| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 104 === Cerebral ischemia can lead to severe brain damage or even death of the patients. Ischemia/reperfusion (I/R)-induced reactive oxygen species (ROS) and brain''s low antioxidant activity all contribute to the increased oxidative stress in ischemic brain that consequently can lead to severe brain injury. How to down regulate the oxidative stress in ischemic brain has continuously been a hot issue in the control of cerebral ischemia. It is known that brain astrocytes have the highest anti-oxidative activity compared to other brain cell types and ALDH3A1 (aldehyde dehydrogenase 3A1 ) in mammalian corneal epithelium plays critical and multifaceted roles in protecting the corneal from oxidative stress. The primary goals of this thesis study were to know whether ALDH3A1 also plays similar roles in protecting astrocytes from ischemia-increased oxidative stress and could the Chinese herb extract from Graptopetalum paraguayense (GP) leafs, ZC008, up-regulate ALDH3A1 activity to promote the protection of ischemic astrocytes against oxidative stress. In the study, the trypan blue dye exclusion assay, Western blotting, zymography assay, DCF (dichlorofluorescein) assay, OPA (o-phthaldialdehyde) assay, GR (glutathione reductase) assay and TBARS (TBA-reactive substances) assay, were respectively used to determine the survival, protein expression and activity of ALDH3A1, intracellular and extracellular level of ROS, the amount of intracellular NADPH, GSH and MDA of the ischemic astrocytes (under the glucose, oxygen and serum deprivation or GOSD condition) , in the absence or presence of DEAB (diethylaminobenzaldehyde; ALDH3A1 inhibitor) or ZC008. The results showed that under the GOSD (6 h) stress, astrocytes have the highest survival rate and ALDH3A1 activity and lowest amount of intracellular and extracellular ROS, compared to microglia and neurons, indicating GOSD-treated astrocytes indeed have the highest anti-oxidative potential among the three brain cell types. When DEAB was added into the GOSD-treated astrocytes, the protein expression level and activity of ALDH3A1and the survival rate of astrocytes were all significantly decreased whereas the intracellular ROS was increased, indicating GOSD-triggered ALDH3A1 is critical for the protection of astrocytes against GOSD-induced oxidative stress. It is known that ALDH3A1 can elevate NADPH and GSH to down regulate ROS level and oxidative rancidity of fat to protect corneal epithelium from oxidative stress. Whether ALDH3A1can act the same in GOSD-treated astrocytes was also investigated. The results showed that intracellular levels of NADPH, GSH and MDA (malondialdehyde; the product of oxidative rancidity of fat) were all significantly decreased in GOSD-treated astrocytes, indicating GOSD-increased oxidative stress is likely due to the reduction of NADPH and GSH but did not cause any oxidative rancidity of fat. DEAB blocking assay further demonstrated that GOSD-triggered ALDH3A1 did not play a part in up-regulating the amount of NADPH and GSH nor in oxidative rancidity of fat in GOSD astrocytes, suggesting that ALDH3A1-decreased intracellular ROS was NADPH and GSH independent. Results from our early study have demonstrated that ZC008 can protect brain from ischemic injury. The underlying mechanisms responsible for ZC008-mediated brain protein however, remain still unknown. The impacts of ZC008 upon ALDH3A1 and the protection of GOSD-treated astrocytes against oxidative stress were therefore examined. The results showed that ZC008 (at medium dose) can further increase the protein expression and activity of ALDH3A1, intracellular ROS (not cytotoxic), NADPH, GSH and the survival rate of GOSD astrocytes but significantly decreased the extracellular ROS level and showed no influence on MDA. ZC008-activated ALDH3A1 appeared to play critical roles in up-regulating GSH (not NADPH) and down regulating intracellular and extracellular ROS to protect astrocytes from GOSD-induced oxidative stress. Neither ZC008 nor ALDH3A1 involved in the oxidative rancidity of fat in GOSD-treated astrocytes. In overall, the role of ALDH3A1 in self- and ZC008-mediated protection of astrocytes against ischemia (GOSD)-induced oxidative stress has been clarified for the first time in an in vitro ischemic model. ZC008-activated ALDH3A1 can effectively down regulate the intracellular and extracellular ROS, in a GSH-dependent manner, to protect astrocytes against GOSD-induced oxidative stress. Through the study, the therapeutic value of ZC008 in the control of cerebral ischemia can be further confirmed mechanistically.
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