no. 9209; dilution, 1:1,000; Cell Signaling Technology, Inc.), S6K antibody (cat. no. 9202; dilution, 1:1,000; Cell Signaling Technology, Inc.), anti-Sestrin2 (ab178518; dilution, 1:1,000; Abcam, Cambridge, UK) and anti-GAPDH (cat. no. ab8245; dilution, 1:1,000; Abcam). GAPDH was used as internal control. Subsequently, the proteins were probed with goat anti-rabbit secondary antibody (HRP) (cat. no. Ab205718, 1:5,000; Abcam) for 1 h at 37°C. The proteins were detected by enhanced chemiluminescence (ECL) reagents (Amersham; GE Healthcare) and X-ray exposure. Finally, the proteins were quantified using Bio-Rad ChemiDoc system with Image Lab software (version 6.0; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Data are presented as mean ± standard deviations, and were statistical analyzed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA), one-way analysis of variance (ANOVA), and Dunnett's test. P<0.05 was considered as indicative of statistical significance.

The morphological structures of the myocardial tissues of rats injected with LPS (10 mg/kg) for 6, 12 and 24 h were determined by H&E staining. The images showed that the fiber bundles in the control group were normal, neatly arranged, without fractures and dissolution, and no edema was observed in the interstitial. The sepsis rat tissues at 6 h after injection were observed as normal and neatly arranged myocardial fiber bundles, with moderate inflammatory cell infiltration. At 12 h, the myocardial fiber bundles were loosely arranged; some myocardial fibers were broken and dissolved, accompanied by mild interstitial edema and moderate inflammatory cell infiltration. At 24 h, myocardial fiber bundles were loosely arranged, some myocardial fibers were broken and dissolved, partial myocardial cells were degenerated and dissolved, accompanied by necrosis, interstitial edema, and moderate inflammatory cell infiltration (Fig. 1A). Then, the levels of inflammatory factors including CTnT, TNF-α and IL-6 in rat blood collected at 6, 12 and 24 h after LPS injection were measured by ELISA. The results showed that serum CTnT levels were increased time-dependently. Levels of TNF-α and IL-6 in the heart were increased by LPS, reached a peak at 6 h after LPS injection and slightly decreased subsequently (P<0.05; Fig. 1B-D). In addition, the lipid peroxidation product MDA levels in the heart were found to be increased time-dependently, as determined by the colorimetric method (P<0.05; Fig. 1E).

The protein levels of S6K, p-S6K, p-AMPK, AMPK and Sestrin2 were determined by western blot analysis in the rats injected with LPS (10 mg/kg) for 6, 12 and 24 h. The results demonstrated that the protein levels of p-S6K were adaptively decreased, and those of p-AMPK and Sestrin2 were adaptively increased by LPS treatment. Moreover, changes at 6 h were the most significant (P<0.01; Fig. 2). Notably, the protein expression levels of total S6K and AMPK were not significantly changed.

The injury effect of LPS on cardiomyocytes was evaluated by CCK-8 assay. Cell viability of the cardiomyocytes treated with LPS (5, 10 and 50 µg/ml) for 24 and 48 h was inhibited time-dependently, and the decrease in cell viability of the cardiomyocytes treated with 50 µg/ml LPS at 48 h was most significant (P<0.05; Fig. 3A). Then, the function of Sestrin2 on cell viability of the cardiomyocytes was detected at 24 and 48 h after LPS treatment. The results showed that the cell viability changed significantly after 48 h of LPS injury. Compared with the control group, cell viability in the LPS and LPS+NC groups was significantly suppressed following treatment with LPS. Cell viability in the LPS+Sestrin2 group was significantly increased, and cell viability in the LPS+siSestrin2 group was significantly decreased, compared with the LPS group. Cell viability in the LPS+Sestrin2 group was lower than that in the Sestrin2 group, and a similar tendency between that in the LPS+siSestrin2 group and in the siSestrin2 group was observed (P<0.05; Fig. 3B). Then, levels of inflammatory factors such as CTnT, TNF-α and IL-6 were detected, showing a significant increase in the LPS and LPS+NC groups, a decrease in the LPS+Sestrin2 group by Sestrin2, and an increase in the LPS+siSestrin2 group by siSestrin2 (Fig. 3C-E). In addition, levels of lipid peroxidation product MDA as determined by colorimetric method, indicated that the MDA levels were increased in the LPS group, and decreased in the LPS+Sestrin2 group (Fig. 3F).

Levels of p-S6K, p-AMPK and Sestrin2 in cardiomyocytes were evaluated by western blot analysis after Sestrin2 overexpression or siSestrin2 transfection or LPS treatment. The results demonstrated that p-S6K levels were significantly decreased in the LPS, LPS+NC and Sestrin2 groups, and markedly increased in the siSestrin2 group, compared with the control group. Meanwhile, p-S6K levels were decreased in the LPS+Sestrin2 group, and increased in the LPS+siSestrin2 group, compared with the LPS group (Fig. 4A and B). In addition, levels of p-AMPK and Sestrin2 were increased in the LPS, LPS+NC and Sestrin2 groups, and decreased in the siSestrin2 group, compared with the control group; while p-AMPK and Sestrin2 levels were increased in the LPS+Sestrin2 group, and decreased in LPS+siSestrin2 group, compared with the LPS group. The present results suggested that there was no significant difference in S6K and AMPK levels between the LPS+siSestrin2 group and the LPS group (Fig. 4A, C-F).