Experimental Research Of Neuroprotective Effects And Mechanisms Of Leptin On Cerebral Ischemia/reperfusion Injury

Aims:Neuroprotective effects of leptin have been implicated in mouse models of cerebral ischemia/reperfusion injury and primary cortical neurons with oxygen-glucose deprivation(OGD).However,limited data are available regarding signaling pathways underlying these neuroprotective effects.Therefore,we investigated whether leptin modulated mitochondrial function through JAK2/STAT3 in an in vivo mouse model of transient middle cerebral artery occlusion(MCAO)and in OGD-challenged primary neuronal cultures.Main methods:Two hundred C57BL/6J and twenty DB/DB mice at 8-10W were randomly separated into groups.Twenty C57BL/6J mice were used by 2,3,5-triphenyltetrazolium chloride(TTC)and six mice were used to observe the size of cerebral infarction and the degree of cerebral edema by magnetic resonance imaging.JAK2/STAT3;mitochondrial biogenesis markers(PGC-1?);and apoptosis-associated proteins(caspase-3,BCL-2,BCL-XL,and cytochrome c)were detected by western blotting and reverse transcription-polymerase chain reaction at 1 h before and after ischemia/reperfusion by seventy-two C57BL/6J and twenty DB/DB mice.P-STAT3 and PGC-1? in neurons and astrocytes were detected by six C57BL/6J mice.Moreover,mitochondrial morphology of the ischemic ipsilateral penumbra was examined using transmission electron microscopy from eight C57BL/6J mice.Primary cerebral cortical neurons were evaluated for viability,mitochondrial membrane potential(MMP),and apoptosis to assess whether dose-dependent neuroprotective effects of leptin during OGD were mitigated by the JAK2/STAT3 inhibitor AG490.Key findings:1.Leptin preconditioning can activate OB-Rb and reduce the area of cerebellar infarction.Immunofluorescence results showed that OB-Rb was widely distributed in cerebral ischemic penumbra and primary cortical neurons.Compared with the saline group,leptin preconditioning reduced the infarct size(The area of leptin group vs sailine group;43.75%± 6.054%vs.21.7%±1.656%,P<0.0001).2.Leptin pretreatment activated the JAK2/STAT3/PGC-la pathway,and P-STAT3 peaked at 1 h of reperfusion.Western blot showed that p-stat3 protein expression increased with the duration of reperfusion and peak P-STAT3 levels observed at 1 h after reperfusion.(P<0.0001,Sham vs 1 h or 24 h).3.Leptin pretreatment can synergistically increase the expression of P-STAT3,PGC-la,Bcl-2 and Bcl-xl proteins.Compared with the Sham group,the protein levels of P-STAT3,PGC-1? were higher in the leptin group(P<0.05;in Fig.3-3C)and Bcl-2,Bcl-xl were higher than the saline group(P<0.05).But there was no statistical difference compared with AG490 group.The expression of CYCS protein in cytoplasm and mitochondria of cerebral ischemia penumbra cortex showed that the expression level of CYCS protein in cytoplasm of leptin group was the lowest(P<0.001;Leptin group vs Saline group),but the expression level of CYCS protein in mitochondria was higher than the saline group and saline+AG490 group(P<0.05;Leptin group vs Saline+AG490 group).The results of RT-PCR also confirmed the above results at the gene level.Compared with heterozygous mice,the expression levels of P-JAK2,PGC-1? and P-STAT3 proteins in homozygous mice were lower(P<0.05).However,the expression levels of Bcl-2 and Bcl-xl proteins were higher(P<0.01),while the expression levels of caspase-3 proteins were lower(P<0.0001).4.Effects of leptin on MPTP.Compared with leptin group,mitochondrial ridges in the saline group were enlarged,swollen and vacuolated.AG490 aggravated ischemia-induced neuronal mitochondrial injury with enlarged,swollen and vacuolated mitochondrial ridges in the leptin+AG490 group.5.P-STAT3 and PGC-1? are expressed in neurons and astrocytes.We observed that P-STAT3 and PGC-la were expressed in both neurons and astrocytes in the leptin group,and the fluorescence intensity was higher than the sham group.However,the fluorescence intensity of P-STAT3 and PGC-1? was decreased in both neurons and astrocytes in the leptin+AG490 group.6.Effects of leptin on cell activity and mitochondrial membrane potential.The optimal pretreatment concentration and reperfusion time of leptin were at 200 ng/mL for 24 h respectively,while the cell activity of neurons was the largest AG490(10 nM)reduced the cell activity of neurons(P<0.001).The results of mitochondrial membrane potential detected by JC-10 showed that the fluorescence intensity was related to the concentration of leptin pretreatment,and the higher the concentration of leptin pretreatment was,the higher the fluorescence intensity of primary cortical neurons was.7.Effects of leptin on apoptosis rate of primary cortical neurons.Leptin preconditioning reduced early apoptotic neurons by 3.2%compared with the saline group.Significance:Our findings suggest that leptin-mediated neuroprotective effects in tMCAO may peak at 1 h to induce the transcription of its target gene PGC-1?,stabilization of MMP,inhibition of MPTP,release of cytochrome c,and apoptosis.