Protective effects of a compound herbal extract (Tong Xin Luo) on free fatty acid induced endothelial injury: Implications of antioxidant system

<p>Abstract</p> <p>Background</p> <p>Tong-Xin-Luo (TXL) – a mixture of herbal extracts, has been used in Chinese medicine with established therapeutic efficacy in patients with coronary artery disease.</p> <p>Methods</p> <p>We investigated the protective role of TXL extracts on endothelial cells injured by a known risk factor – palmitic acid (PA), which is elevated in metabolic syndrome and associated with cardiovascular complications. Human aortic endothelial cells (HAECs) were preconditioned with TXL extracts before exposed to PA for 24 hours.</p> <p>Results</p> <p>We found that PA (0.5 mM) exposure induced 73% apoptosis in endothelial cells. However, when HAECs were preconditioned with ethanol extracted TXL (100 μg/ml), PA induced only 7% of the endothelial cells into apoptosis. Using antibody-based protein microarray, we found that TXL attenuated PA-induced activation of p38-MAPK stress pathway. To investigate the mechanisms involved in TXL's protective effects, we found that TXL reduced PA-induced intracellular oxidative stress. Through AMPK pathway, TXL restored the intracellular antioxidant system, which was depressed by the PA treatment, with an increased expression of thioredoxin and a decreased expression of the thioredoxin interacting protein.</p> <p>Conclusion</p> <p>In summary, our study demonstrates that TXL protects endothelial cells from PA-induced injury. This protection is likely mediated by boosting intracellular antioxidant capacity through AMPK pathway, which may account for the therapeutic efficacy in TXL-mediated cardiovascular protection.</p

Macrophage Replication Screen Identifies a Novel Francisella Hydroperoxide Resistance Protein Involved in Virulence

Francisella tularensis is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance