ABSTRACT
To study trafficking of bulk internalized vesicles such as macropinosome and lysosome in live cells, an efficient and convenient assay was established according to the axon turning assay. By injecting indicator or fluorescent dyes through a micropipette with air pressure into cell cultures to create a stable gradient around the micropipette tip, vesicles were indicated and labeled. With live cell imaging, the whole process was recorded. Without wash-out of fluorescent dyes and transferring, this assay is an effective, fast labeling system for bulk internalized vesicles, and can also be combined with imaging system.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects and mechanism of intracellular 4-hydroxynonenal (4-HNE) accumulation on tumor necrosis factor (TNF)-induced hepatotoxicity in alcoholic liver disease (ALD).</p><p><b>METHODS</b>An ALD model was established in male C57BL/6 mice (6-8 weeks old) by feeding an ethanol-containing diet for 5 weeks; mice given regular (non-ethanol) diet served as controls. ALD-related changes in 4-HNE and TNF levels were detected by western blotting. The underlying mechanisms of this molecular effect were examined by pre-treating HepG2 cells with 4-HNE followed by exposure to various concentrations of TNF. Effects on cell death were evaluated by MTT assay. Effects on TNF-mediated upstream factors' expression were detected by ELISA, western blotting, and real-time PCR. Effects on the TNF-induced inhibitor of NF-kB (IkBa) activity (phosphorylation status) and its formation of adducts were detected by western blotting and immunoprecipitation.</p><p><b>RESULTS</b>ALD mice showed increased hepatic 4-HNE and TNF levels, and the increases were associated with extent of liver injury. Cell culture studies revealed that 4-HNE, at non-toxic concentrations, sensitized hepatocytes to TNF killing, which was associated with suppressed NF-kB trans activity. Furthermore, 4-HNE prevented phosphorylation of IkBa without affecting upstream IkB kinase activity. The ALD-enhanced 4-HNE content was found to associated with increased formation of 4-HNE-IkBa adduction for both the 4-HNE - treated hepatocytes in culture and in the livers of ALD mice.</p><p><b>CONCLUSION</b>Alcohol-induced increase in 4-HNE accumulation represents a potent and clinically relevant mechanism of sensitizing hepatocytes to TNF-induced toxicity. These data support the notion that decreasing or eliminating accumulated intracellular 4-HNE can serve as a potential therapeutic option for ALD.</p>