Twelve adult male Sprague-Dawley (SD) rats (8-weeks old, weighing 180-250 g) were obtained from the Shanghai SLAC Laboratory Animal Co., Ltd.; the rats were housed in groups of three per cage with food and water available ad libitum, and were maintained in controlled room temperature (22±2°C) and humidity (60-80%) under a 12 h/12 h light/dark cycle. All animal protocols were approved by Zhejiang University Animal Committee (Zhejiang, China).

Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). CTnT enzyme-linked immunosorbent assay (ELISA) kits were purchased from R&D systems, Inc. (Minneapolis, MN, USA). MDA levels were determined using the TBA method (Jian Cheng Bioengineering Institute, Nanjing, China). Small interfering RNA targeting TNXIP (siTNXIP) and negative control (TNXIP-NC) were purchased from GenePharma (Shanghai, China).

LPS was dissolved in sterile physiological saline (0.9% NaCl) at a concentration of 1 mg/ml. The rats were injected intraperitoneally with 10 mg LPS/kg. The treatment was performed respectively for 6, 12 and 24 h for pathological analysis. The control group was injected with the same volume of physiological saline in the same manner.

All rats were sacrificed by intraperitoneal injection of sodium pentobarbital (200 mg/kg) following LPS treatment for 6, 12 and 24 h. The rats in the control group were sacrificed using the same method. The absence of sounds of breathing and heartbeat, namely respiratory arrest and cardiac arrest, through a stethoscope confirmed death of the rats. Heart tissues (0.5 g) from the same position were removed from the rats in the four groups and homogenized with physiological saline. Subsequently, 0.2 ml homogenate with 0.2 ml 8.1% SOS, 1.5 ml 20% acetic acid and 1.5 ml 0.8% TBA aqueous solution was placed in a lidded glass test tube and then diluted to 4 ml with triple-distilled water, and placed in a water bath for 60 min at 95°C. The samples were removed and cooled to room temperature, and 1 ml distilled water and 5 ml n-butanol pyridine solution were added to the samples, following which the mixture was shaken for 15 min and separated at 3,000 × g for 15 min at room temperature. The n-butanol phase at 532 µm was measured for colorimetric densities. The value of fluorescence was calculated by comparing with standards prepared from 1,1,3,3-tetraethoxypropane (cat. no. T9889, Sigma-Aldrich; Merck KGaA).

Myocardial tissue was removed and the samples were fixed with 10% paraformaldehyde solution for >48 h, following which they were routinely dehydrated and paraffin-embedded to prepare 5-µm tissue sections. The sections were heated in an incubator at 68°C for 1-2 h, and were then placed in xylene for dewaxing three times for 30 min. The sections were then placed in 100, 95, 85 and 75% gradient alcohol for hydration for 5 min. Following washing the sections for 2 min in tap water, the sections were stained with hematoxylin for 10 min and with eosin for 30 sec at room temperature. The sections were then infiltrated with xylene for 5 sec, and the neutral gum was sealed and observed under a light microscope (BX51, Olympus Corporation, Tokyo, Japan).

The tissue samples obtained from all rats for histological and immunohistochemical analyses were fixed in 10% formalin solution and embedded in paraffin, according to conventional histological methods. The paraffin-embedded tissue samples were cut into 5-µm thick sections and the immunohistochemical expression of TXNIP and NLRP3 in tissue sections were determined using avidin-biotin-peroxidase complex. The tissue sections were deparaffinized, rehydrated and treated with 3% H₂O₂ to block endogenous peroxidase activity. Subsequently, the sections were incubated in citrate buffer (0.1 M, pH 6.0) in a microwave (800 W, 10 min) and washed with a phosphate buffer solution (PBS; 0.1 M, pH 7.2). The sections were then incubated in a blocking buffer (5% bovine serum albumin; cat. no. B2064; Sigma-Aldrich; Merck KGaA) for 10 min at room temperature., washed with PBS and incubated with anti-TXNIP (cat. no. ab231966, dilution 1:500, Abcam, Cambridge, MA, USA) and anti-NLRP3 (cat. no. ab214185, dilution 1:500, Abcam) antibodies for 1 h at room temperature and washed again with PBS. The sections were then incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (1:250; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 30 min at room temperature. Finally, the sections were washed again and treated with a 3,3-diaminobenzidine substrate system (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Negative control (NC) samples were used to determine specific TXNIP and NLRP3 immunoreactivity. The number of positive microvessels in each section was counted in 10 microscopic fields (magnifications, ×100 and ×200) under a light microscope (BX51, Olympus Corporation).

The H9C2 cell line was purchased from American Type Culture Collection (Rockville, MD, USA); the cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 100 µg/ml penicillin and 100 µg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere at 5% CO₂ in air. Subsequently, 2×10⁵ cells were seeded onto culture plates and cultured in medium with LPS (1, 10, 20 and 50 µg/ml) for 24 h at 37°C. siTXNIP was added into the culture medium for transfection 24 h prior to the LPS (20 µg/ml) treatment. Following incubation, the cells were collected for the analysis of cellular viability, mRNA and protein expression, apoptosis and ROS.

To investigate the role of TXNIP in sepsis-induced myocardial dysfunction, the H9C2 cells were divided into five groups, as follows: Control group, LPS group (cells were treated with LPS), NC+LPS group (cells were transfected with empty vector and treated with LPS), siTXNIP+LPS group (cells were transfected with siTXNIP and treated with LPS) and siTXNIP group (cells were transfected with siTXNIP). Lipofectamine™ 3000 (LFN) transfection (Thermo Fisher Scientific, Inc.) was performed according to the manufacturer's instructions. In brief, the lipid complex was prepared by combining the reagent of 4 µl LFN Plus with 2 µg of plasmid DNA and then suspended in 1 ml serum-free medium and incubated at room temperature for 15 min. The solution was then mixed with 40 µl of LFN in serum-free medium and incubated at room temperature for 15 min. The lipid compounds were diluted in serum-free medium to produce a 5-ml volume of the required concentration, and the cells were incubated at 37°C with 5% CO₂ for 24 h.

Following transfection of the H9C2 cells with or without siTXNIP prior to LPS treatment, 10 µl of cell counting kit (CCK)-8 solution was added to the wells and the cell were incubated at 37°C for 2 h tin an incubator with 5% CO₂ in the dark. Subsequently, the OD value in each well from different cell groups at an absorbance of 450 nm was determined using a microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell viability was detected with the CCK-8 assay kit according to the manufacturer's protocol.

Following treatment of the H9C2 cells for 24 h, 1×10⁶ cells were collected and 1 ml of trypsin (trypsin) without ethylenediaminetetraacetic acid was used to digest the cells, which were shaken gently, and trypsin was removed when the wall was wet. After 1 min at room temperature, the digestion was terminated by adding DMEM (Corning) containing 10% FBS. The cells were centrifuged at 1,000 × g for 3 min at room temperature and the supernatant was removed. The cells were washed twice with pre-cooled PBS and resuspended in 1X Annexin V binding buffer. According to the Annexin V-FITC cell apoptosis detection kit (cat. no. K201-100, BioVision, Inc., Milpitas, CA, USA), the cells were stained with 1.25 µl Annexin V-FITC and 10 µl prop-idium iodide and measured by flow cytometry (version 10.0, FlowJo, FACSCalibur™, BD Biosciences, Franklin Lakes, NJ, USA).

The H9C2 cells were transfected with siTNXIP and then treated with LPS. The cells were then incubated with 2′,7′-dichloro dihydrogen fluorescein diacetate ester (Beyotime Institute of Biotechnology, Shanghai, China, 5 µM) for 30 min at 37°C. The excitation wavelength of the flow cytometry (version 10.0, FlowJo, BD Biosciences) was 480 nm and the emission wavelength was 525 nm.

The tissues and the H9C2 cells were flushed with cold PBS three times and placed on the ice with protein lysis buffer (RIPA; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h. The samples were centrifuged at 13,500 × g for 30 min at 4°C and the supernatants were extracted. The concentration of protein was determined using a BCA protein assay kit (Bio-Rad Laboratories, Inc.). Subsequently, SDS-PAGE with 10% running gels was used to separate the proteins (at least 40 µg), which were then transferred onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories, Inc.). To block the nonspecific signals, the membrane was incubated with 5% non-fat milk for at least 2 h at room temperature. The proteins were then incubated with primary antibody overnight at 4°C and then washed with 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) in PBS/0.1% Tween-20 and incubated with secondary antibodies (cat. nos. sc-2004 and sc-2005, 1:2,000; Santa Cruz Biotechnology, Inc. Dallas, TX, USA) for 1 h at room temperature. The protein bands were developed with developer (EZ-ECL kit; Biological Industries) and protein quantity was analyzed using ImageJ software (version 5.0; Bio-Rad Laboratories). The antibodies used were as follows: Anti-GAPDH (mouse; cat. no. sc-47724, 1:1,000; Santa Cruz Biotechnology, Inc.), anti-TXNIP (rabbit, cat. no. ab210826, 1:1,000, Abcam), anti-NLRP3 (rabbit, cat. no. ab214185, 1:1,000, Abcam), anti-caspase-1 (rabbit, cat. no. ab1872, 1:1,000, Abcam), and anti-cleaved caspase-1 (rabbit, cat. no. ab25901, 1:1,000, Abcam), anti-catalase (rabbit, cat. no. ab16731, 1:1,000, Abcam), anti-MnSOD (rabbit, cat. no. ab13533, 1:1,000, Abcam) and secondary antibodies (cat. nos. sc-2004 and sc-2005, 1:2,000; Santa Cruz Biotechnology, Inc.).

According to the program provided by the manufacturer, total RNA of the tissues and H9C2 cells was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Chloroform (Sigma Aldrich; Merck KGaA) was added to the tube and incubated at room temperature for 5 min and centrifuged at 14,000 × g for 20 min at 4°C. The supernatant was transferred to a new tube and isopropanol was added. The aqueous phase was centrifuged at 14,000 × g for 20 min at 4°C. The precipitate was washed with 70% ethanol and suspended again in water treated with 0.1% diethyl carbamate. The purity and concentration of the RNA was assessed using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.), and the absorbance was read at 260 and 280 nm. According to the program provided by the manufacturer (Thermo Fisher Scientific, Inc.), a reverse transcription cDNA kit was used to reverse transcribe 1 µg total RNA for the synthesis of cDNA (at 42°C for 60 min, at 70°C for 5 min, and preserved at 4°C). SYBR-Green PCR Master mix (Roche Diagnostics, Basel, Switzerland) was used to perform the qPCR experiment using the Opticon real-time PCR detection system (ABI 7500; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: 40 cycles at 95°C for 15 sec, at 60°C for 1 min). The relative mRNA quantity was determined using the comparative cycle threshold (2^−ΔΔCq) method (16). The expression of GAPDH was used for normalization. The primer sequences used for RT-qPCR are listed in Table I.

Values are presented as the mean ± standard error of the mean. GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) was used to analyze the values. One-way analysis of variance followed by Turkey's post hoc test was applied to analyze differences among the experimental groups. P<0.05 was considered to indicate a statistically significant difference.

The present study determined CTnT levels in the serum of control rats and in septic rats treated for 6, 12 and 24 h. Quantification of CTnT levels was achieved using the ELISA method, and the results showed that the levels of CTnT were significantly higher in the 6, 12 and 24 h groups, compared with than that in the Con group (Fig. 1A). Detection by RT-qPCR analysis (Fig. 1B) revealed that the expression levels of IL-1β and IL-18 were increased in the 6, 12 and 24 h groups, compared with those in the Con group. MDA, a byproduct of unsaturated fatty acid oxidation, was measured using the TBA method, and the results revealed that the levels of MDA were markedly enhanced in the 6, 12 and 24 h groups, compared with that in the Con group (Fig. 1C). H&E staining was used to examine the changes in myocardial tissues, and it was found that myocardial fiber bundles were loosely arranged, some myocardial fibers were broken and dissolved, and some cardiomyocytes were degenerated and necrotic. Interstitial edema was noted and moderate inflammatory cell infiltration was observed as LPS processing time was prolonged (Fig. 1D).

In view of the importance of the TXNIP/NLRP3 signaling pathway in the development of sepsis-induced myocardial injury, the expression levels of cleaved caspase-1, caspase-1, TXNIP and NLRP3 were determined by western blot analysis or immunohistochemistry. It was found that the levels of cleaved caspase-1 were higher in the 6, 12 and 24 h groups compared with that in the Con group (Fig. 2A and B), and the intensity of staining of the rat myocardial sections treated with LPS was observed in a time-dependent manner (Fig. 2C).

In order to elucidate the mechanisms of sepsis-induced myocardial dysfunction, H9C2 cells were used to establish a model of sepsis-induced myocardial dysfunction using LPS (1, 10, 20 and 50 µg/ml) in vitro. The viability of cells was suppressed by LPS at 20 and 50 µg/ml (Fig. 3A). The mRNA levels of IL-1β, IL-18 (Fig. 3B) and TXNIP (Fig. 3C) were enhanced by LPS, and the protein levels of cleaved caspase-1, TXNIP and NLRP3 were also increased by LPS (Fig. 3D and E).

To verify the role of TXNIP in the H9C2 cell model of sepsis-induced myocardial dysfunction, which was established using LPS (20 µg/ml), the TXNIP gene was knocked down by siRNA. Successful transfection using LFN was observed for TXNIP at the mRNA (Fig. 4A) and protein (Fig. 4B) levels. The viability of cells was decreased by LPS and ameliorated by siTXNIP (Fig. 4C). It was also found that the increase of apoptosis (Fig. 4D) and augmentation of IL-1β and IL-18 (Fig. 4E) were inhibited by siTXNIP in the LPS group.

The levels of cleaved caspase-1, TXNIP and NLRP3 were determined by RT-qPCR or/and western blot analysis, and the results demonstrated that the mRNA level of TXNIP was lower in the siTXNIP+LPS group than in the LPS and NC+LPS groups (Fig. 5A), and that the protein levels of cleaved caspase-1, TXNIP and NLRP3 were inhibited by siTXNIP, compared with levels in the LPS and NC+LPS groups, respectively (Fig. 5B and C).

ROS levels were assessed by flow cytometry, and catalase and MnSOD were detected by RT-qPCR and western blot analyses. The results showed that ROS production was lower in the siTXNIP+LPS group than in the LPS and NC+LPS groups (Fig. 6A), and the levels of catalase and MnSOD were enhanced by siTXNIP, compared with levels in the LPS and NC+LPS groups at the mRNA (Fig. 6B) and protein (Fig. 6C and D) levels.