Effect Of Nitric Oxide On The Injury Of Lung And Heart In Sepsis

AimsSepsis is an increasingly common and lethal medical condition that occurs in stress such as major surgery, severe infection and injury, and its morbidity and mortality rates are still high, especially in the elderly. The elderly patients are more likely to have gram-negative infections, and ready to get multiple organ failure. Much of the associated mortality and morbidity are the result of lung injury and cardiovascular collapse. In sepsis, as important proinflammatory cytokines, the level of tumor necrosis factor a and nitric oxide were high, but the relationship between them and the effect of nitric oxide on lung and heart are still unclear. The purpose of this study was to define the relationship between the two cytokines and organ dysfunctions, to determine the expressions of three types of nitric oxide synthetase in rat heart, and to further elucidate parts of the relevant signaling cascade. Materials and Methods1. Male Wistar rats and D-galactose pretreated Wistar rats were injected intraperitoneally with lipopolysaccharide (Escherichia coli, 10mg/kg). Control groups were treated with the same saline. The clinical manifestations, histological changes, biochemical examinations (BUN, Cr, AST, ALT, CK, CKMB) and tumor necrosis factor-α levels in four major organs (heart, liver, lung, kidney) were recorded at 2 hour or 6 hour after lipopolysaccharide administration. At the designated time points, blood gas analysis, serum tumor necrosis factor-α and nitric oxide were also determined.2. Heart rate, mean arterial blood and the maximal rate of the decrease/increase of left ventricular pressure were recorded on a computer with an analogic/digital transducer. The activities of total and inducible nitric oxidesynthetase in rat heart were analyzed by spectrophotometer before and after lipopolysaccharide administration. The expressions of three types of nitric oxide synthetase in adult rat hearts were assessed by semi-quantitative RT-PCR and Western Blotting. Immunohistochemical analyses were performed to determine the localization of eNOS, nNOS and iNOS proteins in the ventricles of the rats.3. Ventricular myocytes were enzymatically isolated from adult Wistar rats, and cardiac myocyte shortening was assessed with a video-based motion edge-detection system. To record calcium transient, myocytes were loaded with fura-2/AM for 30 min before experiment. Myocyte contraction was induced by an electrical field stimulation, then followed by S-nitroso-N-acetylpenicillamine (SNAP, nitric oxide donor), methylene blue (MB, inhibitor of soluble guanylyl cyclase). Peak twitch amplitude, time to peak shortening, time to 70% relengthening, the maximal velocities of shortening/relengthening and fura-2 fluorescence intensity change were automatically calculated from the cell length data by computer.4. Statistical analysis All values in text, tables and figures are presented as mean±SD. Differences were compared by ANOVA or t test, where appropriate. Probabilities of <0.05 were considered to be statistically significant. All of the statistical tests were performed with the GraphPad Prism software version 4.0. Results1. After intraperitoneally injected with lipopolysaccharide, adult rats andD-galactose pretreated rats showed such manifestations as fever, short breath, dis-spirited, low response, diarrhea, cyanosis and a runny nose. The symptoms and signs of the elderly group were more serious than adult group, and the saline group had no changes. The lung had significant histological changes, and PO₂ got low only 2 h after lipopolysaccharide administration. But histological and biochemical changes (CK, CKMB, AST, ALT, BUN, Cr) of heart, liver, kidney became significant at 6 h, and these indexes were much higher in the D-galactose pretreated rats than in the adult ones at the designated time points. Tumor necrosisfactor-α levels in organs (heart, liver, lung and kidney) peaked at 2 h, and the lung of D-galactose pretreated rats had the maximal raise rate. Serum nitric oxide rose rapidly, especially at 6 h.2. After LPS injection, the systolic and diastolic cardiac functions were impaired; the activities and expression of cNOS were markedly down-regulated, but those of iNOS increased significantly from negligile quantity. eNOS immunoreactivity was identified in cardiomyocytes and endothelium, and nNOS in cardiomyocytes; in sepsis immunoreactivity of eNOS and nNOS in aforementioned cells became weak, but immunoreactivity of iNOS got strong.3. SNAP, nitric oxide donor, significantly decreased peak twitch amplitude and the maximal velocities of shortening/relengthening; increased time to peak shortening and time to 70% relengthening in freshly isolated rat ventricular myocytes. Meanwhile, consistent with its negative contractile effect on myocytes, SNAP also markedly decreased the amplitude of calcium transients. The addition of MB reduced the above response induced by SNAP.Conclusions1. Lung dysfunction precedes heart, liver and kidney dysfunction in sepsis, and the severity of organ dysfunction is related to the level of tumor necrosis factor-α and nitric oxide, especially in D-galactose pretreated group. These results implicate tumor necrosis factor-α and nitric oxide as key to the development of organ dysfunction.2. The expressions of three types of NOS are differentially regulated and iNOS produced in the cardiomyocytes may contribute to the progression of heart failure after LPS injection.3. Nitric oxide exerts negative inotropic effects on rat cardiomyocytes via a cGMP-mediated fashion.