Sodium hydrosulfide (NaHS) was purchased from Sigma-Aldrich (Merck KGaA; Darmstadt, Germany; cat. no. 161527). LY294002 (a PI3K inhibitor) was purchased from ApexBio (Houston, TX, USA; cat. no. 8250–200 mg). Akt, phospho (p)-Akt and caspase-3 antibodies were obtained from Abcam (Cambridge, MA, USA; cat. nos. ab179463, ab192623 and ab13847, respectively). PI3K, p-PI3K and nuclear factor-κB (NF-κB) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA; cat. nos. 4249, 4228 and 8242, respectively). B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) antibodies were obtained from Boster Biological Technology, Ltd. (Wuhan, China; cat. nos. BA0412 and BA0315-2, respectively). Peroxidase-conjugated AffiniPure goat anti-rabbit immunoglobulin G (IgG), peroxidase-conjugated AffiniPure goat anti-mouse IgG and mouse anti-β-actin monoclonal antibodies were obtained from OriGene Technologies, Inc. (Beijing, China; cat. nos. ZB-2301, ZB-2305 and TA-09, respectively). In situ Cell Death Detection kit, POD was obtained from Roche Diagnostics GmbH (Mannheim, Germany; cat. no. 11684817910). Rat troponin I (TnI), interleukin (IL)-10, IL-6 and tumor necrosis factor-α (TNF-α) ELISA kits were provided by Elabscience Biotechnology Co., Ltd. (Wuhan, China; cat. nos. E-EL-R0055c, E-EL-R0016c, E-EL-R0015c and E-EL-R0019c, respectively).

Adult male Sprague Dawley rats (7–8 weeks old; body weight 211.58±11.42 g) were provided by the animal center of Xinjiang Medical University [Urumqi, China; animal use license no. SYXK (Sinkiang) 2011-010101]. The animals were housed in cages with pathogen-free conditions, and free access to food and water, under a 12-h light/dark cycle at a room temperature of 22°C with 45% humidity, and normal air conditions (21% O₂, 78% N₂ and 0.03% CO₂). Chloral hydrate (350 mg/kg; intraperitoneal injection) was used to anesthetize rats for surgery and blood collection. Subsequently, rapid cervical dislocation was used to sacrifice the animals painlessly. All experimental protocols in this study complied with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (1996) and were approved by the Animal Protection and Use Committee of Shihezi University (Shihezi, China).

Prior to surgery, all mice were fasted for 8 h, with water freely available. The CLP procedure was performed as previously described (21). Briefly, the rats were anesthetized with chloral hydrate and then secured to a sterile operating table. Under aseptic conditions, a 2-cm incision was made in the abdomen, and the cecum was exposed layer by layer. Prior to ligation of the cecum, feces were gently squeezed to the distal end of the cecum, then the cecum was ligated with a thin wire, and the cecum was pierced with an 18-gauge needle. Any remaining contents were extruded through the puncture site. Subsequently, the cecum was pushed back into the abdominal cavity, the abdominal cavity was closed and the layers were sutured. The sham group underwent the same procedure without CLP. All rats were resuscitated using 0.9% sodium chloride brine (subcutaneous injection, ~40 ml/kg), and following surgery, rats were returned their cages with free access to food and water.

Rats (n=56) were randomly divided into 7 groups as follows: Sham group, underwent exposure of the cecum, but not ligation and perforation; sham + NaHS group, received the sham procedure and was administered a 2-ml/kg intraperitoneal administration of 8.9 µmol/kg NaHS 1 h following the surgery; sham + LY294002 group, underwent the sham procedure and was administered a 2-ml/kg intraperitoneal injection of 40 mg/kg LY294002 following the surgery; CLP group, treated with the CLP surgery; CLP + NaHS group, underwent CLP and was administered NaHS (dosage and method as in the sham + NaHS group); CLP + LY294002 group, underwent CLP and was administered LY294002 following the surgery (dosage and method as in the sham + LY294002 group); CLP + NaHS + LY294002 group, underwent CLP and was administered an intraperitoneal injection of NaHS and LY294002 at the aforementioned doses. The doses of LY294002 and NaHS were selected according to previous studies (5,12). Following the procedures, all the rats were free to drink and eat, and 12 h later they were anesthetized again. The rat abdominal cavity was opened and blood was obtained from the abdominal aorta. Following coagulation, blood samples were centrifuged (3,500 × g, 15 min) and clear supernatants were collected. A portion of each serum sample was sent to the First Affiliated Hospital of Shihezi University for the measurement of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH). The remaining samples were frozen at −80°C for subsequent analysis. The rats were sacrificed by cervical dislocation immediately after blood collection. Heart tissue was obtained immediately after sacrifice. Half of each heart tissue sample was fixed in paraformaldehyde (4%) for histomorphological analysis, and the other half was stored at −80°C for western blot analysis and other experiments.

Serum CK-MB and LDH levels in the rats were measured using an automatic biochemical analyzer (Modular DPP H7600; Roche Diagnostics, Basel, Switzerland). An ELISA kit was used to measure serum cTnI levels according to the manufacturer's instructions.

ELISA kits were used to determine the TNF-α, IL-6 and IL-10 levels in serum according to the manufacturer's instructions.

H₂S levels in serum were detected using a H₂S testing kit purchased from Nanjing Institute of Bioengineering (Nanjing, China). H₂S reacts with zinc acetate, N,N-dimethylphenylenediamine and ammonium ferric sulfate in the kit to form methylene blue, which has a maximum absorption peak at 665 nm. The H₂S content was calculated by determining the absorbance value of methylene blue, and the plasma H₂S level is expressed in µmol/l.

The tissue fixed in paraformaldehyde was conventionally embedded, sliced (4-µm thick) and dewaxed with xylene, then washed with ethanol at various levels. Subsequently, sections were stained with hematoxylin and eosin (H&E) at room temperature for ~2 min, dehydrated, dewaxed and sealed. Finally, images of sections were captured using an optical microscope (Olympus Corporation, Tokyo, Japan).

The frozen myocardial tissue was lysed with lysis buffer (cat. no. R0030; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) on ice, and following protein extraction, a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to determine the concentration of tissue proteins. A total of 10 µl of protein samples (40 mg/ml) were separated by SDS-PAGE using 10% gels, and then transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% bovine serum albumin (cat. no. 4240GR100; BioFroxx; NeoFROXX GmbH, Hesse, Germany) for 2 h at room temperature. Membranes were incubated with antibodies against Akt (1:800 dilution), p-Akt (1:800 dilution), NF-κB (1:800 dilution), PI3K (1:800 dilution), p-PI3K (1:800 dilution), Bcl-2 (1:200 dilution), Bax (1:200 dilution), caspase-3 (1:500 dilution) and β-actin (1:1,000 dilution) at 4°C for 12–18 h. The membranes were then washed six times in TBS-Tween (5 min each time), and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:5,000 dilution) for 2–3 h at room temperature (β-actin was detected using HRP-conjugated goat anti-mouse secondary antibody under the same conditions). Following incubation, the membranes were washed six times over a total of 30 min. The membranes were reacted with an enhanced chemiluminescence reagent (cat. no. WBKLS0100; EMD Millipore, Billerica, MA, USA) and developed using X-ray film. The band intensities were quantified using ImageJ software (Java 1.6.0.24; version 1.51k; National Institutes of Health, Bethesda, MD, USA).

Apoptosis of myocardial cells was determined by a TUNEL assay using the in situ Cell Death Detection kit, POD. Slices (4-µm thick) of heart tissue were incubated in a 68°C oven for 1 h, and then dewaxed in xylene and gradient alcohol. The sliced tissue was then washed three times with PBS (5 min each wash). After washing, the sections were placed in hydrogen peroxide and soaked for 10 min. Subsequently, further washes with PBS were performed, and then sections were steamed in a pressure cooker for 5 min with citrate buffer at pH 6.0, and washed again three times with PBS (5 min each wash). Samples were incubated with 50 µl TUNEL reaction mixture (1:12) at 37°C for 1 h in the dark. The slides were then rinsed three times with PBS, 50 µl POD substrate was added to each section, and the samples were incubated at 37°C for 30 min in the dark. Following incubation, the sections were washed three times with PBS, and then 50 µl diaminobenzidine coloring solution was added dropwise, and the color development was stopped with distilled water when an appropriate degree of coloration was achieved. The sections were counterstained with hematoxylin for 2 min, then dehydrated in alcohol and sealed with neutral gum. Finally, a light microscope was used to calculate the apoptotic index. Dark brown staining indicated TUNEL-positive cells, and five high power fields of the slices were randomly selected to calculate the proportion of positive cells.

Each experiment was repeated three times. All data were analyzed using SPSS 22.0 software (IBM Corp., Armonk, NY, USA). All data are expressed as the mean ± standard error. The data were analyzed using one-way analysis of variance followed by least significant difference tests. P<0.05 was considered to indicate a statistically significant difference.

The rats in the CLP group exhibited the behavioral characteristics of sepsis, including malaise, fever, chills, piloerection, generalized weakness and reduced gross motor activity, as well as weight loss and increased proinflammatory cytokine levels in the serum. One mortality in the CLP group and two in the CLP+LY294002 group were observed.

As presented in Fig. 1, CK-MB, LDH and cTnI concentrations exhibited no significant differences among the sham, sham + NaHS and sham + LY groups. There was a significant increase in serum CK-MB, LDH and cTnI in the CLP group compared with the sham group, and rats in the NaHS treatment group exhibited reductions in CK-MB, LDH and cTnI levels compared with those in the CLP group. However, these reductions were attenuated by LY294002 (a PI3K/Akt pathway-specific inhibitor).

Inflammatory factors were measured in the serum of model rats. Following CLP, the levels of the inflammatory factors TNF-α, IL-6 and IL-10 in the serum of rats were increased significantly compared with those in the sham group (P<0.05; Fig. 2). The administration of NaHS decreased the levels of TNF-α and IL-6 in the serum compared with those in the CLP group (P<0.05), whereas the level of IL-10 was significantly increased. However, the administration of LY294002 eliminated the effects of NaHS (P<0.05 vs. CLP + NaHS group; Fig. 2).

Although level of H₂S was increased in the CLP group, there was no significant difference in H₂S levels between the CLP and sham groups. Following the administration of NaHS, the serum H₂S content was increased significantly compared with that in the CLP group (P<0.05; Fig. 3). This finding indicated that the administration of NaHS increases serum H₂S content (Fig. 3).

H&E staining demonstrated that the sham group had normal myocardial fiber morphology and a regular arrangement of myocardial cells, with no abnormalities of the interstices and microvessels (Fig. 4). However, myocardial cells were disordered in the CLP group, with broken myocardial fibers, and parts of the myocardium exhibited vacuolization changes and inflammatory cell infiltration. The administration of NaHS reduced the degree of myocardial damage, and LY294002 eliminated the protective effect of NaHS (Fig. 4).

TUNEL staining was performed to investigate the effect of NaHS on myocardial apoptosis in rats with sepsis (Fig. 5). Myocardial apoptosis was significantly increased in the CLP group compared with the sham group (P<0.05). However, following the administration of NaHS, the apoptosis rate of cardiomyocytes was significantly reduced compared with that in the CLP group, and LY294002 treatment reversed these alterations (Fig. 5).

The p-PI3K/PI3K and p-Akt/Akt ratios, and the levels of NF-κB, Bcl-2, Bax and caspase-3 were determined using western blot analysis to further understand the protective mechanism of NaHS against the myocardial injury caused by sepsis. The p-PI3K/PI3K and p-Akt/Akt ratios, and NF-κB, Bax and caspase-3 levels were increased by CLP compared with those in the sham group, whereas the level of Bcl-2 was decreased (Figs. 6 and 7). Following the administration of NaHS, the phosphorylation of PI3K and Akt was further increased and the expression of Bcl-2 was increased compared with that in the CLP group, whereas the expression levels of NF-κB, Bax and caspase-3 were decreased. The addition of LY294002 eliminated these NaHS-induced effects.