The pathophysiological role of autophagy in microglia under the in vitro ischemic condition

Autor: Kang-Yun Lu, 陸康芸
Rok vydání: 2014
Druh dokumentu: 學位論文 ; thesis
Popis: 102
The primary goal of the study was to investigate the pathophysiological roles of autophagy in microglia under the in vitro ischemic condition (glucose-, oxygen-, serum-deprivation or GOSD). Microglia are brain phagocytes and also play a critical role in triggering the secondary brain injury (inflammation) after cerebral ischemia. It is known that autophagy involves the cooperation of autophagosome and lysosome to clean up the excessive proteins, organelles or many other harmful intracellular substances. Autophagy not only plays a role in maintaining the metabolic homeostasis it also regulates the occurrence of apoptosis in different ways. The impact of ischemic stress upon autophagy and the pathophysiological roles of autophagy in ischemic microglia have never been reported before therefore become the major research interests of this thesis. The major concerns of the thesis were to know: 1) the impact of GOSD upon the occurrence of autophagy, inflammatory activities and apoptosis of microglia； 2) would autophagy play a key role in the regulation of apoptosis and inflammatory potential of GOSD-treated microglia； 3) how GOSD regulate the protein expression of leptin and PPARγ (peroxidome proliferator-activated receptor gamma) of microglia and what is the mutual relationship between these two inflammatory adipokines； 4) how leptin and PPARγ respectively regulates the occurrence of autophagy, inflammatory activities and apoptosis of GOSD-treated microglia. Briefly, microglia were isolated from the cortex of newborn (P1) Sprague-Dawley rats, then subjected to the in vitro ischemic condition (GOSD) for times as indicated, in the absence or presence of individual drugs (including autophagy inhibitor 3-MA, leptin and PPARγ agonist (Rosi). At the end of each treatment, the survival rate, occurrence of autophagy (increase of LC3II/LC3I ratio), apoptosis (increase of caspase-3 expression and PS externalization) and the inflammatory potential (the release of ROS, NO, PGE2 and the expression of COX-2 and TNF-α) of GOSD-treated microglia were determined by using respective assays. The techniques applied include, the primary culture technique, trypan blue dye exclusion assay, Annexin V/PI assay, DAPI assay, western blot analysis, DCF assay, nitrite release assay, and ELISA. The current results showed that GOSD can injure microglia and stimulate the autophagy, apoptosis and inflammatory activities (increased release of ROS and PGE2) of microglia. GOSD also stimulated the protein expression of leptin but inhibited that of PPARγ, both factors antagonized each other''s expression in GOSD-treated microglia. Autophagy blocking strategy using 3-MA, further demonstrated that GOSD-increased autophagy can protect microglia from GOSD-induced injury, inhibit the apoptosis of microglia, help to remove part of the inflammatory mediators (COX-2 and TNF-α) but stimulate the oxidative activities (ROS and NO) of microglia. Leptin supplements inhibited the occurrence of autophagy and oxidative potential (ROS and NO release) of GOSD-treated microglia, increased the intracellular levels of COX-2 and TNF-α and the occurrence of apoptosis, but in the end still protected microglia against GOSD. PPARγ increased by Rosi on the other hand, inhibited the autophagy, apoptosis, the expression levels of COX-2 and oxidative potential (ROS and NO) of GOSD-treated microglia and only the intracellular TNF-α was up. In overall, GOSD-induced autophagy had revealed multiple functions in microglia but eventually it favored the survival of microglia under the ischemic stress. The pathophysiological roles of autophagy included, apoptosis inhibition, removal of inflammatory proteins, induction of oxidative stress, and the protection of survival of microglia. It is known that over activation of microglia can lead to tissue inflammation and further exacerbate the brain injury caused by cerebral ischemia. Since blocking of autophagy by 3-MA or PPARγ (not leptin) can further decease the survival of GOSD-treated microglia that consequently suggest a potential value of 3-MA and PPARγ in the prevention of brain inflammation (infarction) caused by cerebral ischemia. Nevertheless, it is worthy of note that not all the autophagy inhibitors can block the survival of microglia, such as leptin, which in fact favored the survival of GOSD-treated microglia likely due to other protective mechanisms independent on autophagy.
Databáze: Networked Digital Library of Theses & Dissertations
Externí odkaz: