| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 104 === It is known that inflammation leads to the secondary injury of ischemic brain. Adiposomes on the other hand, could elevate the inflammatory potential of adipocytes and macrophages. The primary goal of this thesis study was therefore aimed to know whether ischemic stress can act through the adiposome formation to increase the inflammatory activities of microglia and then further exacerbate the infarct volume of ischemic brain. In the study, the trypan blue exclusion assay, Oil red O stain, immunofluorescence stain, Western blotting, ELISA (Enzyme-linked immunosorbent assay), DCF (dichlorofluorescin) assay and TTC (2,3,5-triphenyl tetrazolium chloride) stain, were respectively used to determine the changes of cell survival, adiposome formation and the protein expression and/or release of inflammatory mediators, in microglia under the GOSD (glucose, oxygen, and serum deprivation) or brain tissue under the ischemia/reperfusion (I/R) stress, with or without adiposome, COX-2 (cyclooxygenase-2) or PPARγ (peroxisome proliferator-activated receptor gamma) inhibitor. Results from the in vitro study showed that GOSD (2 h) treatment can significantly decrease the survival of microglia but increase the adiposome formation, protein expression and/or release of PPARγ (adiposome inducer) and inflammatory mediators including, COX-2, TNF-α (tumor necrosis factor alpha), IL-1β (interleukin-1beta) and ROS (reactive oxygen species). In the presence of NS-398 (adiposome and COX-2 inhibitor), GOSD-increased COX-2, TNF-α, IL-1β, ROS and cell death were all significantly inhibited, revealing that GOSD-increased adiposomes were able to potentiate the inflammatory activity (COX-2, TNF-α, IL-1β, ROS) of microglia and lead to cell death. SC-58125 (COX-2 inhibitor) was also applied in the study to further clarify whether adiposome-mediated impacts were COX-2 dependent. The results showed that GOSD-induced COX-2 expression and ROS release were inhibited but TNF-α and IL-1β expression were further increased by SC-58125, revealing that GOSD-induced COX-2 can potentiate the production of ROS but inhibit that of TNF-α and IL-1β in microglia. The above results suggest that GOSD-induced adiposomes may be a depot of inflammatory mediators and can regulate the release of TNF-α and IL-1β from microglia, in a COX-2-independent manner. In the presence of GW9662 (PPARγ inhibitor), GOSD-induced PPARγ expression and adiposome formation were all inhibited; in the presence of ROSI (PPARγ agonist), GOSD-induced adiposome formation and cell death were further elevated. The results suggest that GOSD-induced PPARγ can stimulate adiposome formation and lead to death of microglia. Results from the in vivo ischemic animal study further showed that in response to I/R stress, microglia-derived adiposome formation, adiposome-associated TNF-α and IL-1β expression and the infarct volume of ischemic brain were all significantly increased. I/R-increased brain infarction was, however, significantly decreased by GW9662 and NS-398, indicating PPARγ blockade and adiposome inhibition are both protective to ischemic brain. In overall, adiposome in ischemic microlia may serve as a depot of inflammatory mediators. Ischemic stress can act through PPARγ to stimulate adiposome formation and potentiate the inflammatory activity of microglia that consequently can lead to cell death, tissue inflammation and brain infarction. Adiposome inhibition by either NS-398 or GW9662 has revealed a therapeutic potential in the future control of ischemic stroke.
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