Hemorrhage/resuscitation promotes formation of reactive oxygen and nitrogen species, hepatic injury and proinflammatory cytokine release. Previous studies have shown that production of ROS and RNS increases after hemorrhage and resuscitation and that experimental strategies to scavenge free radicals decrease liver damage and prevent hepatic endothelial leukocyte adherence [7–9, 29–31]. Free radical generation also increases after hepatic ischemia/reperfusion, as shown by electron spin resonance spectroscopy after both warm hepatic ischemia/reperfusion and small for size liver transplantation [13, 20]. Green tea (C. sinenesis) extract with its high content of polyphenols inhibits lipid peroxidation in vitro and increases of serum antioxidative capacity in vivo in human subjects [11, 32]. In the present study, hemorrhage/resuscitation increased hepatic 4-HNE protein adduct formation, signifying lipid peroxidation, and hepatic nitrotyrosine content, signifying peroxynitrite formation. Both changes were prevented by polyphenol treatment (Fig. 4 and 5).
Our work utilized a standardized green tea extract containing 85% total polyphenols, as measured colorimetrically. Major polyphenol species in the extract included epigallocatechin gallate, epigallocatechin, gallocatechin gallate, epicatechin gallate, gallocatechin, epicatechin, and catechin . Future studies will be needed to determine which component or components are responsible for the beneficial treatment effect. Some reports indicate that the combination of polyphenols is more efficacious than a single chemically defined polyphenol, such as epicatechin [13, 19, 33]
Reactive oxygen species, such as hydrogen peroxide, superoxide and hydroxyl radical, promote oxidative stress in vivo after hepatic ischemia/reperfusion. Superoxide reduces ferric iron (Fe3+) to ferrous iron (Fe2+), which in turn reacts with hydrogen peroxide to form highly reactive hydroxyl radical (iron-catalyzed Fenton reaction) [34–36]. Additionally, superoxide reacts with nitric oxide to produce toxic peroxynitrite. Such ROS and RNS promote cell injury by damaging a variety of biologically important macromolecules, including lipids, DNA and proteins . In hemorrhage/resuscitation, NADPH oxidase is a key source for superoxide production, whereas low flow states activate endothelial nitric oxide synthase (eNOS) and augment iNOS expression, resulting in increased NO production [8, 37]. Consistent with these observations, our rat model of hemorrhage/resuscitation led to increased hepatic formation of both ROS and RNS, as evidenced by hepatic 4-HNE adduct formation (Fig. 4), protein nitration (Fig. 5) and increased iNOS expression (Fig 6). These events were associated with increased hepatic proinflammatory cytokine content and hepatic damage (Fig. 2 and 3). Our findings together with earlier studies illustrate the importance of increased oxidative and nitrosative stress in development of organ damage and proinflammatory changes after hemorrhage/resuscitation.
Polyphenols decreased liver injury and increased survival after hemorrhage/resuscitation. Green tea polyphenols are effective scavengers of ROS and RNS, as documented in vitro and in vivo. In the present work, polyphenols were quite effective in blunting increases of hepatic 4-HNE adduct formation and protein nitration after hemorrhage/resuscitation to rats. Polyphenols did not exert these effects by altering the hemodynamic alterations caused by hemorrhage, since shed blood volumes and arterial pressures before, during and after hemorrhage were not different between polyphenol-treated and untreated animals (Table 1). Polyphenols also did not prevent the increase of iNOS expression caused by H/R (Fig. 6). The improvement of survival (Fig. 1), decrease of hepatic injury (Fig. 2) and suppression of proinflammatory cytokine formation (Fig. 3) by polyphenols are most likely the consequence of ROS and RNS scavenging. Recent studies from this laboratory also show that green tea polyphenols decrease liver injury after warm hepatic ischemia/reperfusion and after transplantation of reduced size livers and ethanol-induced fatty livers [13, 19, 20]. Although, the molecular mechanism of polyphenol cytoprotection remains to be determined exactly, our data are consistent with the conclusion that quenching of toxic superoxide and/or peroxynitrite by polyphenols is responsible for cytoprotection. Indeed, since superoxide is required for peroxynitrite formation from NO, superoxide quenching alone might be sufficient for cyoprotection. Taken together, these findings show that green tea polyphenols are potentially an effective therapy in diseases where liver ischemia/reperfusion plays a pathogenic role.
In our non-survival model of hemorrhage/resuscitation, polyphenol treatment improved survival from 20% to 70% (Fig. 1). Consistent with our results, previous studies show that hypotension to 30 mm Hg or less for 1 h or longer leads to mortality in rats . Mortality after hemorrhagic shock has been attributed, at least in part, to TNFα production [24, 38]. Antibodies to TNFα decrease mortality and revert hyporeactivity to epinephrine in isolated rat aortic rings, a surrogate parameter for vasoplegia after hemorrhage . Additionally, recombinant TNF-binding protein decreases rolling adhesion and firm adhesion of leukocytes to the hepatic interstitium after hemorrhage [24, 39]. Similarly, increased IL-1β in liver and other organs is associated with hepatic injury, renal dysfunction and pulmonary leukocyte infiltration after H/R [22, 40, 41]. IL-6 is also associated with organ damage in various hemorrhage models (Fig. 3) [23, 42]. Monoclonal IL-6 antibody protects against trauma-hemorrhage induced cardiac dysfunction, hepatic dysfunction and liver injury, and IL-6 knockout mice are protected from postresuscitation inflammation after hemorrhage . By contrast, IL-6 decreases LPS-induced mortality in mice and LPS-induced TNFα release by human monocytes [44, 45]. In our experiments, radical-scavenging plant polyphenols blunted TNFα, IL-1β and IL-6 production after hemorrhage/resuscitation, indicating that ROS/RNS contribute to cytokine formation and release.