Leptin-mediated protection of ischemic brain: regulation of neutrophil and neuron.

• Main Authors: Wan-Ting Hung, 洪婉婷
• Other Authors: Chi-Mei Hsueh
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/06293573008558701176

碩士 === 國立中興大學 === 生命科學系所 === 97 === Abstract Leptin is a 16 kDa protein encoded by the obese (ob) gene, synthesized primarily in adipose tissue. It has difference function though binding of Leptin receptor on surrounding and brain tissues. Leptin is involved in regulating feeding, energy metabolism, neuroendicrine and immune function. There is evidence that Leptin can protect intestine from ischemia-induced tissue damage. Reports regarding Leptin-mediated protection of ischemic brain are rare and the mechanism(s) involved are unclear yet. The primary goal of the thesis was therefore to assess the protective mechanism(s) of Leptin in cerebral ischemia. We particularly focus on the effects of Leptin on neurons and neutrophils of the cerebral ischemic rats. In the study, primary neurons isolated from cortex of embryo rat (E18) were exposed to glucose-oxygen-serum deprivation (GOSD) stress, followed by analysis protein expression of Leptin and its receptor (ObR) at various time. Additional Leptin was also added to GOSD neuron to assess its protection of GOSD neuron and effect on STAT3 phosphorylation. In the meantime, a reversible focal ischemia was developed in S.D. rats by subjecting animals to 90min-CCAO plus-MCAO followed by reperfusion for various time. Effects of Leptin on brain infarct and neutrophils in brain and periphery were then evaluated in this ischemic animal model. Our results showed that Leptin can protect ischemic brain by increasing survival of neurons, restoring neurological deficit and preventing brain entrance of neutrophils. Leptin expression levels were increased at brain infarct area and within GOSD neurons. The expression of ObR was also increased in GOSD neuron. Leptin (2uM) supplement enhance the phosphorylation of STAT3 and attenuated GOSD induced neuronal death in vitro. Administration of 15 ug/kg Leptin into the ischemic brain not only reduced the infarct volume, partially restored the neurological function but also decreased the neutrophil infiltration at the infarct area. The Leptin effects on splenic neutrophils were further exammed directly by co-culturing the splenic neutrophil with leptin in vitro or indirectly by viewing the activity changes of splenic neutrophil isolated from ischemic rats with brain injected Leptin. Results showed that brain injection of Leptin not just prevent the brain entrance of neutrophil but also stimulated the phagocytic activity and mobility of splenic neutrophil. These observation indicate leptin may trigger other mechanisms (such as decrease the BBB permeability to prevent the brain infiltration of neutrophil). The release of NO from splenic neutrophils however, were not influenced by Leptin. Co-culturing neutrophil with Leptin in vitro suppressed the mobility of and NO release capability of neutrophil whereas increased the survival of splenic neutrophil in vitro. In overall, Leptin can protect the ischemic brain by promoting the survival of neurons and phagocytotic activity of splenic neutrophil, but preventing the brain entrances and pro-inflammatory activity of neutrophil. These results can further strengthen our knowledge about leptin-mediated brain protection against cerebral ischemia. In addition, leptin supplementation or temporary neutrophil depletion may be therapeutic useful in the future treatment of this disease.