Mei-Hui Liao

Pentoxifylline improves circulatory failure and survival in murine models of endotoxaemia.

Pentoxifylline, a methylxanthine derivative, has been widely used to improve erythrocyte deformability and capillary blood circulation in patients with claudication and cerebrovascular disorders as well as in animals with sepsis. Here, we investigate the effects of pentoxifylline on the hypotension, vascular hyporeactivity to noradrenaline, release of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), and inducible NO synthase protein expression in a rat model of circulatory shock induced by bacterial endotoxin (Escherichia coli lipopolysaccharide). In addition, we have evaluated the effect of pentoxifylline on the 36-h survival rate in a murine model of endotoxaemia. Male Wistar-Kyoto rats were anaesthetised and instrumented for the measurement of mean arterial pressure and heart rate. Injection of lipopolysaccharide (10 mg/kg, i.v.) resulted in a significant fall in mean arterial pressure and an increase of heart rate. In contrast, animals pretreated with pentoxifylline (3 mg/kg, i.v., at 30 min prior to lipopolysaccharide) maintained a significantly higher mean arterial pressure but showed no effect on the tachycardia when compared to rats given only lipopolysaccharide (lipopolysaccharide-rats). The pressor effect of noradrenaline (1 microg/kg, i.v.) was also significantly reduced after the treatment of rats with lipopolysaccharide. Similarly, rings of thoracic aorta obtained from lipopolysaccharide-rats showed a significant reduction in the contractile responses elicited by noradrenaline (1 microM). Pretreatment of lipopolysaccharide-rats with pentoxifylline partially, but significantly, prevented this lipopolysaccharide-induced hyporeactivity to noradrenaline in vivo and ex vivo. The injection of lipopolysaccharide resulted in bell-shape changes in plasma TNF-alpha level which reached a peak at 60 min, whereas the effect of lipopolysaccharide on the plasma level of nitrate (an indicator of NO formation) was increased in a time-dependent manner. This increase of both TNF-alpha and nitrate levels induced by lipopolysaccharide was significantly reduced in lipopolysaccharide-rats pretreated with pentoxifylline. Endotoxaemia for 240 min caused a significantly increased protein expression of inducible NO synthase in the lung. In lipopolysaccharide-rats pretreated with pentoxifylline, inducible NO synthase protein expression in lung homogenates was attenuated by 48 +/- 5%. Treatment of conscious mice with a high dose of endotoxin (60 mg/kg, i.p.) resulted in a survival rate of only 10% at 36 h (n = 20). However, therapeutic application of pentoxifylline (3 mg/kg, i.p. at 0, 6, 15 and 24 h after lipopolysaccharide) increased the 36-h survival to 35% (n = 20). Thus, pentoxifylline protects against circulatory failure and improves survival in rodents with severe endotoxaemia. These effects may be due to inhibition of the release of TNF-alpha and of the induction of inducible NO synthase.

Terbutaline prevents circulatory failure and mitigates mortality in rodents with endotoxemia.

Septic shock is characterized by a decrease in systemic vascular resistance. Nevertheless, regional increases in vascular resistance can occur that may predispose mammals to organ dysfunction, including the acute respiratory distress syndrome. In the host infected by endotoxin (lipopolysaccharide, LPS), the expression and release of proinflammatory tumor necrosis factor-alpha (TNFalpha) rapidly increases, and this cytokine production is regulated by agents elevating cyclic AMP. In this report, we present evidence that terbutaline, a beta2-agonist, inhibits TNFalpha production and enhances interleukin-10 (IL-10) release in the anesthetized rat treated with LPS. In addition, an overproduction of nitric oxide (NO, examined by its metabolites nitrite/nitrate) by inducible NO synthase (iNOS, examined by western blot analysis) is attenuated by pretreatment of LPS rats with terbutaline. Overall, pretreatment of rats with terbutaline attenuates the delayed hypotension and prevents vascular hyporeactivity to norepinephrine. In addition, pretreatment of mice with terbutaline also improves the survival in a model of severe endotoxemia. The infiltration of polymorphonuclear neutrophils into organs (e.g., lung and liver) from the surviving LPS mice treated with terbutaline was reduced almost to that seen in the normal controls. These findings suggest that the inhibition of TNFalpha and NO (via iNOS) production as well as the increment of IL-10 production contribute to the beneficial effect of terbutaline in animals with endotoxic shock.

RhoA/Rho-Kinase and Nitric Oxide in Vascular Reactivity in Rats with Endotoxaemia

RhoA/Rho-kinase (RhoA/ROK) pathway promotes vasoconstriction by calcium sensitivity mechanism. LPS causes nitric oxide (NO) overproduction to induce vascular hyporeactivity. Thus, we tried to examine the role of RhoA/ROK and NO in the regulation of vascular reactivity in different time-point of endotoxaemia. Male Wistar rats were intravenously infused for 10 min with saline or E. coli endotoxin (lipopolysaccharide, LPS, 10 mg/kg) and divided to five groups (n = 8 in each group): (i) Control, sacrificed at 6 h after saline infusion; (ii) LPS1h, sacrificed at 1 h after LPS infusion; (iii) LPS2h, sacrificed at 2 h after LPS infusion; (iv) LPS4h, sacrificed at 4 h after LPS infusion; and (v) LPS6h, sacrificed at 6 h after LPS infusion. LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Indeed, our results showed that LPS reproduced a biphasic hypotension and sustained vascular hyporeactivity to noradrenaline (NA) in vivo. Interestingly, this hyporeactivity did not occur in ex vivo during early endotoxaemia. This could be due to increases of aortic RhoA activity (n = 5, P<0.05) and myosin phosphatase targeting subunit 1 phosphorylation (n = 3, P<0.05). In addition, pressor response to NA and vascular reactivity in early endotoxaemia were inhibited by ROK inhibitor, Y27632. Furthermore, plasma bradykinin was increased at 10 min (24.6±13.7 ng/mL, n = 5, P<0.05) and aortic endothelial NO synthase expression was increased at 1 h (+200%. n = 3, P<0.05) after LPS. In late endotoxaemia, the vascular hyporeactivity was associated with aortic inducible NO synthase expression (n = 3, P<0.05) and an increased serum NO level (n = 8, P<0.05). Thus, an increased RhoA activity could compensate vascular hyporeactivity in early endotoxaemia, and the large NO production inhibiting RhoA activity would lead to vascular hyporeactivity eventually.

Application of thrombelastography in liver injury induced by endotoxin in rat.

Liver injury developing in patients with sepsis may lead to an increased risk of mortality. Thrombelastography (TEG) is generally applied to evaluate hemostatic disturbance in patients undergoing liver transplantation or cardiopulmonary bypass. The aim of this study was to investigate the development of liver injury and coagulopathy in a lipopolysaccharide (LPS)-induced animal model and to assess the relationship between TEG variables and liver injury. Male Wistar rats received LPS (30 mg/kg over a 4-h intravenous infusion) to induce experimental liver injury or isotonic saline as a control. Variables of hemodynamics and liver biochemistry were measured during the subsequent 6 h after the start of infusion. TEG variables (R-time, K-time, &agr;-angle and maximal amplitude), thrombin–antithrombin complex and plasminogen activator inhibitor-1 were also measured. After LPS infusion, liver injury [examined by biochemical variables (e.g. alanine aminotransferase, ALT) and histological studies] was developed and inflammatory cytokines (tumor necrosis factor-&agr; and interleukin-6) were raised. At the initial period of LPS infusion, R-time was shortened and &agr;-angle was increased. Thereafter, &agr;-angle and maximal amplitude were decreased progressively, demonstrating that endotoxin induced coagulation disturbances. Furthermore, there were strong positive correlation between K-time and natural log (Ln)(ALT) (r = 0.823, P = 0.001); also, there were strong negative correlations between &agr;-angle and Ln(ALT) (r = −0.762, P = 0.002) as well as maximal amplitude and Ln(ALT) (r = −0.732, P = 0.004) at 6 h after LPS infusion. These results demonstrated that TEG could be a potential tool to evaluate the development of liver injury in endotoxemia.

Electro-acupuncture at 'Neiguan' (PC6) attenuates liver injury in endotoxaemic rats.

Background Intravenous injection of lipopolysaccharide (LPS) stimulates macrophages to release proinflammatory cytokines and nitric oxide (NO). This results in hypotension, vascular hyporeactivity and multiple organ failure (eg, liver injury) in rats. In rats with endotoxin shock, electro-acupuncture (EA) of ‘Neiguan’ (PC6) retrieved blood pressure and reduced plasma concentrations of NO. The authors evaluated whether EA at PC6 could alleviate the development of liver injury and dysfunction in endotoxic rats. Methods A total of 28 male adult Wistar rats were included in this study. Rats received intravenous LPS (10 mg/kg for 4 h) or saline for 4 h followed by EA at PC6 acupuncture point. Results Elevated biochemical parameters of liver injury and marked infiltration of neutrophils into liver tissues caused by LPS were significantly attenuated by EA. However, hypotension, tachycardia and raised production of plasma NO were not suppressed by EA at PC6. Conclusions These results indicate that EA at PC6 should be further investigated as a possible adjuvant therapy for endotoxin-induced liver dysfunction. Its mechanism of action needs further investigation.

Possible biomarkers of early mortality in peritonitis-induced sepsis rats

BACKGROUND Sepsis induced by cecal ligation and puncture (CLP) is accompanied by circulatory failure, multiple organ dysfunction syndrome, metabolic acidosis, and electrolyte imbalance in rats. However, it remains uncertain which parameters can be used to predict the mortality of septic rats. Thus, the aim of this study was to examine which possible biomarkers were associated with mortality in the CLP-induced sepsis model. MATERIALS AND METHODS After the carotid artery and vein were cannulated, rats were subsequently subjected to CLP or sham operation. The changes of hemodynamics, biochemical variables, blood gas, and electrolytes were monitored during the 18-h observation. RESULTS The CLP surgery caused circulatory failure, multiple organ dysfunction syndrome, metabolic acidosis, electrolyte imbalance, and death. Compared with survivors, nonsurvivors showed significant difference in (1) blood glucose; (2) lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, creatinine, and blood urea nitrogen in serum; and (3) base excess, HCO3(-), PaCO2, potassium, and calcium in whole blood at 9 h after CLP. No significant difference in blood pressure, heart rate, pressor response to noradrenaline, rectal temperature, total protein, albumin, PaO2, and sodium was observed between nonsurvivors and survivors. However, after multifactor dimensionality reduction analysis, the union of HCO3(-) and blood glucose had the biggest testing balanced accuracy. CONCLUSIONS These results indicate that HCO3(-) plus blood glucose serves as the best biomarker of early death in rats with CLP-induced sepsis. Thus, these parameters could guide experimental procedures for making the right interventions when utilizing CLP as a sepsis model in rats.

Distinct Patterns of Wnt3a and Wnt5a Signaling Pathway in the Lung from Rats with Endotoxic Shock

Septic shock is a syndrome with severe hypotension and multiple organ dysfunction caused by an imbalance between pro-inflammatory and anti-inflammatory response. The most common risk factor of acute lung injury is severe sepsis. Patients with sepsis-related acute respiratory distress syndrome have higher mortality. Recent studies reveal regulatory roles of Wnt3a and Wnt5a signaling in inflammatory processes. Wnt3a signaling has been implicated in anti-inflammatory effects, whereas Wnt5a signaling has been postulated to have pro-inflammatory properties. However, the balance between Wnt3a and Wnt5a signaling pathway in the lung of rats with endotoxic shock has not been determined. Thus, we investigated the major components of Wnt3a and Wnt5a signaling pathway in the lung of endotoxemic rats. Male Wistar rats were intravenously infused with saline or lipopolysaccharide (LPS, 10 mg/kg). The changes of hemodynamics, biochemical variables, and arterial blood gas were examined during the experimental period. At 6 h after saline or LPS, animals were sacrificed, and lungs were obtained for analyzing superoxide production, water accumulation, histologic assessment, and protein expressions of Wnt3a and Wnt5a signaling pathway. Animals that received LPS showed circulatory failure, multiple organ dysfunction, metabolic acidosis, hyperventilation, lung edema, and high mortality. The lung from rats with endotoxic shock exhibited significant decreases in the levels of Wnt3a, Fzd1, Dsh1, phosphorylated GSK-3β at Ser9, and β-catenin. In contrast, the expressions of Wnt5a, Fzd5, and CaMKII were up-regulated in the lung of endotoxemic rats. These findings indicate the major components of Wnt3a and Wnt5a signaling in the lung are disturbed under endotoxic insult.

Procainamide Inhibits DNA Methylation and Alleviates Multiple Organ Dysfunction in Rats with Endotoxic Shock

Excessive inflammatory and oxidative stress lead to circulatory failure, multiple organ dysfunction, and high mortality in patients with sepsis. Microbial infection-induced DNA hypermethylation is associated with the augmentation of inflammation and oxidative stress. In our previous study, the antiarrhythmic drug procainamide inhibits the expression of DNA methyltransferase 1 (DNMT1) and diminishes IL-6 levels in rats with rhabdomyolysis. Thus, we further evaluated the effects of procainamide on the development of circulatory failure and multiple organ dysfunction in rats with endotoxic shock. Male Wistar rats were intravenously infused with saline or lipopolysaccharide (LPS) followed by procainamide administration. The changes of hemodynamics, blood glucose, biochemical variables, and plasma nitric oxide (NO) levels were analyzed during the experimental period. At the end of experiments, animal organs were also obtained for examining superoxide production, neutrophil infiltration, and DNA methylation status. Our results showed that LPS induced circulatory failure, multiple organ dysfunction, and high mortality rate in endotoxemic rats. Overt neutrophil infiltration and superoxide production, accompanied by the elevations of DNMT1 and 5-methylcytosine levels in the lung of endotoxemic rats were also observed. Treatment of endotoxemic animals with procainamide not only inhibited the increased levels of DNMT1 and 5-methylcytosine but also ameliorated neutrophil infiltration and superoxide production in the lung. In addition, the anti-inflammatory gene, IL27RA, was down-regulated in the LPS group and up-regulated in the LPS + Procainamide group. Procainamide also diminished IL27RA methylation in the lung of endotoxemic rat. Moreover, both DNMT inhibitors procainamide and hydralazine improved hypotension, hypoglycemia, and multiple organ dysfunction of LPS-treated rats. Thus, we suggest that the beneficial effects of procainamide could be attributed to the suppression of DNA methylation, neutrophil infiltration, superoxide production, and NO formation. It seems that this old drug may have new potential uses in infectious diseases, in particular, associated with endotoxemia.

Effects of gabexate mesilate on coagulopathy and organ dysfunction in rats with endotoxemia: a potential use of thrombelastography in endotoxin-induced sepsis.

Sepsis and its associated multiple organ failure are related to high mortality in critical patients. Several studies have reported that gabexate mesilate, a synthetic inhibitor of trypsin-like serine protease, protects tissues/organs against injury in the models of endotoxemia. The aim of this study was to examine whether gabexate mesilate could attenuate coagulopathy and organ dysfunction in lipopolysaccharide (LPS)-induced sepsis model by using thrombelastography (TEG). LPS (7.5 mg/kg/h, intravenouly for 4 h) was administered to male adult Wistar rats. Some of the LPS rats received a continuous infusion of gabexate mesilate (10 mg/kg/h, intravenously for 8.5 h) for 30 min before the LPS administration. Variable parameters of hemodynamics, biochemistry, hemostasis and inflammatory response were measured for 6 h after the LPS infusion. TEG variables (R-time, K-time, &agr;-angle, and maximal amplitude) were also measured. The pretreatment of LPS rats with gabexate mesilate significantly attenuated the lung, liver and kidney dysfunction, consumptive coagulopathy, the increases in serum tumor necrosis factor-&agr;, interleukin-6, plasma thrombin–antithrombin complex and plasminogen activator inhibitor-1, and neutrophils infiltration score in lung, liver and kidney, compared with the LPS alone group. In addition, TEG parameters correlated with tissue and liver injury in the late phase of endotoxemia. In particular, a strong negative correlation between maximal amplitude at 4 h and Ln (lactate dehydrogenase) at 6 h after LPS infusion was noted (r = −0.752, P < 0.001, R2 = 0.566). These results indicate that beneficial effects of anticoagulants (e.g. gabexate mesilate) in endotoxemia could be monitored by TEG per se.

Therapeutic Effects of Procainamide on Endotoxin-Induced Rhabdomyolysis in Rats

Overt systemic inflammatory response is a predisposing mechanism for infection-induced skeletal muscle damage and rhabdomyolysis. Aberrant DNA methylation plays a crucial role in the pathophysiology of excessive inflammatory response. The antiarrhythmic drug procainamide is a non-nucleoside inhibitor of DNA methyltransferase 1 (DNMT1) used to alleviate DNA hypermethylation. Therefore, we evaluated the effects of procainamide on the syndromes and complications of rhabdomyolysis rats induced by lipopolysaccharide (LPS). Rhabdomyolysis animal model was established by intravenous infusion of LPS (5 mg/kg) accompanied by procainamide therapy (50 mg/kg). During the experimental period, the changes of hemodynamics, muscle injury index, kidney function, blood gas, blood electrolytes, blood glucose, and plasma interleukin-6 (IL-6) levels were examined. Kidneys and lungs were exercised to analyze superoxide production, neutrophil infiltration, and DNMTs expression. The rats in this model showed similar clinical syndromes and complications of rhabdomyolysis including high levels of plasma creatine kinase, acute kidney injury, hyperkalemia, hypocalcemia, metabolic acidosis, hypotension, tachycardia, and hypoglycemia. The increases of lung DNMT1 expression and plasma IL-6 concentration were also observed in rhabdomyolysis animals induced by LPS. Treatment with procainamide not only inhibited the overexpression of DNMT1 but also diminished the overproduction of IL-6 in rhabdomyolysis rats. In addition, procainamide improved muscle damage, renal dysfunction, electrolytes disturbance, metabolic acidosis, hypotension, and hypoglycemia in the rats with rhabdomyolysis. Moreover, another DNMT inhibitor hydralazine mitigated hypoglycemia, muscle damage, and renal dysfunction in rhabdomyolysis rats. These findings reveal that therapeutic effects of procainamide could be based on the suppression of DNMT1 and pro-inflammatory cytokine in endotoxin-induced rhabdomyolysis.