| Summary: | 博士 === 慈濟大學 === 藥理暨毒理學碩士班/博士班 === 101 === Background and purposes
Caring for patients with severe sepsis is costly and must confront great risk. Because the complete mechanism of severe sepsis is still vague, and the mortality rate of sepsis is very high even various treatment strategies were developed. Lipopolysaccharide (LPS) triggers innate immunity mainly via TLR 4 signaling causing sepsis, thus LPS induced sepsis can be used as an animal model. ATF 3 is a negative regulator of TLR 4 signaling. HMGB1 plays a critical role in the final step of sepsis. However, the mechanisms of ATF 3 and the role of HMGB1 in regulating innate immunity-induced sepsis are incompletely understood. We try to clarify the role of ATF3 and HMGB1 in endotoxemic TLR4 pathway to advance the understanding of human sepsis.
Methods and materials
Eight to ten-week-old male B6 mice and ATF3 knockout mice were divided randomly into several groups. Control groups were administered with saline, and experimental groups were administered with 5 mg/kg of LPS intraperitoneally (i.p.) at different time points to induce endotoxemia. Another ATF3 knockout mice were pretreated with AAV-ATF3 to replenish ATF3 expression before LPS treatment. Blood sample and organs were collected at 0,3,6,12,24 hours post-treatment. Mortality rate was calculated at 24 hours. Blood samples (0.5 ml) were centrifuged and frozen at -80°C for subsequent assay. The liver and lung tissues were embedded in paraffin and studied using immunofluorescence and immunoblotting of cytosolic inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), HMGB1 and ATF3 proteins. RAW264.7 cells (a mouse macrophage cell line) were treated with LPS (200 ng/ml), 24 hours later culture median and cells were harvested for further assay.
Results
We found that serum HMGB1 levels were 10-fold higher in patients with sepsis than normal controls. We further demonstrated that ATF 3 gene knockout in mice subjected to LPS-induced endotoxemia correlates with an increase in the mortality rate and the elevated expression of IL-6, TNF-α, NO, MCP-1 and HMGB1 in the lung tissues or serum. The biochemical effects of ATF3 were observed in in vitro macrophages, and blocked by ATF3 siRNA treatment. We have also shown that adeno-associated virus mediated ATF3 gene transfer protected ATF3 knockout mice from LPS-induced mortality and lung injury. In addition, ATF3 knock down increased LPS-induced release of HMGB1with aggravation of the tissue injury on sepsis.
Conclusion
In conclusion, the existence of ATF3 can offer protection against mortality and organs dysfunction in endotoxemia. Upregulation of ATF3 not only contributes to the reduced release of inflammatory molecules, especially HMGB1 which induced lung injury, but also increased the survival rate of mice after LPS challenge. Therefore, suppressing LPS-induced inflammation with ATF3 induction or ATF3 mimetics may be an important strategy for sepsis therapy.
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