Effect of dobutamine on intrinsic myocardial function and myocardial apoptosis in septic rats with myocardial dysfunction

Dobutamine (DOB) has been recommended as the rst-line inotrope for septic patients with low cardiac output, but its long-term impact on intrinsic myocardial dysfunction during sepsis remains unclear. This study investigated the long-term effect of DOB on intrinsic myocardial function and cardiomyocyte apoptosis in sepsis. the and biomarkers In the of advisability usefulness dobutamine a new inotrope for the treatment of septic patients with low cardiac


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Background Sepsis,the life-threatening organ dysfunction induced by a dysregulated body response to infection, is a leading cause of critical illness and hospital mortality worldwide [1]. Myocardial dysfunction is a common complication in septic patients, about 50% of patients with severe sepsis and septic shock may exhibit pronounced myocardial dysfunction [2], which is associated with a higher mortality [3]. According to the current evidence, the pathogenesis of septic myocardial dysfunction involves a complex interaction of many factors, including in ammatory cytokines, apoptosis and neuroimmunomodulation [4][5][6][7]. For example, myocardial inhibitory factors, such as tumor necrosis factor (TNF) -α and interleukin (IL) -1β, could mediate myocardial dysfunction in experimental sepsis models [4]; Lipopolysaccharide (LPS) and cecal ligation and puncture (CLP) were found to induce myocardial vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 expression, blockade of VCAM-1 or ICAM-1 ameliorated myocardial dysfunction induced by sepsis [8,9]; LPS also activated myocardial caspase-3 and apoptosis, broad-spectrum caspase inhibitors or caspase-3 inhibitors not only reduced LPS-induced myocardial caspase activation and nuclear apoptosis, but also improved LPS-evoked myocardial dysfunction [10,11]. Although a large number of experimental studies have focused on myocardial dysfunction and associated mechanisms during sepsis over the last 50 years, pathophysiology of septic myocardial dysfunction is not completely understood and it remains a clinical enigma. The majority of clinical studies determined ventricular function in a global manner using indices, such as cardiac index and ejection fraction (EF) [4,5,12]. Indeed, left ventricular EF is a load-dependent index, which re ects the coupling between left ventricular contractility and afterload, rather than the left ventricular intrinsic myocardial contractile function. In sepsis, when the afterload is severely decreased due to reduced systemic resistance, left ventricular EF may be normal, despite seriously depressed left ventricular intrinsic contractility [5,12,13]. As a result of these limitations, the time pattern of intrinsic myocardial dysfunction progression during sepsis still needs clari cation. Although recent great advancements have been made in echocardiography, by which the myocardial dysfunction is unmasked even in the presence of preserved left ventricular EF [14], the clinical relevance of intrinsic myocardial dysfunction is also still underestimated and no speci c effective therapies for septic myocardial dysfunction exist.
In 2016, Surviving Sepsis Campaign Guidelines recommended that dobutamine, a β 1 -adrenoceptor (AR) agonist, was the rst-line inotrope for septic patients with low cardiac output despite adequate uid resuscitation and the use of vasopressors [15]. However, recent studies have shown that dobutamine may increase myocardial oxygen consumption and the incidence of arrhythmias events in sepsis [16].
Furthermore, our recent studies found that exhaustion of cardiac norepinephrine or blockade of β 1 -AR almost completely inhibited LPS-induced myocardial apoptosis [17], whereas activation of β 1 -AR promoted LPS-induced cardiomyocyte apoptosis [18]. Evidently, there is no direct evidence to demonstrate if dobutamine treatment in the presence of intrinsic myocardial dysfunction improves intrinsic myocardial function at the later stage of sepsis.
Therefore, the purpose of the present study was to establish a rat polymicrobial sepsis model by the CLP approach and to determine: 1) when intrinsic myocardial systolic and diastolic dysfunction occurred during sepsis, 2) and whether treatment with dobutamine in the presence of intrinsic myocardial dysfunction affected intrinsic myocardial function, cardiac in ammation and apoptosis at the later stage of sepsis.

Experimental animals
Male Sprague-Dawley (SD) rats, 8-10 weeks old (weight 250-300 g), were obtained from the medical laboratory animal center of Guangzhou University of Chinese Medicine and fed in the speci c-pathogenfree laboratory environment for at least 10 days, in the conditions of temperature (24˚C), humidity (48%), and 12 h light/dark circadian cycle. All studies were conducted in compliance with the guide for the Care and Use of Laboratory animals published by US national institutes of health, and approved by the Animal Care and Use Committee at Jinan University. All efforts were made to minimize the number of rats used and their suffering.

Sepsis model
The model of sepsis was induced by CLP, as described previously [19]. Brie y, rats were anesthetized with iso urane inhalation (3% iso urane in 97% O 2 ) and then xed on the operating table. A small midline abdominal surgical incision was performed under sterile conditions. The cecum was exposed and tightly ligated at 1.5 cm length of the distal cecum using a 4.0-silk suture, and then punctured once with an 18gauge needle following with carefully squeezing to extrude a small amount of bowel content through the puncture site. Then, the cecum was repositioned into the abdominal cavity and the surgical incision was closed layer by layer. Sham-operated rats were performed using the same surgical procedure but without ligation or puncture of the cecum. Meanwhile, the catheters full of heparinized saline (50 IU heparin/ml) was inserted the right jugular vein for drug administration [20]. The normal saline (3 ml/100 g) were administered subcutaneously for post-surgery uid resuscitation immediately after surgery, and buprenorphine (0.05 mg/kg body weight) was injected subcutaneously immediately and 12 h separately after CLP. All rats had free access to food and water when they were returned to their cages after recovery from anesthesia.

Experimental design
Firstly, male SD rats were randomly divided into sham and CLP groups. The survival rates were recorded for 10 days after CLP or sham operation. In a separate experiment, at 6 h, 9 h, 12 h after CLP or sham surgery, the rat hearts were harvested,the intrinsic myocardial functions were determined by the Langendorff perfusion system and left ventricle tissue was collected after heart perfusion. Meanwhile, the lungs were removed and the lung wet-dry weight (W/D) ratios were calculated, the serum in ammatory cytokine, cardiac troponin I (cTnI), N-terminal pro-brain natriuretic peptide (NT-proBNP) and heart-type fatty acid-binding protein (H-FABP) concentrations, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities as well as blood urea nitrogen (BUN) and creatinine (Cr) contents were analyzed.
Secondly, male SD rats were randomly divided into six groups, sham, CLP, CLP + 5 µg/kg DOB, CLP + 10 µg/kg DOB, sham + 5 µg/kg DOB and sham + 10 µg/kg DOB groups. At 6 h after CLP or sham operation, DOB (5 or 10 µg/kg/min, dobutamine hydrochloride, Sigma, USA) or normal saline (NS) was administered by the jugular vein for 2 h. At 20 h after sham operation or CLP surgery, the rat hearts were harvested and the intrinsic myocardial functions were determined by the Langendorff apparatus, meanwhile the serum samples were collected for enzyme-linked immunosorbent assay (ELISA) analysis.
After heart perfusion, the left ventricular tissues were xed in 4% paraformaldehyde for terminal deoxynucleotidy1 transferase dUTP nick-end labeling (TUNEL) assay, the total protein from the left ventricular tissues were extracted for Western blotting. Furthermore, serum and cardiac TNF-α, IL-6 and IL-1β were detected at 20 h after CLP or sham surgery by ELISA in a separate experiment.
Lastly, rats were randomly divided into ve groups, sham, CLP group, CLP + 2.5 µg/kg DOB, CLP + 5 µg/kg DOB and CLP + 10 µg/kg DOB groups. At 6 h after sham operation or CLP, DOB (2.5, 5 or 10 µg/kg/min, dobutamine hydrochloride, Sigma, USA) or normal saline was administered by the jugular vein for 2 h. The survival rates were recorded for 10 d after CLP or sham operation. At 20 h after CLP exposure, serum IL-10 concentration was determined by ELISA in a separate experiment.

Langendorff perfusion
Male SD rats were anaesthetized with 3% iso urane, and the myocardial functions were measured by modi ed method as described previously [21]. The hearts were rapidly harvested and immediately transferred to an ice-cold Krebs solution followed by cannulating the aorta. Then, the hearts were mounted in the Langendorff system (AD Instruments, USA) and subjugated to retrograde perfusion at 10 mL/min constant ow with modi ed warm Krebs-Henseleit solution (37℃), which consisted of NaCl 6.896 g / L, KCl 0.35 g / L, MgSO 4 0.296 g / L, KH2PO 2 0.16 g / L, NaHCO 3 2.1 g / L, glucose 2.18 g / L, CaCl 2 0.277 g / L and equilibrated with 95% O 2 and 5% CO 2 at a pH of 7.35. A uid-lled latex balloon was inserted into the left ventricle, and the initial end-diastolic pressure was adjusted at 4-10 mmHg quickly. After a 10 min stabilization period, myocardial functions, including left ventricular developed pressure, as well as the maximal rate of left ventricular pressure rise and fall (± dP/dt), were continuously recorded for up to 40 min by BL-420 F bio-transduction system.

Western blotting
The protein specimen of left ventricular tissues were extracted on ice using RIPA lysis buffer containing protease inhibitors according to the manufacturer's instructions. Then, the samples were centrifugation at 12000 rpm for 10 min at 4 ℃. Protein concentration was measured using Pierce™ BCA Protein Assay Kit (23225, Thermo sher). Equal volume of protein (10 µg) were loaded and separated by running 10% SDS-PAGE gels, and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk at room temperature for 1 h, and then incubated with primary antibodies against GAPDH (2118S, Cell Signaling Technology), VCAM-1 (ab115135, Abcom), cleaved caspase-3 (9664S, Cell Signaling Technology), caspase-3 (9665S, Cell Signaling Technology) for overnight at 4 ℃. The membranes were washed three times for 10 min in 1x TBST and then incubated with the respective peroxidase labeled secondary antibodies at room temperature for 1 h. The membranes were then washed with 1X TBST for 3 times and developed using the Clarity Western ECL substrate. Finally, the density of the bands was quanti ed.
The levels of TNF-α, IL-1β and IL-6 in serum and myocardial as well as serum IL-10 were detected using ELISA kits from R&D systems (USA). The levels of serum cTnI, heart-type fatty acid-binding protein (H-FABP) were measured using ELISA kits from Life Diagnostics (USA). The serum N-terminal pro-brain natriuretic peptide (NT-proBNP) levels were determined using the speci c rat quantikine ELISA kits from Cloud-Clone Corp (China), according to the manufacture's protocol.

TUNEL assay
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay was performed on cardiac tissue sections (4 µm) to detected cardiomyocyte apoptosis according to the in situ cell death detection kit 's instructions (11684817910, Roche). Brie y, the cardiac tissues were xed with 4% paraformaldehyde at 4℃ for overnight, and then placed in 20%, 30%, 40% sucrose in PBS for gradient dehydration. After washing in 1X TBST, the sections were incubated with primary antibodies against cardiac troponin I (1:200, abcam, USA) at 4℃ for overnight, and then with prepared TUNEL reaction mixture in the dark at 37℃ for 1 h. The sections were rinsed with 1X TBST and incubated with uorescent secondary antibody (1: 200) at room temperature for 1 h in the dark. Finally, the tissue sections were incubated with DAPI (1:150) for 15 min and mounted with coverslips. Then, the sections were visualized with laser-scanning confocal microscopy. The TUNEL-positive cells were determined by green staining in the nucleus of apoptotic cells.

Statistical analysis
Quantitative data were expressed as the mean ± standard error of the mean (SEM) and analyzed using the statistical software SPSS 20.0. The statistically signi cant difference was used by a two-tailed independent Student's t-test for two groups, the One-Way ANOVA test followed by Bonferroni post hoc test was performed to analyse the normally distributed data for the comparisons among the groups and nonparametric Mann-Whitney U tests were used for the data that was not normally distributed. The survival rate of rats was analyzed by Kaplan-Meier survival analysis with log-rank test. Statistical signi cance was accepted at p < 0.05.

Results
The intrinsic myocardial dysfunction occurred at 6 h after CLP surgery in septic rats.
We initially ascertained when intrinsic myocardial dysfunction was present in rats with CLP-induced sepsis. As shown in Fig. 1A, the mortality rate of septic rats was 70% on day 10 after CLP surgery. In order to determine left ventricular intrinsic systolic and diastolic function, the Langendorff technique of isolated heart perfusion was performed. The results revealed that left ventricular ± dP/dt markedly decreased in septic rats at 6 h, 9 h and 12 h after CLP exposure compared to sham-operated rats (P < 0.05) (Fig. 1B  and 1C). The activities of serum ALT and AST were increased at 9 h and 12 h after CLP surgery in CLP rats compared with sham group (P < 0.05) (Fig. 1D and 1E). However, there was no signi cant difference in serum ALT and AST activities at 6 h after surgery between CLP rats and sham-operated rats, shamoperated and CLP rats did not substantially differ in serum BUN and Cr concentrations as well as lung W-D ratios at 6 h, 9 h and 12 h after surgery (P > 0.05) (Fig. 1D-1H). These results indicate that left ventricular intrinsic systolic and diastolic dysfunction are present at 6 h after CLP surgery in CLP rats, which is earlier than hepatic and renal dysfunction as well as lung edema in this condition.

Cardiac in ammation and injury in CLP rats
As mentioned above, myocardial TNF-α and adhesion molecules, such as ICAM-1and VCAM-1, contribute to the sepsis-induced myocardial dysfunction [4][5][6][7][8]. Real-time PCR and Western blot analyses showed that CLP rats had marked elevations in cardiac levels of TNF-α, ICAM-1 and VCAM-1 mRNAs as well as VCAM-1 protein at 6-12 h after CLP-induced sepsis compared with sham-operated controls, respectively (P < 0.05, Fig. 2A-2D). Additionally, greatly increased TNF-α concentration in plasma was observed at 6-12 h after CLP surgery in septic rats in comparison with sham-operated control animals (Fig. 2E). However, there was no signi cant difference in the levels of cTnI, NT-proBNP and H-FABP in plasma, clinical markers of cardiac injury, between CLP and sham-operated control rats at 6 h, 9 h and 12 h after surgery (Fig. 2F-2H).

DOB did not affect intrinsic myocardial dysfunction at the late stage of sepsis in CLP rats
Surviving sepsis campaign guidelines suggested administering DOB in the presence of myocardial dysfunction. Sakai M, et al. demonstrated that intravenous administration of 10 µg/kg dobutamine had a marked rapid positive inotropic response on the sham-operated mouse heart, which lasted for 5 min, but not on CLP-treated mouse heart at 18 h after CLP exposure [22]. However, the long-term effect of dobutamine on cardiac dysfunction in sepsis remains unclear. We found CLP induced a signi cant intrinsic myocardial dysfunction at 6 h after CLP surgery. Therefore, we further observed the effect of DOB administered at 6 h after CLP induction on intrinsic myocardial function at the late stage of sepsis in septic rats with myocardial dysfunction. The CLP signi cantly reduced ± dP/dt of the left ventricle at 20 h after CLP compared to sham group, but intravenous administration of DOB (5, 10 µg/kg) at 6 h after CLP induction did not increase left ventricular ± dP/dt at 20 h after CLP exposure in septic rats. In addition, intravenous administration of DOB (5, 10 µg/kg) at 6 h after sham surgery did not affect left ventricular ± dP/dt at 20 h after surgery in sham-operated control rats (Fig. 3A, 3B).
DOB did not affect cardiac in ammation and myocardial apoptosis in CLP rats with myocardial dysfunction.
In ammatory cytokines, such as TNF-α, IL-1β and IL-6, as well as myocardial apoptosis contribute to the sepsis-induced myocardial dysfunction. We further investigated the long-term effect of DOB administered at 6 h after CLP induction on cardiac cytokines and myocardial apoptosis at 20 h after CLP surgery in septic rats. Compared with sham group, CLP markedly increased the concentrations of serum and cardiac TNF-α, IL-1β and IL-6 as well as myocardial caspase-3 activation and apoptosis at 20 h after CLP, all of which were not affected by treatment with DOB (5, 10 µg/kg) at 6 h after CLP (Fig. 4, 5). DOB (10 µg/kg) increased serum IL-10 concentration and improved survival in CLP rats.
Intriguingly, serum IL-10 concentration was signi cantly increased at 20 h after CLP and further elevated by DOB (10 µg/kg) administered at 6 h after CLP in septic rats (P < 0.05), which was not the case in septic rats treated with 5 µg/kg DOB (Fig. 6B). Importantly, DOB (10 µg/kg) administered at 6 h after CLP improved survival in CLP rats compared with CLP group (P < 0.05) (Fig. 6A).

Discussion
Dobutamine has been used in septic patients with low cardiac output for many years as a rst-line therapy recommended by the Surviving Sepsis Campaign guidelines [15]. However, clinical outcome estimation is limited for advisability of the usefulness of dobutamine in the treatment of septic shock patients in the presence of intrinsic myocardial dysfunction. Recently, Sakai M, et al. found that positive inotropic action of dobutamine was markedly impaired in sepsis due to increased cAMP breakdown caused by myocardial phosphodiesterase 4 upregulation, intravenous injection of dobutamine (0.01 mg/kg) had a signi cant fast positive inotropic effect on the heart in sham-operated mice, but no fast positive inotropic response in CLP mice [22]. On the other hand, it has been demonstrated that activation of β 1 -AR promoted LPS-induced cardiomyocyte apoptosis [18]. Therefore, it is necessary to further observe the effect of dobutamine, a β 1 -AR agonist, on intrinsic myocardial function during sepsis.
In order to investigate the long-term effect of dobutamine on intrinsic myocardial dysfunction in sepsis, we rst determined when intrinsic myocardial systolic and diastolic dysfunction occurred during severe sepsis in the present study. It is well known that left ventricular ejection fraction is a load-dependent indicator, which did not accurately re ect the intrinsic contraction and diastolic function during sepsis [13]. We utilized the Langendorff perfusion system to measure the intrinsic myocardial function that less affected by vascular loading conditions in sepsis. In this study, we found that the mortality rate was 70% in CLP rats on day 10 after CLP induction. In this condition, the CLP rats had signi cant intrinsic myocardial dysfunction at 6 h after CLP exposure. However, the liver dysfunction occurred at 9 h after CLP induction, but the serum Cr, BUN and lung W-D ratios did not change at 6 h, 9 h and 12 h after CLP surgery. These data indicated that severe CLP rats had intrinsic myocardial dysfunction in the early stage of sepsis, which was earlier than the dysfunction of liver and kidney and lung edema in the severe CLP rats.
It is well known that the production of cytokines (e.g. TNF-α) and adhesion molecules (e.g. ICAM-1 and VCAM-1) are signi cantly increased in sepsis-induced myocardial dysfunction, inhibition of TNF-α, ICAM-1 or VCAM-1 improves left ventricular function in sepsis [7,8,23,24]. In the present study, we observed that serum TNF-α level, the mRNA expression of cardiac TNF-α, ICAM-1 and VCAM-1, and VCAM-1 protein levels were signi cantly increased in CLP rats at 6 h, 9 h, and 12 h after CLP. These results further demonstrated these in ammatory molecules contributed to pathogenesis of intrinsic myocardial dysfunction at 6-12 h after CLP. It was reported that some serum myocardial injury markers, such as NT-proBNP, cTnI and H-FABP, might re ect sepsis-induced myocardial dysfunction [25][26][27][28]. Sakai M, et al. demonstrated that serum cardiac troponin-I (cTnI) levels were signi cantly increased at 18 h after CLP induction in septic mice with cardiac dysfunction [22]. In this study, we found that the levels of serum cTnI, NT-proBNP and H-FABP did not increase at 6-12 h after CLP in CLP rats compared with shamoperated rats. These results indicate serum cTnI, NT-proBNP and H-FABP are not suitable as early biomarkers for this kind of intrinsic myocardial dysfunction in sepsis.
As mentioned above, DOB was suggested to be administered in the presence of myocardial dysfunction in septic patients [15]. According to our study that showed intrinsic myocardial dysfunction occurred at 6 h after CLP exposure, DOB was injected at 6 h after CLP induction in CLP rats. We found that DOB treatment ( 5 or 10 µg/kg/min for 2 h) had no signi cant impact on the intrinsic contraction and diastolic dysfunction at 20 h after CLP exposure in CLP rats. We further found that DOB treatment ( 5 or 10 µg/kg/min for 2 h) did not change the levels of serum and myocardial TNF-α,IL-1β and IL-6 in CLP rats. In addition, our previous study con rmed that DOB promoted LPS-induced cardiomyocyte apoptosis in vitro through activating cAMP-dependent protein kinase and enhancing calmodulin-dependent protein kinase II and IκBα phosphorylation [18]. However, in the present study, we found that intravenous administration of DOB have no signi cant impact on cardiac caspase-3 activation and cardiomyocyte apoptosis 20 h after CLP surgery.
It was reported that pretreatment with reserpine that exhausts cardiac norepinephrine without affecting the circulating norepinephrine concentration signi cantly inhibited cardiomyocyte apoptosis in septic rats [29] and β 1 -AR antagonist attenuated LPS-caused cardiomyocyte apoptosis [17]. These results indicate that activation of myocardial β 1 -AR by norepinephrine derived from cardiac sympathetic nerve, rather than circulating norepinephrine, promoted cardiomyocyte apoptosis in sepsis. This may explain why intravenous administration of DOB did not affect cardiomyocyte apoptosis in sepsis in the present study. In addition, the previous studies demonstrated that the downregulation /desensitization of β 1 -AR signal and decreased response of myo lament to Ca 2+ occurred in sepsis [30][31][32][33]. These ndings may also explain why DOB treatment after CLP surgery did not affect cardiomyocyte apoptosis and intrinsic myocardial dysfunction in sepsis.
Furthermore, we found that intravenous dobutamine at a dose up to 10 µg/kg signi cantly improved the survival in septic rats with myocardial dysfunction. This nding dovetails well with other studies which demonstrated that DOB had improved the survival in patients with septic shock and animal models of sepsis [34,35]. We also found intravenous dobutamine at a dose of 10 µg/kg markedly increased serum IL-10 level in CLP rats. It has been demonstrated that activation of β receptors ampli es the release of IL-10 by LPS-induced macrophages, IL-10-de cient mice increases mortality in Escherichia coli-treated mice [36,37], and interleukin-10 administered after the onset of CLP protected against the lethality of septic rats [38]. Thus, the reason that treatment with DOB a dose of 10 µg/kg improved the survival may be related with the increased levels of IL-10 in CLP rats.

Conclusions
In the present study, we demonstrated that intrinsic myocardial dysfunction occurred earlier than hepatic and renal dysfunction in severe sepsis and serum cTnI, NT-proBNP and H-FABP were not suitable as early biomarkers for the intrinsic cardiac depression in sepsis. Treatment with DOB in the presence of intrinsic myocardial dysfunction (at 6 h after CLP) did not attenuate the intrinsic myocardial dysfunction, in ammation and cardiomyocyte apoptosis at the later stage (at 20 h after CLP) of septic rats. These results strongly suggest that it is necessary to reevaluate advisability of the usefulness of dobutamine and to seek a new inotrope for the treatment of septic patients with low cardiac output.

Ethics approval
All animal studies were conducted in compliance with the guide for the Care and Use of Laboratory animals published by US national institutes of health, and approved by the Animal Care and Use Committee at Jinan University.

Consent for publication
Not applicable.

Competing interests
The authors declare no con ict of interest.

Funding
This study was supported by grants from the National Natural Science Foundation of China (81372028,81670359).

Authors' contributions
XXT conducted the study, analyzed the data, and drafted the manuscript. YQX made ELISA analysis and prepared the manuscript. DXL analyzed the data. HDW designed the study, analyzed the data and made revisions throughout the manuscript. The remaining authors contributed for the experiments and acquisition of data. All authors read and approved the nal manuscript to be published.   The long-term effect of dobutamine (DOB, 5, 10 g/kg) administered at 6 h after CLP on cardiac dysfunction in septic rats. Sprague-Dawley rats were randomized to undergo CLP or sham surgery, DOB at a dose of 5 g/kg (D5), 10 g/kg (D10) or normal saline (NS) was administered intravenously at 6 h after CLP or sham surgery, left ventricular ±dP/dt were detected at 20 h after CLP or sham surgery by Langendorff system (A, B). n=8-10. *P<0.05.

Figure 4
The long-term effect of dobutamine (DOB, 5, 10 g/kg) administered at 6 h after CLP on serum and cardiac cytokines in septic rats. Sprague-Dawley rats were randomized to undergo CLP or sham surgery, DOB at a dose of 5 g/kg (D5), 10 g/kg (D10) or normal saline (NS) was administered intravenously at 6 h after CLP or sham surgery, serum (A-C) and cardiac (D-F) TNF-, interleukin (IL)-6 and IL-1 were detected at 20 h after CLP or sham surgery by ELISA. n=6-13. *P<0.05.