The pathogenesis of multiple organ dysfunction syndrome (MODS) in septic shock is complicated and not fully understood. Some studies show that an overproduction of nitric oxide (NO) leads to the refractory hypotension and multiple organ failure, while other studies suggest that free radicals, e.g. superoxide (O2−), contribute to the detrimental effect on vascular responsiveness and tissue/organ damage. Thus, this study was performed on the Wistar rat by using cecal ligation and puncture (CLP) to induce septic shock‐associated MODS. We evaluated the effect of an antioxidant melatonin in CLP‐induced septic rats and demonstrated that melatonin (3 mg/kg, i.v. at 3, 6, 12 hr after CLP) significantly (a) attenuated hyporeactivity to norepinephrine and delayed hypotension, (b) reduced plasma index of hepatic and renal dysfunction, (c) diminished plasma NO and interleukin‐1β (IL‐1β) concentrations as well as aortic O2− levels, (d) reduced marked infiltration of polymorphonuclear neutrophils (PMNs) in the lung and liver tissues, and (e) promoted the survival rate at 18 hr to twofold compared with the CLP alone group. The current study underlined the inhibition of plasma NO and IL‐1β as well as aortic O2− production and the reduction of PMN infiltration may lead to the amelioration of MODS, which may contribute to the beneficial effect of antioxidants (e.g. melatonin in this study) in conscious rats with peritonitis‐induced lethality. Thus, the antioxidant could be a novel agent for the treatment of septic animals or patients in the early stage.

There is good evidence that endotoxemia, sepsis, and septic shock are associated with the generation and release of reactive oxygen species (ROS) such as superoxide anion (O2), indicating that oxygen-derived free radicals play an important role in the pathogenesis of sepsis/shock. Studies on the application of free oxygen radical scavengers to limit the damage to tissues and organs have been recently attempted. A stable piperidine nitroxide of low molecular weight (Tempol) can permeate biological membranes and scavenge O2 in vitro and in vivo. Thus, we investigated effects of Tempol on the circulatory failure and multiple organ injuries caused by a clinically relevant polymicrobial sepsis model in the rat-cecal ligation and puncture (CLP). CLP not only successfully induced circulatory failure but also substantially increased plasma concentrations of glutamate-oxalate-transferase and glutamate-pyruvate-transferase (indicators of liver injury), creatinine and blood urea nitrogen (indicators of kidney injury), and decreased base excess in arterial blood in the late stage, indicating the development of multiple organ injury in this study. These were also confirmed by a histologic examination showing that the CLP-induced sepsis accompanied increase of polymorphonuclear neutrophil (PMN) infiltration in the lung and sequestration in the liver. Our results demonstrated that Tempol not only ameliorated the deterioration of hemodynamic changes and renal and liver injuries but also attenuated PMN infiltration in the lung and sequestration in the liver (histology). In addition, Tempol improved the survival in CLP-induced septic rats. Moreover, Tempol reduced the plasma NO. and interleukin-1beta and organ O2 levels in CLP-treated rats. In conclusion, Tempol prevented circulatory failure and attenuated organ dysfunction/injury as well as decreased the mortality rate in CLP-treated animals. These beneficial effects of Tempol may be attributed to inhibition of ROS formation (e.g., NO. and O2), suggesting antioxidant (e.g., Tempol) is a potential therapeutic agent in the treatment of intraperitoneal septic shock.

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.

Perivascular adipose tissue (PVAT)-derived factors have been proposed to play an important role in the pathogenesis of atherosclerosis. Caveolin-1 (Cav-1), occupying the calcium/calmodulin binding site of endothelial NO synthase (eNOS) and then inhibiting nitric oxide (NO) production, is also involved in the development of atherosclerosis. Thus, we investigated whether PVAT regulated vascular tone via Cav-1 and/or endothelial NO pathways. Isometric tension studies were carried out in isolated thoracic aortas from Wistar rats in the presence and absence of PVAT. Concentration-response curves of phenylephrine, acetylcholine, and sodium nitroprusside were illustrated to examine the vascular reactivity and endothelial function. The protein expressions of eNOS and Cav-1 were also examined in aortic homogenates. Our results demonstrated that PVAT significantly enhanced vasoconstriction and inhibited vasodilatation via endothelium-dependent mechanism. The aortic NO production was diminished after PVAT treatment, whereas protein expression and activity of eNOS were not significantly affected. In addition, Cav-1 protein expression was significantly increased in aortas with PVAT transfer. Furthermore, a caveolae depleter methyl-β-cyclodextrin abolished the effect of PVAT on the enhancement of vasoconstriction, and reversed the impairment of aortic NO production. In conclusion, unknown factor(s) released from PVAT may inhibit endothelial NO production and induce vasocontraction via an increase of Cav-1 protein expression.