All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (11), and approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (Wuhan, China). A total of 45 specific pathogen-free male Sprague-Dawley rats (8–10 weeks-old; 180–220 g) were purchased from the Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). All animals were housed in a light-controlled room with a 12 h light/dark cycle, at a temperature of 25°C and a humidity of 45–50%. All animals had free access to food and water for 1 week prior to the commencement of the study to allow them to acclimatize to the laboratory environment. Rats were randomly divided into three groups (15 rats per group), including the control, LPS and LPS+GLN groups. Rats in the LPS+GLN group were intragastrically administered with GLN (cat. no. V900419; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a dose of 1 g/kg once per day for 5 days, while rats in the control and LPS groups received the same volume of normal saline. On day 6, cardiac dysfunction was induced by a single intraperitoneal injection of 10 mg/kg LPS (cat. no. L2880; Sigma-Aldrich; Merck KGaA), as described previously (12). The rats in the control group were injected with the same volume of saline. At 24 h following LPS injection the echocardiography and catheter-based analyses were performed on the surviving rats (control group, n=15; LPS group, n=9; and LPS+GLN group, n=13) under 1.5% inhaled isoflurane anesthesia (13).

The time of LPS injection was assigned as the baseline (time point 0). The survival of rats was observed at 6, 12, 18 or 24 h, respectively, following LPS injection and the mortality rates of the three groups were analyzed.

Transthoracic echocardiography was performed at 24 h after the animals were injected with LPS or normal saline. Echocardiography was performed on anesthetized (inhaled 1.5% isoflurane) rats using a MyLab 30CV (Esaote SpA, Genoa, Italy) equipped with a 10-MHz linear array ultrasound transducer. The left ventricular (LV) dimensions were assessed in parasternal short-axis view during systole or diastole, with a sweep speed of 50 mm/s at the mid-papillary muscle level from the LV M-mode tracing. The LV end-systolic diameter (LVESD), LV end-diastolic dimension L (LVEDD) and fractional shortening (FS) were also measured. Hemodynamic variables were analyzed using a microtip transducer catheter (SPR-839; Millar, Inc., Houston, TX, USA) and a Millar Pressure-Volume system (MPVS-400; Millar, Inc.). The maximal rate of pressure development (dP/dt max), and minimal rate of pressure decay (dP/dt min) were then processed with the PVAN data analysis software LabChart version 7.3.7 (Millar, Inc.; ADInstruments, Dunedin, New Zealand).

Following the collection of echocardiography and catheter-based measurements, blood was collected from the retro orbital plexus in the rats eyes. All blood samples were centrifuged at 4,200 × g for 10 min at room temperature, and the supernatants were subsequently collected and stored at −20°C. The levels of TNF-α (cat. no. KRC3011), IL-1β (cat. no. BMS630) and IL-6 (cat. no. KRC0061) in the blood samples were measured using ELISA kits (eBioscience; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocols.

The animals were sacrificed following the collection of the blood samples and the hearts were excised, washed with 10% KCl and fixed with 10% formalin. Next, the heart tissues were embedded in paraffin and then cut transversely close to the apex to visualize the left and right ventricles. Subsequently, 4–5-µm cardiac tissue sections were stained with hematoxylin and eosin (HE) for histopathological examination, and the pathological changes were evaluated under a light microscope.

In order to investigate the relative mRNA expression of B-type natriuretic peptide (BNP), TLR4, TNF-α, IL-1β and IL-6, pulverized heart tissues were homogenized and total RNA was isolated using TRIzol reagent (cat. no. 15596-026; Invitrogen; Thermo Fisher Scientific, Inc.). A DU 730 Series UV/Vis spectrophotometer (Beckman Coulter, Inc., Brea, CA, USA) was used to determine the quality of RNA using the A260/A280 ratio. Total mRNA (2 µg of each sample) was reverse transcribed using a PrimeScript™ RT reagent kit (cat. no. RR047A; Takara Bio, Inc., Otsu, Japan). According to the manufacturer's protocol, PCR amplification was conducted using a SYBR^® Green Master Mix kit (cat. no. RR820A; Takara Bio, Inc.) and a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The thermocycling conditions for PCR were as follows: Initial denaturation step at 95°C for 30 sec; followed by 40 cycles of denaturation at 95°C for 5 sec, annealing at 60°C for 34 sec; and a dissociation stage at 95°C for 15 sec, followed by 60°C for 1 min and 95°C for 15 sec. The primers used for amplification of the respective genes are listed in Table I. All results were normalized against GAPDH gene expression by using the 2^−ΔΔCq method (14).

In order to determine the activation state of the TLR4/MAPK/NF-κB signaling pathway, the nuclear and cytoplasmic proteins were extracted from the cardiac tissues. Cardiac tissues were harvested and homogenized on ice for 15 min using a radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology, Haimen, China), a cOmplete Protease Inhibitor Cocktail (cat. no. 4693159001; Roche Diagnostics, Basel, Switzerland) and PhosStop Phosphatase Inhibitor Cocktail (cat. no. 4906837001; Roche Diagnostics). The homogenates were subsequently collected and transferred to microcentrifugation tubes for centrifugation at 12,000 × g for 30 min at 4°C. The supernatant was collected and the protein concentration of samples was measured using a BCA kit (cat. no. 23227; Thermo Fisher Scientific, Inc.). The nuclear proteins were extracted using Nuclear and Cytoplasmic Extraction Reagents (cat. no. P0027; Beyotime Institute of Biotechnology), according to the manufacturer's protocol. A total of 50 µg of protein was loaded per lane, subjected to 10% SDS-PAGE and subsequently transferred onto polyvinylidene difluoride membranes (IPFL00010; EMD Millipore, Billerica, MA, USA). The membranes were subsequently blocked with 5% non-fat milk for 1 h at room temperature and washed with PBS 3 times for 5–10 min each time. The membranes were incubated overnight at 4°C with the following primary antibodies directed against TLR4 (dilution, 1:1,000; cat. no. 14358), total extracellular signal-regulated kinase 1/2 (dilution, 1:1,000; ERK1/2; cat. no. 4695), phosphorylated (p-)ERK1/2 (dilution, 1:2,000; cat. no. 4370), total p38 (dilution, 1:1,000; cat. no. 8690), p-p38 (dilution, 1:1,000; cat. no. 4511), total c-Jun N-terminal kinase (dilution, 1:1,000; JNK; cat. no. 9592), p-JNK (dilution, 1:1,000; cat. no. 4668), p65 (dilution, 1:1,000; cat. no. 8242), IκBα (dilution, 1:1,000; cat. no. 4814) and GAPDH (dilution, 1:1,000; cat. no. 97166; all purchased from Cell Signaling Technology, Inc., Danvers, MA, USA), as well as Lamin B1 (dilution, 1:300; cat. no. BA1228; Wuhan Boster Biological Technology, Ltd., Wuhan, China). This was followed by incubation with the following secondary antibodies for 1 h at room temperature in the dark: IRDye 800CW goat anti-mouse 926-32210 (dilution, 1:10,000; cat. no. C50316-03) and IRDye 800CW goat anti-rabbit 926-32211 (dilution 1:10,000; cat. no. C50602-08) (both LI-COR Biosciences, Lincoln, NE, USA). The blots were scanned by a two-color infrared imaging system (Odyssey; LI-COR Biosciences, Lincoln, NE, USA). Specific protein expression levels were normalized to the GAPDH protein expression for total tissues lysates and the membranes were also probed for Lamin B1 as additional loading controls for nuclear protein expression levels. Densitometry analysis was performed using Odyssey Software, version 3.0.29 (LI-COR Biosciences).

Data are expressed as the means ± standard deviation. Differences among groups were determined by two-way analysis of variance followed by a post hoc Tukey's test. Comparisons between two groups were performed by an unpaired Student's t-test. Kaplan-Meier curves were used to analyze the mortality rate of the three groups and the log-rank test was used to compare the survival distributions of two groups and determine the efficacy of GLN treatment. All data were analyzed by SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA), and P<0.05 was considered to be an indicator of a statistically significant difference.

To evaluate the protective effect of GLN pretreatment on the progression of LPS-induced cardiac dysfunction, the rats were assessed by echocardiography and hemodynamic analysis at 24 h after LPS administration. The results revealed that LVEDD, and LVESD were significantly increased in the LPS group compared with the control group, whereas the dP/dt max, dP/dt min and FS were significantly decreased (Fig. 1A and B). These results suggest that LPS aggravated cardiac dysfunction. Pretreatment of the rats with GLN resulted in a significant decrease in the level of LVEDD and LVESD compared with the LPS group, and also caused a significant increase in the dP/dt max, dP/dt min and FS. These results indicate that treatment with GLN has a beneficial effect on the impaired cardiac function in LPS-induced sepsis in rats. As an indicator of cardiac function, the mRNA level of BNP was also investigated (Fig. 1C). The results demonstrated that the mRNA level of BNP in the LPS+GLN group was significantly decreased compared with the LPS only group, but still significantly increased compared with the control group.

Compared with the control group, the LPS rats demonstrated numerous necrotic alterations in the myofibril and neutrophil granulocyte infiltration, which were confirmed by HE staining. The necrotic alterations in the myofibrils and neutrophil granulocyte infiltration were attenuated in the LPS+GLN group compared with the LPS only group (Fig. 2A). RT-qPCR revealed that the mRNA expression levels of inflammatory cytokines, including TNF-α, IL-1β and IL-6, in the LPS+GLN group were significantly decreased compared with the LPS only group, however, they were still significantly increased compared with the control group (Fig. 2B). Similarly, the levels of inflammatory cytokines in the serum were also markedly reduced in the LPS+GLN group compared with the LPS only group and significantly higher compared with the control group (Fig. 2C). These results suggest that GLN may alleviate inflammation and the expression levels of inflammatory cytokines induced by LPS.

Subsequent to stimulation for 24 h with LPS, the level of TLR4 protein expression was evidently higher compared with that in the control group. By contrast, upon pretreatment with GLN, TLR4 protein expression was significantly decreased compared with the LPS only group, however, TLR4 protein expression was still significantly higher in the LPS+GLN group compared with the control group (Fig. 3A and B). This trend was also detected in the level of TLR4 mRNA expression (Fig. 3C).

In order to elucidate the molecular mechanisms underlying the anti-inflammatory effect of GLN, the activation state of the TLR4 downstream signaling pathways, including MAPK and NF-κB pathways, were examined. As revealed in Fig. 4A and B, following the activation of TLR4 induced by LPS, the protein expression levels of p-ERK, p-P38 and p-JNK were significantly increased in the LPS group compared with the control group. GLN pretreatment regulated the MAPK signaling pathway by significantly decreasing the LPS-induced expression of p-ERK, p-P38 and p-JNK compared with the LPS only group. The protein expression levels of p-ERK, p-P38 and JNK in the LPS+GLN group were also significantly increased compared with the control group. Similarly, the protein expression of nuclear p65 (normalized to Lamin B1 protein expression) was significantly increased in the LPS group compared with the control group, but significantly decreased in the LPS+GLN group compared with the LPS only group (Fig. 4C and D). The expression of nuclear p65 in the LPS+GLN group was still significantly increased compared with the control group. Conversely, the protein expression of IκBα was significantly decreased in the LPS group compared with the control group, but was significantly increased in the LPS+GLN group compared with the LPS group. The expression of IκBα in the LPS+GLN group was still significantly decreased compared with the control group. These results suggest that the activation of the MAPK and NF-κB signaling pathways may be partially inhibited by GLN.

Kaplan-Meier survival curves (Fig. 5) demonstrated that the rats with LPS-induced sepsis without GLN pretreatment presented a 24 h mortality rate of up to 40%. However, GLN pretreatment reduced the 24 h mortality rate to 13.3% of the sepsis group rats. The result of the log-rank test revealed a statistically significant difference between the LPS group and the LPS+GLN group (χ²=8.769; P=0.012). After 24 h, the rats were examined by echocardiography and hemodynamic analysis; therefore, further observation of the mortality rate was not possible.