The Anti-apoptosis Effects Of ST2-104 Peptide On Neurons And Its Mechanism After Cerebral Ischemia In Rats

Cerebral ischemia,an acute cerebrovascular disease,is mainly manifested as brain tissue damage and neurological deficits.It is one of the main fatal diseases in the world with high morbidity,mortality,disability and recurrence.The pathological process of cerebral ischemia is very complicated,and apoptosis is the main form of damage.Studies have shown that within a few hours to a few days after the occurrence of ischemia,there is a large amount of apoptosis cells in the ischemic area,especially the penumbra area.Effective anti-apoptotic measures given within a period of time after cerebral ischemia have the potential protective effects.Autophagy is an important catabolic mechanism of eukaryotic cells,which is essential for cell homeostasis.Studies have found that autophagy is involved in cell development,aging,starvation,inflammation,oxidative stress,programmed death,and immune response.Apoptosis is related to autophagy and they have many common regulatory points.Studies have found that autophagy can be activated after cerebral ischemia,destroy the internal homeostasis of cells,disrupt normal cell metabolism,promote cell death through apoptosis-dependent pathways,and aggravate brain tissue damage.Therefore,regulating autophagy and inhibiting apoptosis may be a new target for the treatment of cerebral ischemia.In recent years,many studies have shown that there is a close relationship between autophagy and Ca²⁺.As an important second messenger in cells,Ca²⁺plays a very critical role in signal pathway.CaMKKβis closely related to the concentration of[Ca²⁺]i.Under normal circumstances,the[Ca²⁺]i concentration is maintained at a low level.Under stress,the[Ca²⁺]i concentration rises rapidly,then activate CaMKKβ.CaMKKβcan activate CaMKI,CaMKIV and AMP-activated protein kinase（AMPK）.Activated AMPK can inhibit mTOR activity and activate ULK1 and finally induce autophagy.The CaMKKβinhibitor STO-609 can inhibit the activity of CaMKKβ,thereby inhibiting the downstream AMPK/mTOR signaling pathway.Studies have shown that the expression of CaMKKβin brain tissuesignificantly increased during cerebral ischemia.ST2-104 peptide is a peptide derived from CRMP2 with a penetrating structure.It is a new type of polypeptide fused with a penetrating peptide with nine arginine structures.It can pass through the blood brain barrier,reach the brain tissue and antagonize CRMP2,then bind to the N-methyl-D aspartate receptor,and inhibitthe excessive activation of the NMDAR.Studies have shown that ST2-104 peptide plays an active protective role in Alzheimer’s disease,brain trauma and other diseases.However,there are few reports on the effect of ST2-104 peptide on cerebral ischemia injury and its mechanism.Methods:The MCAO rats were established by the thread plug method.The rats were divided into Sham group,MCAO group,L-ST2-104 group and H-ST2-104 group,the neurological deficit of the rats was examined by the Longa score method,and the cerebral infarction area was determined by TTC staining.The ischemic brain tissues were taken,and the expression levels of proteins were determined by WB.The SH-SY5Y cell injury model induced by Glu was established.The cells were divided into Con group,Glu group,ST2-104 group and Glu+ST2-104 group.The autophagy agonist rapamycin,the autophagy inhibitor 3-MA and CaMKKβinhibitor STO-609were used for intervention,cell viability was detected by MTT method,autophagy was detected by MDC staining,apoptosis was detected by Hochest 33258 staining,concentration of intracellular Ca²⁺was detected by Flow Cytometry and WB method was used to detect the expression levels of all proteins.Results:Compared with the Sham group,the MCAO group significantly increased the Longa score（P<0.01）and the infarct volumes of rats（P<0.01）;compared with the MCAO group,the H-ST2-104 group significantly decreased the score（P<0.01）and reduced the infarct volumes（P<0.01）.Compared with Sham group,the expression of Bax and C-caspase-3 protein in MCAO group increased significantly（P<0.01）;compared with the MCAO group,C-caspase-3 protein expression in the H-ST2-104group reduced significantly（P<0.01）;CaMKKβ,p-AMPK protein expression reduced significantly（P<0.01）,p-mTOR protein expression increased significantly（P<0.01）.The MTT results showed that,compared with the Con group,the cells treated with Glu under the condition of 20 m M for 24 h,the cell viability reduced significantly（P<0.01）;Hoechst 33258 showed that the fluorescence increased,densely stained,and fragmented in the Glu group.MDC observed that the fluorescence intensity increased and the nuclei fragmented into dots and stained into green particles.FCM detected a significant up-ward in the concentration of Ca²⁺,WB results showed that the expression autophagy-related protein LC3-II protein expression significantly increased（P<0.01）.Compared with the Glu group,the cell viability of the Glu+ST2-104 group significantly increased（P<0.01）,Hoechst 33258 staining showed that,the fluorescence intensity reduced,and the cell morphology significantly improved;MDC fluorescent staining showed that,the intensity weakened,and the dot-like structure dyed into green particles significantly reduced;concentration of Ca²⁺decreased remarkably,WB results show autophagy-related protein LC3-II,Beclin-1 protein expression significantly reduced（P<0.01）.The autophagy agonist rapamycin significantly reversed the protective effects of ST2-104 peptide,and the autophagy inhibitor 3-MA significantly enhanced the protective effects of ST2-104peptide.CaMKKβinhibitor STO-609 was used to pretreat cells.Compared with Glu group,Hoechst 33258 fluorescence staining in Glu+STO-609 group showed that the phenomenon of apoptosis significantly reduced;MDC results showed that the fluorescence intensity and the dot-like structures stained into green particles were reduced;WB results showed that the expression of Bax and C-caspase-3 protein significantly reduced（P<0.01）,CaMKKβ,p-AMPK protein expression significantly reduced（P<0.01）,p-mTOR protein expressionsignificantly increased（P<0.05）.Conclusion:ST2-104 peptide has a protective effect on cerebral ischemia in rats.The mechanism may be related to ST2-104 peptide inhibiting CaMKKβ/AMPK/mTOR signaling pathway,inhibiting autophagy,and then inhibiting neuronal apoptosis.