Our study has shown that activation of LXRα with the synthetic ligand T0901317 improves the hemodynamic profile of rats subjected to severe hemorrhage and resuscitation. Furthermore, lung injury and neutrophil infiltration are reduced with LXRα activation. This pulmonary protection is seen in conjunction with a decrease in plasma cytokine and chemokines levels. These anti-inflammatory effects are likely due to an inhibition of the NF-κB pathway. These findings all suggest that LXRα regulates inflammation after a hemorrhagic insult on a systemic scale, though its effects on organs other than the lungs remains to be explored.
In our study, we used a pressure-controlled model of hemorrhagic shock to standardize the level of hypoperfusion during the hemorrhage phase. After resuscitation, LXRα activation significantly decreased heart rate while also increasing stroke volume and cardiac index. Interestingly, there was no significant change in the MABP post-resuscitation despite these positive findings. One possible explanation for this is the interaction between LXRα and the Renin-Angiotensin System (RAS). Several studies have shown that activated LXRα interacts with the RAS, leading to decreased renin levels and a decreased response to Angiotensin II (25
). This interaction would limit the ability of animals to vasoconstrict and increase their blood pressure. Concordant with this hypothesis, we did not observe any changes in total peripheral resistance (data not shown) in rats treated with T0901317, despite improvements in other hemodynamic parameters.
Improvement of hemodynamic parameters such as CI and SVI implies that systemic oxygen demand has decreased. However, systemic markers of acidosis are used in conjunction with hemodynamic parameters to clinically assess cellular hypoxia (28
). Among the values used are plasma lactate, base deficit and bicarbonate. In our study, we found that activation of LXRα significantly improved plasma lactate and showed a trend towards improvement of bicarbonate and base deficit as compared to vehicle-treated animals. This indicates that therapy with T0901317 improved end organ perfusion and abrogated cellular hypoxia, leading to less metabolic acidosis. However, the role of LXRα activation in glucose utilization cannot be ignored. Several studies have shown that activation of LXRα leads to increased pancreatic insulin secretion, decreased hepatic gluconeogensis and improved peripheral glucose uptake (7
). This improved glucose utilization could lead to improved cellular metabolism and improvement in metabolic parameters of acidosis, especially lactate. In our study, we monitored glucose levels during hemorrhage and resuscitation and found elevated plasma glucose levels after hemorrhage, with reduction of these levels to baseline 180 minutes after resuscitation. However, there was no significant difference in these plasma glucose levels at any time point in rats treated with T0901317 versus
vehicle-treated rats (data not shown). This implies that LXRα activation did not significantly change peripheral glucose utilization and the improvement in lactate was indeed a reflection of reduced cellular hypoxia in those rats treated with T0901317.
Decreased concentrations of total cholesterol occur early in course of critical illness, including major trauma and hemorrhage. Low cholesterol levels correlate with the severity of the systemic inflammatory response and high concentrations of pro-inflammatory cytokines (29
). Since LXRα is a master regulator of cholesterol synthesis, transport and catabolism (31
), we also assessed plasma total cholesterol levels in rats subjected to severe hemorrhage and resuscitation. In contrast to clinical data, which report occurrence of hypocholesterolemia in critically ill patients, we observed that there was an increase of total cholesterol after hemorrhage and resuscitation as compared to basal levels of sham control rats. However, we found no significant impact on total cholesterol levels as a result of treatment with T0901317. This suggests that the anti-inflammatory effects of T0901317 are independent of its cholesterol regulating properties. The increase in total plasma cholesterol seen after hemorrhage and resuscitation in vehicle-treated rats is likely a stress response to severe hemorrhage, aimed at increasing the pre-cursors of cortisol biosynthesis. In support of this hypothesis, a previous study by Daull et al (32
) showed a similar elevation of plasma cholesterol during the immediate resuscitative period following autologous blood transfusion amongst rats subjected to hemorrhage and resuscitation. Furthermore, a study by Abarca et al. (33
) also showed an increase in plasma cholesterol levels of rats during the early phase of reversible circulatory shock induced by endotoxin injection.
Hemorrhage and resuscitation induces an inflammatory response that includes the infiltration of neutrophils into tissue (2
). This infiltration is followed by the release of reactive oxygen species and subsequent tissue damage, which can ultimately lead to acute lung injury and even death (34
). Reduction of leukocyte adherence and infiltration into lung tissue has been found to improve survival after severe hemorrhage and resuscitation (36
). In our study, we have shown a reduction in hemorrhage-induced neutrophil infiltration and lung injury as assessed by both histology and myeloperoxidase assay. Multiple studies have previously shown LXRα activation to reduce lung injury and neutrophil infiltration following infectious, LPS, or chemical insult to rodent lung (15
). Our study is the first report that LXRα activation affords lung protection following hemorrhagic shock. Thus, our findings suggest that LXRα activation not only limits inflammation in lung-specific models, but also exerts pulmonary protective properties during sterile non-infectious systemic inflammation.
Pro-inflammatory cytokine and chemokine release is a hallmark of the inflammatory response after hemorrhage and resuscitation (2
). This elevation leads to a systemic inflammatory response and organ dysfunction, including acute lung injury (34
). Reduction of cytokine levels are therefore a therapeutic goal aimed at reduction of organ injury, especially pulmonary injury. Several previous studies have shown that LXRα activation reduces levels of these pro-inflammatory cytokines and chemokines after LPS or irritant insult both in vivo and in vitro. A recent study by Crisafulli et al. also shows the same reduction after splanchnic ischemia and reperfusion (11
). Our study confirms these results in a model of rodent hemorrhagic shock, with significant reductions in plasma levels of MCP-1, MIP-1α, TNF-α and KC. High levels of TNF-α have been associated with increased acute lung injury (34
). MCP-1, MIP-1α and KC are chemokines that induce chemotaxis of inflammatory cells (40
). In our study, treatment with T0901317 induced a significant decrease of TNFα and several chemokines, in particular KC, which correlates with the observed reduction of lung neutrophilia and injury. Therefore, we hypothesize that LXRα activation abrogates neutrophil infiltration into the lung and subsequently limits parenchymal damage following hemorrhagic shock by limiting the production of crucial pro-inflammatory cytokines and chemokines.
NF-κB is a nuclear transcription factor that regulates the expression of a multitude of pro-inflammatory genes and has been implicated as a factor in the development of acute lung injury (17
). Amongst the pro-inflammatory cytokines up-regulated by NF-κB activation are TNF-α, KC, MIP-1α, MCP-1 and IL-6. In a previous study, we demonstrated that LXRα activation inhibits the LPS-induced inflammatory response of murine macrophages by inhibiting the NF-κB pathway (19
). Recent in vivo models of spinal cord trauma and splanchnic ischemia and reperfusion have also shown a reduction in NF-κB activation after treatment with a synthetic LXRα agonist (39
). In our current model of hemorrhagic shock and resuscitation, we have demonstrated that treatment with T0901317 inhibits the early onset (i.e. at 60 minutes post-resuscitation) of NF-κB binding to DNA in the lung. The exact mechanism by which LXRα inhibits NF-κB activation remains unclear and appears complex. Activated LXRα seems to inhibit NF-κB/DNA binding while also inhibiting the transactivation of NF-κB already bound to DNA. For example, earlier in vitro studies have shown that activation of the NF-κB pathway in LPS-challenged macrophages is inhibited by LXRα activation, but failed to show a decrease in NF-κB binding to DNA (10
). Ghisletti et al. support this finding in their study, showing that LXRα activation of macrophages prevents the clearance of co-repressors in a SUMOylation dependent manner, thus preventing the transcription of several pro-inflammatory genes (45
). This trans-repression would decrease the transcriptional activity of NF-κB without altering its binding profile. However, in vivo studies of splanchnic ischemia and reperfusion did show a reduction of NF-κB binding to DNA, similar to the findings we report in this study (39
). This suggests that LXRα does block NF-κB binding to DNA and subsequent transcription of pro-inflammatory genes in in vivo models or in models of ischemia and reperfusion. However, the exact mechanism remains unclear and further investigation is needed to define the precise role activated LXRα plays in inhibition of the NF-κB pathway in vivo.
In conclusion, we have shown that activation of LXRα with the synthetic agonist T0901317 improves the hemodynamic profile and reduces lung inflammation after hemorrhage and resuscitation in a rodent model. The reduction of inflammation is consistent with inhibition of NF-κB binding during the early phase of shock. Further studies using inhibitors of LXRα, such as GSK 2033 (46
), or transgenic mice deprived of the functional gene for LXRα (14
) will establish the precise mechanisms of LXRα and NF-κB interaction.