Effects Of Somatostatin On Acute Plasma Levels Of TNF-α,IL-6and IL-10Undergoing Cardiopulmonary Bypass In Pigs

Cardiopulmonary bypass (CPB) is a very important auxiliary means of cardiac surgical operation, which also, however, is an unphysiological mode of circulation. During CPB, a large amount of possible causes such as non-physiological perfusion, blood direct contact with the non-biological substance of artificial devices of the CPB ducts, organ ischemia-reperfusion, the rapid changes in hemodynamics, surgical trauma, intraoperative temperature diversity and bacterial translocation from intestine, etc. can quickly activate the immune system which delivers a series of inflammatory mediators. The interaction of these inflammatory mediators can start the inflammatory cascade reaction, which causes inflammatory reaction out of control and even result in systemic inflammatory response syndrome (SIRS). The release of inflammatory mediators, enzymes, oxygen radicals and endotoxin affect the function of organs, even lead to or accelerate single or multiple organ dysfunction syndrome, which may contribute to the development of postoperative complications, including acute pulmonary injury, myocardial dysfunction, renal dysfunction, and postoperative infection, and even raise the mortality of postoperative patients. So it is significant to the theory and practice of cardiac surgical operation that researching the measures of palliating systemic inflammatory response and related complications during peri-operative.Most of study, recently, indicated that the inflammatory mediators are the main factors of inflammatory response during cardiopulmonary bypass. The major functional mediators of inflammation during CPB are tumor necrosis factor-a (TNF-a), interleukin-6(IL-6), and interleukin-10(IL-10), which acting the center components on inducing SIRS. TNF-a is the incipient factor and the most important leukocyte endogenous mediator of this inflammatory reaction, which can stimulate the delivery of IL-6、IL-8and IL-10. IL-6is the excessive activator of acute phase proteins, which also can be regarded as a marker of acute phase reaction. Expression of IL-10during cardiopulmonary bypass was regarded as an auto-protection mechanism which can inhibit the activity of histoleucocyte and macrophage, and inhibit the composition of TNF-a and IL-6. The balance of anti-inflammatory and Pro-inflammatory interleukins is considered significant to inflammation extent and clinical prognosis. Thus, the following study aimed at surveying the changed tendency of inflammatory and anti-inflammatory response by examining the concentration’s diversify of inflammatory factors (TNF-a, IL-6) and anti-inflammatory factors (IL-10).In recent years, the research that reduce the systemic inflammatory response syndrome initiated by CPB were continuous. There are also a lot of intervening measures applied to palliate the SIRS, such as embrocating the CPB ducts with heparin, modified ultrafiltration, besides using some drugs including aprotinin, glucocorticoid, ulinastatin, etc. Related research indicated these methods could prevent the inflammatory response of CPB partly, but no final conclusion has yet been reached, especially this kind of drugs using in CPB procedure also have many side-effects or complications. Stilamin is a type of somatostatin (SST), which is a synthetic cyclic14amino-acid peptide. Somatostatin can availably inhibit the excessive secretion of kinds of digestive enzymes and inflammatory mediators, abounding in pancreatic D cells, gastrointestinal neurosis, and etc. thereby, it can alleviate the injury of main organs dysfunction caused by enzymes and inflammatory mediators, and avoid multiple organ dysfunction syndrome (MODS). In other ways, it has been comfirmed that, as acting on the somatostatin’s receptor, somatostatin and the similar somatostatin neuropeptide (cortistatin) can prevent inflammatory response, and be used to treat severe sepsis and septic shock. It will have the same function in similar mechanism using somatostatin during CPB. Somatostatin has been mainly used in gastrointestinal bleeding, acute pancreatitis and etc. There are no relevant reports of somatostatin in the cardiovascular surgical research up to now. We will investigate the effects of somatostatin on CPB-induced systemic inflammatory response in the pig model, thus evaluating the regulation of inflammatory response.Objective:This study will investigate the protective effects of somatostatin on the acute inflammatory cytokines (TNF-a, IL-6and IL-10) expression in pigs undergoing cardiopulmonary bypass, then accumulate experiences and provide scientific basis for the relevant following clinical trial.Materials and Methods:1. Animal testTwenty-four healthy domestic pigs (21±2.5kg) were randomized into four groups of6each, control group(SS00) and Three experimental groups, which respectively received somatostatin (Stilamin)5μg/kg(SS05),10μg/kg(SS10)and20μg/kg(SS20) by venous pump through the central vein15min before the CPB, and throughout the60min procedure. The control group infused equal physiological saline instead. All the four groups using endotracheal intubation and intravenous anesthetic techniques, after anesthesia and heparinized, cardiopulmonary bypass was instituted with cardiac arrest. In the CPB, hold systemic hypothermia and moderate hemodilution. The pre-washed liquid selected25～30ml/kg colloid and crystalloid solution (ringer’s solution, sodium bicarbonate). The mid-sternal thoracotomy was performed among four groups, arterial cannulation was thru ascending aorta while the venous cannulation was thru the right atrium, and ascending aorta was clamped when the temperature decreased to about30℃, then antegrade perfuse the1:4St. Thomas cardioplegia solution (4℃) in the aortic root, cardiopulmonary bypass was instituted with the average perfusion rate of80-120ml/(kg-min). Intraoperative attention to the left ventricular drainage was adequate to avoid heart postings. The aortic cross clamping time of all pigs was45min, then released the aortic clamp, after the heart successfully restored beating, routinely given to various drugs, and then rewarming and weaning from the CPB, the hemodynamics were observed and monitored in6hours. All tests were achieved by the same group of surgeons.2. Observation indexs and Sample collection:Recorded some indexs such as the CPB assist circulation time (min), the auto-beating rate (%), the dose of inotropic drugs. Measured and monitored the hemodynamics (mean arterial pressure, MAP; heart rate, HR), blood oxygen saturation (SpO2), hematocrit(HCT), temperature, and etc.3ml arterial blood was sampled individually15min before CPB (TO), the time when aortic cannulation was removing (T1), and at the2nd (T2),4th(T3) and6th(T4)hour after the end of CPB, then centrifuged (3000rev/min,15min) within2h and extracted the plasma, which was conserved at-20℃before being assayed.3. Assay method: Serum levels of TNF-a, IL-6and IL-10were measured by enzyme-linked immunosorbent assay technique (ELISA), the ELISA kits were supplied by Hermes Criterion Biotechnology (Canada).4.Statistical analysis:Statistically analysis all the data with SPSS13.0. The data were expressed in the form of (x±s), and analysised by statistical treatment of ANOVA of repeated measure or multiple comparisons (LSD-t or SNK analysis). If P<0.05means that the different of data has significant statistically.Results:1. There were no significant differences among four groups in gender, age, weight, temperature and MAP, HR, SpO2before operation (P>0.05).2. There were no significant differences between each two group in CPB assist circulation time, the auto-beating rate(%), the dose of inotropic drugs and the midazolam, sufentanil, rocuronium bromide and propofol during the operations (P>0.05), while the ascending aorta clamping time and the duration of mechanical ventilation of every testing animals were the same.3. There were no significant differences between two groups in MAP, HR, SpO2and T during the cardiopulmonary bypass (P>0.05), and no severe complication occurred after operation.4. There were no statistical differences between each two group of the four groups in TNF-a levels at the point of TO (P>0.05). The levels of TNF-a were increased rapidly after CPB, at T1, T2and T3, the TNF-a levels were higher than TO among control group(SS00group) and3experimental groups (P<0.05), and reached its peak at the point of T2, then decreased gradually. However, the TNF-a concentration was still higher at T4than pre-operation(P<0.05). The levels of TNF-a in SS10group and SS20group, at T1, T2, T3and T4, were lower than SS00 group(P<0.05), while there were no differences in TNF-a levels between SS00group and SS05group at each time point measured during CPB (P>0.05), there were also no differences between SS10group and SS20group(P>0.05).5. There were no significant differences between each two group of the four groups in IL-6levels at the point of TO (P>0.05). The levels of IL-6were increased rapidly after CPB, at T1, T2and T3, the IL-6levels were higher than TO in all groups (P<0.05), and reached its peak at the point of T2, then decreased gradually. However, the IL-6concentration was still higher at T4than15min before CPB(P<0.05). The levels of IL-6in SS10group and SS20group, at T1, T2, T3and T4, were lower than SS00group(P<0.05), while no difference was found between SS00group and SS05group (P>0.05).The IL-6peak concentrations in SS05group, SSIO group and SS20group were20%,40%and46%respectively lower than the SS00group, and there were statistical differences between SS00group and SS10group or SS20group in the peak IL-6levels(P<0.05), while no differences comparing with SS05at the point of T2(P>0.05). No difference was found between SS10group and SS20group in the IL-6levels at each time point (P>0.05).6. There were no statistical differences between each two group of the four groups in IL-10levels at the point of TO (P>0.05). The levels of IL-10were also increased rapidly after CPB, at T1, T2, T3and T4, the IL-10levels were higher than TO among control group(SS00group) and3experimental groups (P<0.05). The levels of IL-10in SS10group and SS20group were lower than SS00group at Tl, T2, T3and T4(P<0.05), however, it was rapider to reach the peak, higher concentration and longer duration, compared with SS00group and SS05group, of IL-10levels in SSIO group and SS20group (P<0.05), while there were no differences in IL-10levels between SS00group and SS05group at each time point measured during CPB (P>0.05), and no differences between SSIO group and SS20 group(P>0.05).Conclusions:1. The pig model of CPB was easy to establish and facilitate the investigation of the pathophysiological processes concerning CPB-related inflammatory response and possible protective interventions. Its anatomy of heart and blood vascular system is similar to human, the circulatory system during CPB keeps steady and the automatic recovery rate of heart beats is high relatively.2. The results also indicate that no complication and side-effect and no impact on operative safety caused by somatostatin are found after the operation which administered the routine dose or ultra-routine dose of somatostatin (Stilamin) during CPB.3. Systemic inflammatory reaction due to unphysiological circulation, ischemia-reperfusion injury are induced by cardiopulmonary bypass, which leading to the significantly increasion of tumor necrosis factor, interleukin-6, etc. At the same time, the anti-inflammatory response is also activated, which demonstrates the inflammatory induced by cardiopulmonary bypass is a network complex process.4. Infused by venous pump through the central vein at15min before the CPB with5μg/kg of somatostatin can not reduce the increased amplitude of plasma levels of IL-6, TNF-a and enhance the increased amplitude of the plasma IL-10levels that result from CPB procedure, so this dose of somatostatin could not regulate the CPB-related inflammatory response.5. Infused10μg/kg of somatostatin by venous pump at15min before the CPB can reduce the increased amplitude of plasma levels of IL-6and TNF-a, whereas enhance the increased amplitude of the plasma IL-10levels that result from CPB procedure. Thus, somatostatin possesses the effect to down-regulate acute inflammatory response during CPB, however, such inhibitory effect is found to be not greatly enhanced following increasing the dosage to20μg/kg, we consider10μg/kg of somatostatin may be the better choice.To summarize, serum levels of TNF-α、IL-6and IL-10increase rapidly after CPB beginning, and reach the peak at about2hours after the CPB, then decreased gradually, and remain higher than the normal level at6hours after the CPB. Presentation of this result is that systemic inflammatory response takes place during CPB and it is severe at2hours after operation, so the best opportunity of adopting drastic and continuous measures to oppose systemic inflammatory response is since the beginning of CPB until the end of CPB. Comparatively, high dose of somatostatin can prevent this unavoidable inflammatory response effectively, and no significant impact on the operative safety.