Emodin Mediates Neuroprotection Through The PTEN/PI3K/AKT Pathway After Cerebral Ischemia-reperfusion Injury Of Rats

Objective:The aim of this study was to study the neuroprotective effects of Emodin middle cerebral artery occlusion(MCAO)with reperfusion in rats,and to investigate the potential role of PTEN/PI3K/AKT signaling pathway in Emodin-mediated neuroprotective effects.Methods:(1)30 healthy adult male SD rats(270±20g)were randomly divided into sham-operated group(6 rats)and MCAO group(24 rats).Left MCAO was applied for 2 h,following by reperfusion for 1 h;.Sham animals were operated as for MCAO rats except that the middle cerebral artery was not occluded.The five-point scale described by Longa et al was used to assess behavioral neurological deficit.Rats with behavioral neurological deficit scores of 2-4 were randomly assigned to four groups,which received vehicle(MCAO group,6 rats),25 mg/kg/d Emodin(MCAO+ LEmodin group,6 rats),50mg/kg/d(MCAO+MEmodin group,6 rats)and 100mg/kg/d(MCAO+ HEmodin group,6 rats)respectively,by gavage,for 1d after MCAO with reperfusion.TTC staining was used to stain the brain of rats a,and the volume of cerebral infarction was analyzed.(2)27 healthy adult male SD rats(270±20g)were randomly divided into sham-operated group(9 rats)and MCAO group(18 rats).Left MCAO was applied for 2 h,following by reperfusion for 1 h.Sham animals were operated as for MCAO rats except that the middle cerebral artery was not occluded.The five-point scale described by Longa et al was used to assess behavioral neurological deficit.Rats with behavioral neurological deficit scores of 2-4 were randomly assigned to two groups,which received vehicle(MCAO group,9 rats),50 mg/kg/d Emodin(MCAO+ Emodin group,9 rats),respectively,by gavage,for 1d after MCAO with reperfusion.Nissl staining was used to stain the Nissl bodies in brain of rats.The immunofluorescence method was used to stain NeuN?C3?GFAP in brain of rats.RT-qPCR was used to detect the mRNA expression levels of IL-1? and TNF-? in the ischemic side of rats.The protein levels of PTEN?p-AKT,AKT,E-selectin and GFAP in ischemic was analyzed by Western blot.Neutrophils were stained with(NAPHTHOL AS-D CHLOROACETATE ESTERASE AND a-NAPHTHYL ACETATR ESTERASE)in brain sections.(3)18 healthy adult male SD rats(270±20g)were randomly divided into two groups,(9 rats per group).One group was intracerebroventricularly injected with artificial cerebrospinal fluid,another group was injected with the PI3K inhibitor LY294002.6 rats from each group were randomly selected and subjected to MCAO/reperfusion operation,the remaining animals were subjected to sham operation.3 animals from each group subsequently administrated with Emodin,by gavage,for Id after MCAO with reperfusion.The protein levels of p-AKT.AKT,p65,Bcl-xl,Bax were measured by Western blotting in the ischemic brain tissue of rats.RT-qPCR was used to detect the mRNA levels of IL-1? in ischemic brain.Results:(1)TTC results showed that 50 and 100 mg/kg/d emodin reduced the infarct volume,but 25mg/kg/d emodin didn't significantly reduce the infract volume,compared to the MCAO group.Nissl staining results showed,Nissl positive cells in the MCAO group was decreased,compared with the sham model group;and the number of Nissl positive cells were significantly increased by Emodin.NeuN staining results showed a decrease in NeuN positive cells in MCAO group,compared with sham group,and this decrease was reversed by Emodin.The mRNA expression of TNF-? and IL-1? were significantly increased in the MCAO group,compared with the sham group,and these increases were reversed by Emodin.(2)Compared with the sham group,the levels of PTEN,E-selectin were increased in the MCAO group,and emodin reversed these decreases.At the same time,the ratio of p-AKT/AKT was decreased after MCAO/reperfusion,and Emodin increased the ratio of p-AKT/AKT.The immunofluorescence staining results showed that the expression of C3 and GFAP was significantly increased after MCAO/reperfusion injury,compared with the sham group;,and these increases were reversed by Emodin.was significantly reduced compared with the model group.The neutrophil staining results showed that neutrophils were significantly increased in the lesion area after MCAO/reperfusion,and Emodin reduced neutrophils in the lesion area.(3)PI3K inhibitor Ly294002 reversed Emodin-mediated increase of the ratio of p-AKT/AKT.Compared to the sham group,the levels of nuclear PTEN,NF-?B p65,Bax and IL-1? were increased,whereas Bcl-xl was decreased after MCAO/reperfusion.These changes were reversed by Eomdin.Although LY294002 didn't affect levels of these proteins,compared to the MCAO/reperfusion group,Ly294002 reversed the effects of Emodin on the ratio of p-AKT/AKT,NF-?B p65 and Bax as well as Bcl-xl in ischemic brain.Conclusion:(1)Emodin not only effectively reduces the cerebral infarction volume of MCAO reperfusion rats,but also decreases neuronal cell apoptosis and inhibits inflammation.Thus Emodin plays a critical role neuroprotection after MCAO/reperfusion injury of the rats.(2)Inhibition of apoptosis,complement and inflammation by Emodin at least partially depends on the regulation of the PTEN/PI3K/AKT signaling pathway.