| ObjectiveSepsis is defined as the presence (probable or documented) of infection together with systemic inflammatory response syndrome (SIRS). It may lead to severe sepsis, septic shock and even death. Severe sepsis is defined as a merger in organ dysfunction manifestations of sepsis. Septic shock is defined as sepsis with cardiovascular organ dysfunction or sepsis induced sustained hypotension, whereas fully liquid recovery cannot reverse it. Different organ failure has been recognized as the main reason of sepsis-induced death, especially the cardiovascular failure. It is well documented that myocardium dysfunction was common in sepsis and septic shock which affected 94% patients. It involved a variety of mechanisms such as immune system activation, myocardial mitochondrial dysfunction, nitric oxide (NO) and peroxynitrite, and so on. In adult patients with septic shock, patients with myocardial dysfunction had a higher mortality rate than thouse patients without myocardial dysfunction. And children patients with septic shock were more likely to develop vasomotor dysfunction and require blood pressure treatment, they often tend to have reaction to systolic and diastolic medicine treatment, prompting a more important role of myocardial dysfunction in children patients than that in adult patients. In sepsis, myocardial mitochondria damage caused an increase in oxidative stress nitride and also caused cardiac mitochondrial morphology changes. In fact, catecholamine can reduce oxidative stress levels and improve mitochondrial respiratory function. Isoproterenol (ISO) is a classic synthetic catecholamine, it is similar in structure to endogenous catecholamine such as epinephrine and norephrine. ISO is a strong non-select agonist of β receptor (β1-and β2-AR). Bothβ1- and β2- AR are G-protein-coupled receptors (GPCRs), they play important role in the regulation of cardiovascular function. It has previously been shown that apart from it’s hemodynamic effects, ISO may also play anti-inflammation and -shock effects via regulating LPS-induced inflammation response. However, there was few research about the role of ISO on myocardial tissues. In this paper, we speculated that ISO may reduce the myocardial injury by means of decreasing the levels of pro-inflammatory factors and oxidative stress factors, increasing anti-inflammatory factors and anti-oxidative stress factors. By observing the influences of continuous intravenous infusion different doses ISO on biochemical indices, mitochondria structure and function, inflammation factors and oxidative stress factors of rat heart, this study aimed at investigating whether ISO is protective for rat heart and the corresponding mechanisms. In addition, this study also attempted to identify the appropriate dose for ISO, thereby provide experimental and theoretical foundations for the clinical application of ISO to early protective treatment of sepsis.Methods1. Animal preparation 30 specific pathogen free (SPF) lwver male SD rats (provided by medical experimental animal center of Sun Yat-sen university:animals batch number 44008500003278), the animals were all body quality of 250～300 g.12 hours before the right external carotid vein catheter, all animals had a fast but free from drinking water.1.1 Sepsis model:24 hours before the experiment, right external carotid vein catheter was inserted to establish intravenous infusion and blood sampling channel. Septic model or control group was established by receiving lipopolysaccharide (LPS) or 0.9% saline 10mg/kg intraperitoneally.1.2 Grouping:using the simple random method,30 Sprague Dawley (SD) rats were randomly divided into five groups (n=6 per group):control group, endotoxin group and ISO intervene (small-dose, medium-dose and large-dose) group. Endotoxin group received intravenous injection of LPS 10mg/kg followed by an continuous intravenous infusion of 0.9% saline lml/h; ISO intervene group received LPS 10mg/kg followed by an continuous intravenous infusion of ISO 0.06μg/(kg-min),0.3μg/(kg-min) and 0.6μg/(kg-min); the control group received intraperitoneal injection and continuous intravenous infusion same amount of 0.9% saline.1.3 Endpoint:the endpoint of the study was 24 h after intraperitoneal injection of 9 g/L saline or LPS.2. Sample collection, preparation and test2.1 Blood samples collection, preparation and testAt 2 h and 6 h after intraperitoneal injection of LPS or saline, lml intravenous blood was collect, thereafter same amount of saline was infused back. At the primary endpoint of the study (24 h after intraperitoneal injection of LPS or saline),3-4ml intravenous blood was collect. After standing for 1-2 h at room temperature, the blood samples were centrifuged (4℃,3000rpm, 10min) to obtain serum. The serum samples were kept portions in -80℃ refrigerator. The serum levels of creatine kinase (CK) and creatine kinase isoenzyme (CK-MB) at 24 h intraperitoneal injection of LPS or saline were detected by fully automatic biochemical analyzer. The levels of serum inflammatory cytokines (TNF-a, IL-1β and IL-10) at 2 h,6 h and 24 h after intraperitoneal injection of LPS or saline were detected by enzyme-linked immune sorbent assay (ELISA).2.2 Myocardium samples collection, preparation and testAt primary endpoint of the study (24 h after intraperitoneal injection of LPS or saline), rats were anesthesia by 10%(100g/L) chloral hydrate. After that, open the abdominal cavity and gathered the heart rapidly. The heart was rinsed at pre-cooling saline, the blood and large vessels were removed at the same time. 100mg myocardium were used for extracting mitochondria through differential centrifugation method, the obtained mitochondria suspension was JC-1 stained, and then the degree of mitochondrial swelling and the membrane potential were detected by flow cytometry (FCM).Take a small piece of myocardium (1cm3), cutting into 1mm3 pieces for preparation of electron microscopic specimens to observe the ultrastructure of the mitochondria.In addition, take another small piece of myocardium for preparation of light microscopy specimens (HE stains) to observe the pathological changes of the mitochondria.The remaining myocardium was kept portions in -80℃ refrigerator for detection of myocardium mitochondria oxidative stress factors. Oxidative stress factors (SOD and iNOS activities, MDA and NO contents) were detected by colorimetry assay.3. Statistical analysisData were analyzed by SPSS 20.0, all results were reported as mean± standard deviation (x±s). Differences between groups were determined by one-way ANOVA. If there was statistical significance and homogeneity of variance between groups, Least-significant difference (LSD) was used, while if there was statistical significance and heterogeneity of variance between groups, Dunnett T3 was used. P less than 0.05 was considered to be sigificant.Results1. Serum levels of biochemical indices (CK and CK-MB) at 24 h after intraperitoneal injection of LPS or salineLevels of CK and CK-MB at 24 h after intraperitoneal injection of LPS or saline in endotoxin group were increased compared with control group (P<0.05); ISO intervene significantly decreased the levels of CK and CK-MB (P<0.05) compared with endotoxin group.2. Degrees of myocardium mitochondria oxidative stress factors2.1 NO contentDegrees of NO content at 24 h after intraperitoneal injection of LPS in endotoxin group were increased compared with control group (8.300±0.740 & 10.823±2.240, P<0.05); ISO intervene significantly decreased the degrees of NO content compared with endotoxin group (P<0.05).2.2 iNOS activityDegrees of iNOS activity at 24 h after intraperitoneal injection of LPS in endotoxin group were increased compared with control group (0.031±0.008 & 0.045±0.008, P<0.05); degrees of iNOS activity significantly decreased in small-dose group compared with endotoxin group (0.045±0.008 & 0.033±0.003, P< 0.05); degrees of iNOS activity significantly increased in medium- and large-dose group compared with endotoxin group and small-dose group (P<0.05).2.3 SOD activityDegrees of SOD activity at 24 h after intraperitoneal injection of LPS in endotoxin group significantly decreased compared with control group (11.543± 1.080 & 9.892±0.815, P<0.05); ISO intervene significantly increased the degrees of SOD activity compared with endotoxin group (P<0.05); degrees of SOD activity significantly increased in medium- and large-dose group compared with small-dose group (11.822±0.460 & 19.802±1.309’11.822±0.460 & 19.737±1.215,P<0.05).2.4 MDA contentDegrees of MDA content at 24 h after intraperitoneal injection of LPS in endotoxin group significantly increased compared with control group (0.950±0.195 & 1.663±0.618,P<0.05); degrees of MDA content significantly decreased in small-and medium-dose group compared with endotoxin group (1.663±0.618 & 0.768±0.312,1.663±0.618 & 1.203±0.167, P<0.05); degrees of MDA content significantly increased in medium-dose group compared with small-dose group (0.768±0.312 & 1.203±0.167, P<0.05); degrees of MDA content significantly increased in large-dose group compared with control group, small-and medium-dose group (P<0.05).3. Degrees of mitochondrial swelling and the membrane potential3.1 Degrees of mitochondria swellingISO intervene (small-, medium-and large-dose) significantly decreased the degrees of mitochondria swelling compared with endotoxin group (P<0.05). Degrees of mitochondria swelling in medium-dose group (1.160±0.186) significantly decreased compared with control group (1.537±0.109) and small-dose group (1.506±0.116) (P<0.05). Degrees of mitochondria swelling significantly increased in large-dose group compared with medium-dose group (1.160±0.186 & 1.393±0.128,P<0.05).3.2 Mitochondria membrane potentialDegrees of the mitochondria membrane potential were decreased in endotoxin-(0.190±0.074), small-(0.175±0.05), medium-(0.198±0.086) and large-dose (0.251±0.10) group compared with control group (0.825±0.058)(P <0.05).4. Ultrastructure changes of the myocardium mitochondria between groupsMyocardium mitochondria in control group were normal in size and neatly arranged; crista fragmentation, membrane fusion, swelling and vacuolus were seen in myocardium mitochondria of endotoxin group; the ultrastructure of myocardium mitochondria significantly improved in small-and medium-dose group compared with endotoxin group, while myocardium mitochondria in large-dose group showed abnormal changes such as swelling and crista fragmentation.5. Pathological changes of the myocardium mitochondria (HE stains) between groupsMyocardium in control group arranged normally without any abnormal; in endotoxin group, myocardium were loose structured and edema with lymphocytes infiltration and erythrocyte diapedesis, indicating inflammatory responses; in small-and medium-dose group, mild edema and regularly arrayed cells were seen while no erythrocytes were seen, revealing that inflammation response were reduced; in large-dose group, mild edema were seen, indicating inflammatory response.6. The levels of serum inflammatory cytokines at three points (2 h,6 h and 24 h) between groups6.1 Levels of serum TNF-α at three points (2 h,6 h and 24 h) between groups6.1.1 Levels of serum TNF-a at 2 h after intraperitoneal injection of LPS or saline between groupsLevels of serum TNF-a significantly increased in endotoxin group and ISO intervene group (small-, medium-and large-dose group) compared with control group (P<0.05); levels of serum TNF-a significantly decreased in medium -and large-dose group compared with endotoxin group (P<0.05); levels of serum TNF-a significantly decreased in medium-and large-dose group compared with small-dose group (P<0.05).6.1.2 Levels of serum TNF-a at 6 h after intraperitoneal injection of LPS or salineLevels of serum TNF-a significantly increased in endotoxin group and ISO intervene group (small-, medium-and large-dose group) compared with control group (P<0.05); levels of serum TNF-a significantly decreased in medium-and large-dose group compared with endotoxin group (P<0.05); levels of serum TNF-a significantly decreased in large-dose group compared with small-dose group (P< 0.05).6.1.3 Levels of serum TNF-a at 24 h after intraperitoneal inj ection of LPS or salineLevels of serum TNF-a significantly increased in endotoxin group, small-and medium-dose group compared with control group (P<0.05); levels of serum TNF-a significantly decreased in large-dose group compared with endotoxin group (P< 0.05); levels of serum TNF-a significantly decreased in large-dose group compared with small-and medium-dose group (P<0.05).6.2 Levels of serum IL-1β at three points (2 h,6 h and 24 h) between groups6.2.1 Levels of serum IL-1β at 2 h after intraperitoneal injection of LPS or salineLevels of serum IL-1β significantly increased in endotoxin group, small-and medium-dose group compared with control group (P<0.05).6.2.2 Levels of serum IL-1β at 6 h after intraperitoneal injection of LPS or salineLevels of serum IL-1β significantly increased in endotoxin group, small-and medium-dose group compared with control group (P<0.05).6.2.3 Levels of serum IL-1β at 24 h after intraperitoneal injection of LPS or salineLevels of serum IL-1β significantly increased in endotoxin group, small- and medium-dose group compared with control group (P<0.05).6.3 Levels of serum IL-10 at three points (2 h,6 h and 24 h) between groups6.3.1 Levels of serum IL-10 at 2 h after intraperitoneal injection of LPS or saline between groupsLevels of serum IL-10 was not detect in control group; levels of serum IL-10 significantly decreased in large-dose group compared with small-dose group (P< 0.05).6.3.2 Levels of serum IL-10 at 6 h after intraperitoneal injection of LPS or saline between groupsLevels of serum IL-10 was not detect in control group; levels of serum IL-10 significantly increased in small-dose group compared with endotoxin group (P< 0.05); levels of serum IL-10 significantly decreased in medium-dose grout compared with small-dose group (P<0.05).6.3.3 Levels of serum IL-10 at 24 h after intraperitoneal injection of LPS or saline between groupsLevels of serum IL-10 was not detect in control group; levels of serum IL-10 significantly increased in small-dose group and decreased in large-dose group compared with endotoxin group (?<0.05); levels of serum IL-10 significantly decreased in medium-and large-dose group compared with small-dose group (P< 0.05).Conclusion1. The myocardium and myocardial mitochondria of early septic rats were damaged;2. Continuous intravenous infusion low-dose ISO [0.06μg/(kg-min)] revealed protective effect on these damages, the corresponding mechanism may relate to the decrease of inflammation response and the oxidative/nitrosative stress. |
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