OMT was purchased from Ningxia Qi Yuan Pharmaceutical Co., Ltd. (Yinchuan, China). The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) kit was obtained from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Immunoprecipitation cell lysis buffer and the bicinchoninic acid (BCA) protein concentration assay kit were purchased from Jiangsu Green Biotechnology Co., Ltd. (Xuzhou, China). The ¹²⁵I TNF-α radioimmunoassay kit was obtained from Beijing Chemclin Biotech Co., Ltd. (Beijing, China). TRIzol^® was purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Antibodies targeting lipopolysaccharide binding protein (LBP; goat polyclonal; cat. no. sc-70072), CD14 (goat polyclonal; cat. no. sc-5749), NF-κB (rabbit poly-clonal; cat. no. sc-372), phosphorylated (p)-NF-κB inhibitor (IκB)-α (goat polyclonal; cat. no. sc-7977), p-p38-MAPK (mouse monoclonal; cat. no. sc-7973) and caspase-3 (mouse monoclonal; cat. no. sc-7272)were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Anti-β-actin (cat. no. A4700) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The animal experimental procedure used in the present study was approved by the Ningxia Medical University Animal Care Committee (Yinchuan, China). Male Sprague-Dawley specific-pathogen-free rats [Animal Center, Ningxia Medical University, Yinchuan, China; SCXK (Ning) 2005-001], weighing 200–250 g, were randomly divided into six groups (n=8/group): Sham operation (CON) group, OMT control group, CLP model group, and CLP + OMT (high dose, 52 mg/kg; medium dose, 26 mg/kg; low dose, 13 mg/kg) groups. The septic shock model was induced by CLP, as previously described (17). The rats were maintained in light- and temperature-controlled conditions (12 h light/dark cycle; 22±2°C) and were given ad libitum access to food and water. Briefly, rats were anesthetized with 40 mg/kg pentobarbital (2%; Sigma-Aldrich) by intraperitoneal injection. A 2–3 cm midventral abdominal incision was made to expose the intestines. The ileocecal valve was ligated with 3–0 silk and three perforations were made in the cecum using a 20-gauge needle. To ensure consistent cecal damage among the animals, the perforated cecum was squeezed until 50–80 µl feces extruded onto both surfaces, the bowel was reinserted into the abdomen and the incision was closed. In the sham group, the abdomen was opened to expose the intestines, and then closed. A single post-operative saline bolus was provided (30 ml/kg subcutaneous) for fluid support. Caudal artery blood pressure was monitored; once it had decreased to 2/3 basic blood pressure and <20 mmHg, the model was considered successful. Rats received the drugs via tail vein injection (volume, 5 ml/kg). In the sham and OMT control groups, rats received intravenous injection of normal saline and OMT (26 ml/kg), respectively, and the CLP group received normal saline (26 ml/kg). After treatment, the cecum was exposed under sterile conditions. The rats' tail artery pressure dropped to 2/3 of the baseline blood pressure and pulse pressure was reduced to <20 mmHg as a judgment of sepsis model standards.

To examine the cardiac function of mice in response to sepsis, the right common carotid artery was used to determine heart rate (HR), mean arterial pressure (MAP), left intraventricular pressure change rate (LVdp/dt_max), left ventricular end systolic pressure (LVESP) and left ventricular end diastolic pressure (LVEDP) using cardiac catheterization. Rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.), and were then sacrificed by cervical dislocation. Subsequently, the heart was removed for histological analyses. Apical tissue blocks (~2 mm³) were collected, fixed in 10% formalin, and embedded in paraffin. After staining with hematoxylin and eosin, pathological alterations to the myocardial tissue were observed under a light microscope (CHC-212; Olympus Corporation, Tokyo, Japan). Other myocardial tissues (~2 mm³) were placed in 2% glutaraldehyde, and were sectioned as electron microscopy specimens, in order to observe ultrastructural changes.

Cardiac tissues (~100 mg) were collected and homogenized using a Polytron PT1200E (Kinematica, Shanghai, China). Total RNA was extracted using TRIzol^® reagent. The PCR reaction volume was 25 µl, and RT-PCR was conducted using the Promega One-Step RT-PCR kit (Promega Corporation, Madison, WI, USA). According to the protocol, 5 µl DEPC water, 5 µl AMV/TfI 5X buffer, 0.5 µl dNTP mixture, 1 µl 25 mM MgSO₄, 0.5 µl Tf1 DNA polymerase, 0.5 µl AMV-RT, 12.5 µl Master Mix, 2.5 µl upstream primer (50 pmol), 2.5 µl downstream primer (50 pmol) and RNA (0.5–1 µg) were mixed in a microcentrifuge tube. RT-PCR was performed using GeneAmp 9700 thermal cycler (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). First strand cDNA synthesis was conducted as follows: 1 cycle at 48°C for 45 min for RT, 1 cycle at 94°C for 2 min for AMV-RT inactivation and RNA/cDNA/primer denaturation. Second strand cDNA synthesis and PCR amplification were conducted as follows: 35 cycles at 94°C for 30 sec for denaturation, annealing at the primer-specific temperatures for 1 min, 68°C for 2 min for extension, and 1 cycle at 68°C for 7 min for final extension. β-actin was used as an internal control. The primers and annealing temperatures used are listed in Table I. Primers were designed using Primer 5.0 software (Premier Biosoft, Palo Alto, CA, USA), and were synthesized by Baisheng Company (Beijing, China). The PCR products were analyzed by 2% agarose gel electrophoresis using a Gel Doc™ EZ system purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The results were semi-quantified and expressed as relative optical density x surface area (mm²). Relative mRNA expression levels were expressed as optical density normalized to β-actin.

Cardiac tissues (~100 mg) were collected and homogenized in radioimmunoprecipitation assay buffer (Biovision, Inc., Wuhan, China). Total protein concentration was measured using the BCA kit. Equal amounts of protein (40 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and were then transferred to 0.45 µm polyvinylidene difluoride membranes (KeHaoJia Company, Beijing, China). Blots were soaked in blocking buffer (5% non-fat milk) and were then incubated with the various primary antibodies (LBP, 1:200; anti-CD14, 1:200; NF-κB, 1:500; p-IκB-α, 1:200; p-p38-MAPK, 1:300; caspase-3, 1:300; β-actin, 1:400) at 4°C overnight. After thorough washing with Tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated anti-goat (cat. no. BA1060), anti-rabbit (cat. no. BA1054) and anti-mouse (cat. no. BA1051) secondary antibodies (1:10,000; Wuhan Boster Biological Technology Co., Ltd. (Wuhan, China), and immune complexes were visualized using an enhanced chemiluminescence detection system (Pierce Biotechnology, Inc., Rockford, IL, USA). The results were analyzed using a Gel Doc™ EZ system from Bio-Rad Laboratories, Inc. Expression levels were semi-quantified relative to the optical density of β-actin.

Apical sections of rat myocardial tissue (~2 mm³) were collected and fixed in 2% glutaraldehyde for electron microscopy (H-600; Hitachi, Tokyo, Japan) in order to observe ultrastructural alterations to the myocardial tissue.

The levels of TNF-α in the myocardial tissue were determined by radioimmunoassay, according to the manufacturer's protocol. Myocardial tissue (100 mg) was mixed with a threefold volume of phosphate-buffered saline and was homogenized. Subsequently, the homogenate was centrifuged at 35,616 × g for 20 min at 4°C, and the protein levels of TNF-α in the supernatant were quantified using the radioimmunoassay assay kit.

The TUNEL assay was conducted according to the manufacturer's protocol. Briefly, myocardial tissue was fixed in 4% paraformaldehyde for 1 h, and was processed for antigen retrieval with 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. The tissue was subsequently incubated with a TUNEL reaction mixture containing terminal deoxynucleotidyl transferase in a humidified chamber for 1 h at 37°C. Apoptosis of myocardial cells was characterized as reduced cell volume and visible brown particles within the nuclei. Myocardial cell apoptotic index (AI) was determined according to the following equation: AI = (total number of apoptotic cells/total number of cells) × 100%.

Data are presented as the mean ± standard error of the mean (n=3). Data were analyzed by one-way analysis of variance followed by Student-Newman-Keuls post-hoc test for multiple comparisons. Statistical analyses were conducted using SPSS 11.5 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Once the rats awoke from anesthesia, they started drinking and cleaning their fur. Rats in the CON and OMT groups exhibited a normal manner and appearance, as evidenced by a smooth and shiny coat and regular food intake. In addition, the rats in these groups did not appear dispirited or restless, and piloerection or chills were not observed. The abdominal organs also appeared normal. Rats in the CLP group gradually appeared listless, and exhibited reduced activity, food intake and responsiveness. In addition, the coat of the CLP rats appeared to have lost its luster. After 2–4 h, sickness gradually increased and the rats appeared slumped and restless, with increased piloerection, chills, diarrhea and eye secretions. After 9 h, their condition worsened. Following an intraperitoneal cesarean section, there was visible liquid turbidity in the abdominal cavity; and the cecum emitted a foul-smelling odor, alongside swelling, necrosis, adhesion, and jejunal bowel distension. These findings are consistent with the literature (18), thus indicating that a rat model of sepsis was successfully generated. Rats in the CLP + OMT high and medium dose groups exhibited increased activity, and reduced dispiritedness, restlessness, piloerection and chills, as compared with the CLP group, following a laparotomy. In addition, there was no obvious liquid abnormality in the abdominal cavity, no cecal swelling, and no observation of gangrene and adhesion; however, improvement was not as obvious in the CLP + OMT low dose group.

The CLP rat model was used to evaluate the effects of OMT on myocardial diastolic dysfunction, which is an important characteristic of septic shock in rats. As shown in Fig. 1, there was no difference in the cardiac function parameters, including HR, MAP, LVESP, LVEDP, LVdp/dt_max and -LVdp/dt_max indices, between the OMT control group and the CON group. In the CLP group, each index was significantly altered compared with the CON group. HR was increased by 18%, MAP was reduced by 37%, LVESP was reduced by 27%, LVEDP was increased by 39%, LVdp/dt_max was reduced by 38%, and -LVdp/dt_max was reduced by 37%. Following treatment with various doses of OMT, cardiac function improved. Compared with the CLP group, CLP + high dose OMT reduced HR by 10% (P<0.05), increased MAP by 35% (P<0.01), increased LVESP by 28% (P<0.01), reduced LVEDP by 25% (P<0.05), increased LVdp/dt_max by 45% (P<0.01), and increased -LVdp/dt_max by 29% (P<0.01). CLP + medium dose OMT reduced HR by 8% (P<0.05), increased MAP by 22% (P<0.05), increased LVESP by 16% (P<0.05), reduced LVEDP by 20% (P<0.01), increased LVdp/dt_max by 32% (P<0.01), and increased-LVdp/dt_max by 23% (P<0.01). CLP + low dose OMT reduced HR by 4% (P>0.05), increased MAP by 11% (P<0.05), increased LVESP by 4% (P>0.05), reduced LVEDP by 7% (P>0.05), increased LVdp/dt_max by 13% (P<0.05), and increased -LVdp/dt_max by 10% (P<0.05).

Myocardial histology appeared no different between the CON and OMT control groups. The endocardial membrane was complete, and edema and fibrous connective tissue hyperplasia were not detected. The myocardial stripes were clear and the nucleus was centered, with no vasodilation or inflammatory cell infiltration detected. Epicardial membrane integrity was intact, and was not covered with inflammatory exudate (Fig. 2A and B). The myocardial tissue of the CLP group exhibited an extensively disordered subendocardial structure compared with the CON group. A wide range of inflammatory cells infiltrated the tissue, and large numbers of mononuclear cells, intermingled with a few lymphocytes and neutrophils were detected. In addition, telangiectasia and bleeding were observed (Fig. 2C). The CLP group also displayed interstitial edema and fibroblast proliferation, and various degrees of cell necrosis and fibrosis. Myocardial tissue damage in the CLP + OMT low dose group was reduced to some degree; however, the myocardial structure remained slightly disorganized, with inflammatory cell infiltration, telangiectasia, bleeding, cell necrosis and fibrosis all detected (Fig. 2D). Myocardial tissue damage in the CLP + OMT medium and high dose groups was markedly reduced, and normal basic cardiac structure was observed. Edema, degeneration and necrosis were notably reduced; however, a small amount of inflammatory cell infiltrate and a few exudative changes remained (Fig. 2E and F). These results indicate that OMT may reduce myocardial injury and exert protective effects on cardiac structure and function in rats with septic shock.

The myocardial tissue, including myofilaments, sarcomeres, capillaries, mitochondria, sarcoplasmic reticulum and nuclei, was not altered in the OMT control group compared with the CON group, and all structures appeared normal (Fig. 3A and B). The myofilament and sarcomere arrangement was normal, and the capillaries opened well. In addition, mitochondrial structures were normal, and mitochondria exhibited complete membranes, dense ridges and clear matrix. The structure of the intercalated disks was continuous. Sarcoplasmic reticulum was smooth and continuous. Nuclear structure was clear, with slightly visible nuclear pyknosis and uniform chromatin. Conversely, the ultramicrohistological structure of the CLP myocardial tissue was markedly degraded. As shown in Fig. 3C, mitochondria appeared markedly swollen, resulting in damaged membranes and disordered cristae. Disordered myofilaments and sarcomeres subsequently dissolved, leading to the generation of vacuoles. The nuclei shrunk in size and the chromatin was marginated. The structure of the intercalated disks was not continuous, and dissolution was observed. However, the ultramicrohistological injuries of the myocardial tissue were markedly improved in the CLP + OMT groups (Fig. 3D–F). The myocardial fibers were normally arranged, and the majority of mitochondria exhibited a complete structure with ordered dense ridges. Although the structure of the mitochondria cristae appeared vague, arrangement remained regular. In addition, mitochondrial swelling was reduced; however, in some cases mitochondrial damage was not yet fully recovered.

The mRNA expression levels of LBP, CD14, NF-κB (p65), TNF-α, p38-MAPK and caspase-3 were similar in the myocardial tissue of the OMT control and CON groups. However, expression levels were significantly increased in the CLP group (P<0.05). Compared with the CLP group, the mRNA expression levels of LBP, CD14, NF-κB (p65), TNF-α, p38-MAPK and caspase-3 were markedly decreased in the CLP + OMT groups (P<0.05; Fig. 4).

Western blotting results indicated that NF-κB (p65), p-IκB-α, p-p38-MAPK and caspase-3 protein expression levels were similar in the myocardial tissue of the OMT control and CON groups. However, expression levels were significantly increased in the CLP group (P<0.05). Conversely, NF-κB (p65), p-IκB-α, p-p38-MAPK and caspase-3 protein expression levels were markedly decreased in the CLP + OMT groups compared with in the CLP group (P<0.05; Fig. 5).

The results of a radioimmunoassay demonstrated that TNF-α levels were significantly increased in the CLP group compared with in the CON and OMT control groups (P<0.05). However, compared with the CLP group, the TNF-α levels were significantly decreased in the CLP + OMT groups (P<0.05) (Fig. 6A).

No significant differences were detected in myocardial cell AI between the CON and OMT control groups. Compared with the CON group, myocardial AI was significantly increased in the CLP group (P<0.01). Conversely, myocardial AI was significantly reduced following treatment with various doses of OMT compared with in the CLP group (P<0.05) (Fig. 6B).