Oxymatrine was purchased from Ningxia Qi Yuan Pharmaceutical Co. (Yinchuan, China). Dexamethasone was from Hubei Day Drug Pharmaceutical Co. (Hubei, China). Coomassie Brilliant Blue protein quantification kit was purchased from Nanjing Jiancheng Bioengineering Company (Nanjing, China). The ¹²⁵I tumor necrosis factor-α and ¹²⁵I interleukin-1β radiation immunoassay kits were purchased from the Beijing Chemclin Bioengineering Company (Beijing, China). TRIzol was obtained from Invitrogen Life Technologies (Carlsbad, CA. USA). PrimeScript RT reagent kit was from Takara Bio (Takara Bio, Inc., Dalian, China). RT-PCR kit was from Promega (Promega Corporation, Madison, WI, USA). Rat monoclonal antibodies against JAK2 and STAT3 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and IP cell lystates and the BCA protein concentration assay kit were from Jiangsu Green Biotechnology Company (Jiangsu, China).

Animal studies were approved by the Ningxia Medical University Animal Care Committee (Ningxia, China). Male Sprague-Dawley rats, specific pathogen free, weighing 200–250 g, were obtained from the Animal Center of Ningxia Medical University [SCXK (Ning) 2005-001]. Rats were randomly divided into 7 groups (n=8): sham surgery, OMT control, CLP model, positive control [CLP + dexamethasone (DEX), 10 mg/kg] and CLP + OMT 52, 26 and 13 mg/kg. The septic shock model was induced by CLP as described previously (15). Rats received tail vein injection with drugs (volume, 5 ml/kg). In the sham-operated and OMT control groups, the rats received intravenous injection with normal saline and OMT (26 ml/kg) and the CLP group received normal saline (26 ml/kg) only. Following treatment, the cecum was exposed under sterile conditions. Postoperative monitoring rat tail artery pressure was performed, and rat blood pressure dropped to 2/3 of base blood pressure and pulse pressure <20 mmHg was the standard used to judge the success of the sepsis model.

To examine cardiac function, the right common carotid artery was used to determine the rat 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) by inserting the cardiac catheterization. The heart was then removed for histological analysis. Apical tissue blocks (~2 mm³) were collected and fixed in 10% formalin, then embedded in paraffin. Following haematoxylin-eosin staining, pathological changes of myocardial tissue were observed under the light microscope. Additional myocardial tissues ~2 mm³ were placed in 2% glutaraldehyde and sectioned as electron microscopy specimens to determine ultrastructural changes.

Total RNA was prepared using TRIzol and was reverse-transcribed to cDNA using a PrimeScript RT reagent kit. RT-PCR was performed using a commercially available kit as follows: 35 cycles of denaturation at 94°C for 60 sec, annealing at 58°C for 60 sec and extension at 72°C for 50 sec. β-actin was used as an internal control to evaluate relative expression of TNF-α and IL-1β. The primers used were: TNF-α (355 bp) 5′-CAATGGCATGGATCTCAAAG-3′ and 5′-CAGAGCAATGACTCCAAAGT-3′, IL-1β (399 bp) 5′-AGAAGCTGTGGCAGCTACCT-3′ and 5′-TTGGGA TCCACACTCTCCAG-3′, β-actin (299 bp) 5′-AGGTGAGAG GGAAATCGTGCG-3′ and 5′-GTGCCACCAGACAGC ACTGTGC-3′.

Total protein concentration was measured using a BCA kit. Equal amounts of protein (40 μg) were separated electrophoretically using 10% SDS-PAGE and the gel was then transferred to a 0.45 μm PVDF membrane. Blots were soaked in blocking buffer (5% non-fat milk) and then incubated with primary antibodies (anti-JAK, 1:200; anti-STAT3, 1:200; anti-β-actin, 1:1,000) overnight at 4°C. Following thorough washing with TBST buffer, horseradish peroxidase conjugated secondary antibodies (1:10,000) were applied and immune complexes were then visualized using the enhanced chemiluminescence detection system.

Levels of TNF-α and IL-1β in myocardial tissue were determined by radioimmunoassay. Myocardial tissue (100 mg) was mixed with a 3-fold volume of PBS and homogenized, then centrifuged at 12900 x g for 20 min at 4°C. Protein levels of TNF-α and IL-1β in the supernatant were quantified using a radioimmunoassay assay kit.

Results were presented as the mean ± SEM of at least three separate experiments. Data were analyzed by one-way ANOVA, followed by the Student-Newman-Keuls post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Changes in myocardial diastolic function are an important characteristic of septic shock (16). A rat model of CLP was used to evaluate the effect of OMT on cardiac function in rats with septic shock. As demonstrated in Table I and Fig. 1, cardiac function parameters, including HR, MAP, LVSP, LVEDP, LVdp/dtmax and -LVdp/dtmax indices were not affected in the OMT control group compared with the control (CON) group. In the CLP group, significant changes in all indices were identified (P<0.01) compared with CON. HR increased by 15%, MAP reduced by 33%, LVSP reduced by 24%, LVEDP increased by 47%, LVdp/dtmax reduced by 38% and -LVdp/dtmax reduced by 32%. Following treatment with various doses of OMT, the CLP + high-dose OMT group was observed to reduce HR by 11% (P<0.05), increase MAP by 26% (P<0.01), increase LVSP by 20% (P<0.01), reduce LVEDP by 20% (P<0.01) and increase LVdp/dtmax by 50% (P<0.01) and -LVdp/dtmax by 25% (P<0.01) compared with CLP. The CLP + middle dose OMT group was observed to have a reduced HR by 7% (P<0.05), increased MAP by 17% (P< 0.05), increased LVSP in 10% (P<0.05), reduced LVEDP by 14% (P<0.05) and increased LVdp/dtmax by 35% (P<0.01) and -LVdp/dtmax by 25% (P<0.01). HR was reduced by 3% (P>0.05), MAP was increased by 9% (P<0.05), LVSP was increased by 3% (P>0.05), LVEDP was reduced by 2% (P>0.05), LVdp/dtmax was increased by 13% (P<0.05) and -LVdp/dtmax was increased by 9% (P<0.05) in the CLP + low-dose OMT group. The positive control CLP + DEX group did not reveal marked differences in various indices compared with CON, CLP + high-dose OMT and CLP + middle-dose OMT, however a significant difference in all indices was found when compared with CLP and CLP + low-dose OMT.

Differences in myocardial tissue were not identified between the CON and OMT control group. The endocardial membrane was complete, with no edema and fibrous connective tissue hyperplasia. The myocardial stripes were clear, the nucleus centered and no vasodilation and inflammatory cell infiltration were observed. Epicardial membrane demonstrated integrity in the stroma without inflammatory exudate (Fig. 2A and B). As shown in Fig. 2C, CLP rat myocardial tissue revealed marked subendocardial myocardial structural disorder compared with the CON group. Infiltration of a number of inflammatory cells with a considerable number of mononuclear cells and a few lymphocytes and neutrophils and telangiectasia and bleeding were observed. The CLP group also exhibited interstitial edema, fibroblast proliferation and cell necrosis and fibrosis to various degrees. Following various doses of OMT treatment, myocardial tissue damage in the CLP + OMT-L group was reduced compared with CLP, however, low levels of disorganized myocardial structures with inflammatory cell infiltration, telangiectasia and bleeding, as well as cell necrosis and fibrosis were identified (Fig. 2D). However, myocardial tissue damage in the CLP + OMT-M and CLP + OMT-H groups was markedly reduced and a normal basic cardiac structure was observed (Fig. 2E and F). Edema, degeneration and necrosis were significantly reduced but remained accompanied by a small amount of inflammatory cell infiltration and exudative changes. Compared with the CLP group, myocardial injury was significantly reduced in the CLP + DEX group and myocardial cells were organized in rows, accompanied by a small amount of inflammatory cell infiltration. However, no significant changes in cell swelling, degeneration and necrosis were identified (Fig. 2G).

The results indicate that OMT may reduce myocardial injury and have a protective effect on cardiac structure and function in rats with septic shock. Results in the CLP + OMT groups with high and middle doses and the positive control (CLP + DEX group) were consistent.

Compared with CON, myocardial tissue, including myofilaments, sarcomere, capillaries, mitochondrion, sarcoplasmic reticulum and nucleolus in the OMT control group were observed to exhibit no significant changes and all had normal shapes and clear structures (Fig. 3A and B). Myofilaments and sarcomere arrangement was normal and blood flow volume was normal. Mitochondrial structure was normal and clear with complete membranes, dense ridges and a clear matrix. The intercalated disc was in order and successive. The sarcoplasmic reticulum was smooth and continuous. The nucleolus was clear with visible light nucleus pycnosis and the chromatin was uniform. However, the ultramicro-histological myocardial tissue of the CLP group was observed to be significantly damaged. As is evident in Fig. 3C, mitochondria were markedly swollen, leading to membrane damage and disordered cristae. In addition, myofilaments were dissolved and sarcomeres were disordered, leading to vacuoles. The nucleus was significantly reduces in size and the chromatin margination was observed. Dissolution of intercalated disc demonstrated discontinuity and uneven distribution. However, compared with the CLP, the injuries of ultramicro-histological myocardial tissue in CLP + OMT groups were significantly reduced (Fig. 3D-F). Myocardial fiber arrangement was normal, the majority of the mitochondrial structure was complete and ridge dense arrangement had a regular pattern. Although a section of the ridge was undefined, the arrangement was still regular, mitochondrial swelling was reduced and specific incidences of damage were not recovered fully. The CLP + OMT-L group was found to exhibit the highest levels of damage. The ultramicro-histological myocardial tissue in the CLP + DEX group was almost normal and rarely injured (Fig. 3G). Myocardial fibers were aligned, the majority of mitochondrial structures were complete, ridges were dense, regions of the ridge were undefined, fibers were arranged in an organized manner, specific incidences of damage were not recovered fully and intercalated disc demonstrated clear continuity. The sarcoplasmic reticulum was smooth and continuous. The nucleolus was clear with light nucleus pycnosis and the chromatin was uniform.

Western blot analysis revealed that JAK2 and STAT3 protein levels in OMT control myocardial tissue was similar to that of the CON group but was observed to be significantly higher in the CLP group (P<0.05). However, JAK2 and STAT3 protein levels in CLP + OMT and positive control groups were markedly decreased compared with CLP (P<0.05; Fig. 4).

Radioimmunoassay revealed that TNF-α and IL-1β protein levels in the CLP group was significantly increased compared with the CON and OMT control (P<0.05). However, compared with the CLP group, TNF-α and IL-1β protein levels in CLP + OMT and the positive control were found to be significantly decreased (P<0.05; Fig. 5).

TNF-α and IL-1β mRNA levels in OMT control and CON myocardial tissue were normal, however, levels were identified to be significantly increased in the CLP group (P<0.05). Compared with the CLP group, TNF-α and IL-1β mRNA levels in CLP + OMT and the positive control were markedly decreased (P<0.05; Fig. 6).