| Leptin is a secretory polypeptide hormone, which performe effects via acting on specific receptors distribution in central nervous system (CNS). Our previous studies have demonstrated that Leptin is affinitive with improving of the brain infarct locus. However, the mechanisms by which Leptin mediates its neuroprotective activities are not clear. It is well known that the damage mechanisms of cerebral infarction were irreversible neuron damage and death which were induced by over production of oxygen radicals, excitatory neurotransmitter, [Ca2-]i et al. Due to the damage of neuron is irreversible, we hypothesis that other types of nerve cells may be involved in the protective effects of Leptin against cerebral infarction.Gap junction channel is an important manner in direct cell-to-cell communication, which allows the exchange of nutrition, metabolite, ions and small molecules, and subsequently performes important regulation of cellular pathophysiologic changes. One of the most abundant connexin in the central nervous system (CNS) is connexin43 (Cx43) which are considered performing notable effects on injury. Cx43 mainly express in astrocytes. Moreover, astrocytes are protective and sustentive cells for neuron. Therefor, we conjecture that astrocyte’s Cx43 expression may be involved in the protective effects of Leptin against cerebral infarction.In this research, brain, organotypic brain slice and cell ischemia/reperfusion (I/R) injury models were performed to exploring the neuroprotective effects of Leptin and its mechanisms. The 3 parts of this research include:Part Ⅰ The neuroprotective effects of Leptin against brain I/R injury, and the roles of Leptin on Cx43 expression in brain; Part Ⅱ Identifying the roles of Leptin on Cx43 and the mechanisms of Leptin’s neuroprotective effects in organotypic brain slice culture; Part III exploring the signalling through which Leptin inhibits ischemia induced Cx43 elevation. The results:1 Leptin significantly improved murine brain I/R injury, and inhibit Cx43 elevation in brain(1) The model of focal cerebral ischemia/reperfusion injury was performed by unilateral middle cerebral artery occlusion (MCAO). The infarct volume and neurological deficit were both improved in a concentration dependent manner by Leptin treatmen;(2) Leptin administration significantly enhanced Bcl-2 expression, but inhibit Caspase-3 expression;(3) Brain I/R injury significantly induced Cx43 elevation in ischemic penumbra. Treatments with Leptin significantly inhibit Cx43 elevation. Moreover, Cx43 expression levels were higher in the brain of ob gene deficit mice than wild type.2 In the level of organotypic brain slice culture, Leptin inhibits ischemia induced Cx43 elevation and subsequent inhibiting [Ca2+]i overload(1) Brain slice culture ischemic injury induced notable Cx43 expression in ob/ob mice than wt mice. Leptin administration significantly inhibit Cx43 elevation which could be reversed by the blocker of Leptin’receptors;(2) Leptin treatment notablely reduced neuronal cell death, [Ca2+]i overload and Calpain-1 over activation;(3) Immunostaining of Cx43 expression located in astrocytes.3 By researches on cells in vitro, we demonstrated that Leptin inhibits ischemia induced Cx43 elevation in astrocytes via ERK1/2 signal pathway(1) Application of the Cx43 siRNA dramaticly decreased the cells death rate after H/R injury(2) Leptin administration reduced the normal cells or H/R-induced increase in Cx43 expression at a concentration dependent manner in astrocytes. Leptin treatment also notablely reduced [Ca2+]i overload and Calpain-1 over activation in astrocytes. Cx43 inhibitor-Cbx has similar roles of Leptin;(3) The neuroprotective effects of Leptin appear to be mediated by upregulated ERK1/2 phosphorylation, with subsequent inhibition of the ischemia induced astrocyte Cx43 elevation and a resultant attenuation of intracellular calcium overload.Above all, our results demonstrated that:(1) Leptin can improve murine brain I/R injury, and performe important neuroprotective effects;(2) Inhibition of I/R injury induced Cx43 elevation in astrocytes may be involved in the neuroprotective effects of Leptin;(3) Leptin treatment inhibits astrocyte Cx43 elevation and a resultant attenuation of intracellular calcium overload;(4) Leptin inhibition I/R induced Cx43 elevation mainly through ERK1/2 signal pathway. |
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