| Background and Objectives:Cardiac surgery with cardiopulmonary bypass for neonates with complex congenital heart diseases is occasionally followed by post-operative complications that may contribute to post-operative mortality, such as low cardiac output syndromes. Both cardiopulmonary and surgical operations per se are trauma to the heart, and may also induce a systemic inflammatory response. Moreover, cardiopulmonary bypass is related to sera endotoxin elevation during surgery. Endotoxin can cause pro-inflammatory cytokines secretion in the neonatal mouse blood, such as TNF-a and IL-6. Furthermore, the increase of circulating cytokine levels was closely correlated with impaired cardiac function.Accumulating in vivo and in vitro evidences have demonstrated that, LXR is important modulator in innate immunity and inflammatory gene expression. LXRs were expressed in murine heart, and exert protective effects according to published studies. When perfused in Langendorff model, heart of myocardial ischemia mice pretreated by GW3965 showed less post-ischemic increase of left ventricular end diastolic pressure and post-ischemic depression of left ventricular developed pressure. In cell culture experiments, LXRs activation exerts anti-hypertrophic effect in cardiomyocytes through inhibiting NF-?B signaling pathway in response to LPS or angiotension-? challenge.Autophagy is considered as a highly conserved process that is responsible for degradation and recycling of organelles and long-lived proteins. It has been reported that autophagy was involved in the modulation of pathogenesis in the cardiac disorders, for example, myocardial ischemia. Importantly, autophagy exerts protective effect on myocardium during the inflammatory status initiated by ischemia. Moreover, LPS was reported to stimulate autophagic activity in neonatal rat cardiomyocytes, which was associated with cytoprotection via inhibiting apoptosis. Our group previously demonstrated that the activation of endogenous LXRs by LXR agonist, TO901317, resulted in the inhibition of ethanol-induced microglial activation, thus protecting Purkinje cells from apoptosis in the neonatal mice. Whether the role of LXR on inflammation induced myocardial injury is related to the induction of cardiac autophagy by LPS in neonatal mice needs to be explored.Our present study can be divided into two parts:(1) neonatal mice with TO901317 pretreatment were followed by acute LPS exposure to evaluate the anti-inflammatory effects of TO901317; (2) and to evaluate its effects on autophagy and apoptosis in hearts from microstructure, ultrastructure, and protein levels. Meanwhile, we further explored the potential mechanism underlies in the association of TO901317 and cardiac inflammation, autophagy and apoptosis.Methods:1. TO901317 and neonatal mouse myocardial inflammation:(1) Building the LPS induced systemic inflammatory model and TO pretreatment model in neonatal mice; (2) HE staining and counting for inflammatory cells in neonatal mice myocardium; (3) Detection for IL-6 and TNF-a mRNA expression in myocardium by quantative RT-PCR; (4) Western Blot analyses of NF-kB p65 and phosphorylated NF-kB p65, IkB-(x and phosphorylated NF-kB p65 protein levels.2. TO901317 and neonatal mouse myocardial autophagy and apoptosis:(1) Detection of LC3 expression by Western Blot; (2) Immunofluorescence labeling for LC3-anchofed autophagosomes; (3) Transmission Electron Microscopic observation for autophagosomes; (4) Apoptosis detection in neonatal myocardium by TUNEL assay; (5) Akt phosphorylation level evaluation by Western Blot.Results:1. Single high dose LPS intraperitoneal injection can lead to myocardial inflammation in neonatal mice, characterized by infiltration of inflammatory cell infiltration, and proinflammatory cytokines release presented as TNF-aand IL-6 mRNA elevation in neonatal mice myocardium (elevated by 2.26 and 1.48 folds, p< 0.01, respectively.).2. After Single high dose LPS intraperitoneal injection, myocardium of neonatal mice pretreated by TO901317 showed less inflammatory cell infiltration, and lower TNF-a (decreased by 40%, p<0.01)and IL-6 (decreased by 36%, p<0.05) mRNA expression level, when compared to mice with only LPS injection.3. The LPS induced NF-?B p65 phosphorylation was decreased by 56.5%(p< 0.01). Similarily, IkB-? degradation in neonatal mice myocardium can also be attenuated by TO pretreatment (decreased by 28.2%, p< 0.01). Suggesting the involvement of NF-?B signaling pathway in the attenuation of inflammation by TO.4. During the acute phase of LPS challenge, neonatal mice myocardium showed higher LC3-II/GAPDH ratio (increased by 3.32-folds, p< 0.01). Quantification of immunofluorescence labeling revealed 5.34-folds (p< 0.01) more LC3 puncta were presented in LPS stimulated mice myocardium.5. After Single high dose LPS intraperitoneal injection, myocardium of neonatal mice pretreated by TO901317 showed enhanced LC3 expression (increased by 1.15-folds, p< 0.01) and increased LC3 puncta (by 0.68-fold, p< 0.05) than myocardium of mice with LPS treatment alone. Transmission Electron Microscopy visioned both autophagosome and autolysome in the same field, further demonstrate the occurrence of autophagic activation.6. Apoptotic body was showed under Transmission Electron Microscopy in myocardium of neonatal mice treated only by LPS. TUNEL-POD immunohistological staining showed apoptotic cardiac cells in myocardium of mice treated by LPS. Statistical analysis of TUNEL-positive cells count demonstrated a decrease (by 31%, p< 0.05) in number in TO901317 pretreated mice myocardium after LPS challenge, suggesting the anti-apoptotic effect of TO901317.7. Results from immunolabeling indicated that, LPS challenge decrease the Akt phosphorylation level (by 37%, p< 0.01) in neonatal mice myocardium. However, TO pretreatment can further reduce the decrement of Akt phosphorylation level (by 27%, p< 0.05) in neonatal mice myocardium.Conclusions:1. LXR agonist TO901317 attenuates myocardial inflammation via inhibition of NF-kB signaling pathway in neonatal mice challenged by LPS.2. LXR agonist TO901317 enhances LPS induced autophagic activity in neonatal mice myocardium. Moreover, it attenuates myocardial apoptosis in neonatal mice with LPS injection.3. The anti-inflammatory effect, pro-autophagic effect and anti-apoptotic effect can be explained by the change of Akt phosphorylation level. However, the detailed mechanisms need further investigations. |
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