A Study On The Effect And Mechanism Of Apelin-13 On Cerebral Ischemia/reperfusion Injury By Inhibiting Apoptosis And Autophagy

Background:Ischemic stroke is caused by insufficient blood supply to the brain.The incidence of ischemic stroke remains high worldwide.And the prevention and treatment of ischemic stroke are still facing huge challenges.Intravenous thrombolysis is currently the most effective treatment for ischemic stroke.However,it has many limitations and complications.And it will exacerbate brain damage after reperfusion.Therefore,improving the resistance of nerve cells after cerebral ischemia/reperfusion(I/R)and reducing their damage have become the current research focus on the treatment of ischemic stroke.Studies have shown that the occurrence and development of cerebral I/R injury may be related to many physiological processes such as endoplasmic reticulum stress,neuroinflammation,oxidative stress,autophagy and apoptosis.Apelin is a neuropeptide which is widely present in the central nervous system and has neuroprotective functions.Among the different peptides of Apelin,Apelin-13 has extremely strong biological activity.In this study,the rat models of cerebral I/R injury were used to investigate the protective effect and mechanisms of Apelin-13 on cerebral I/R injury,which may provide new ideas and theoretical basis for drug research.Objective:Study the mechanism of apoptosis and autophagy in Apelin-13 inhibiting cerebral I/R injury.Methods:Spragu-Dawley(SD)rats aged 8-10 weeks(weighting 260-280 g)were selected to establish the middle cerebral artery occlusion(MCAO)model by suture method.SD rats were divided into sham,MCAO,Apelin-13,and MCAO+Apelin-13 groups according to simple randomization using a random number table.Rats in the sham and Apelin-13 groups were treated the same as those in the MCAO group,but the monofilament was not advanced into the common carotid artery.For the drug treatment,rats in the sham and MCAO groups were injected with vehicle,while rats in the Apelin-13 and MCAO+Apelin-13 groups were injected with Apelin-13.The suture was removed after 2 hours.After 24 hours of reperfusion,the rats were killed by overdose chloral hydrate anesthesia.TTC staining was used to detect the infarct areas.Neurological score was evaluated to detect the behavioral function.Lactate dehydrogenase(LDH)was determined to detect the damage of rat hippocampal neurons.The expression of caspase-3,LC3B and p62 in rat hippocampal neurons were determined by immunofluorescence double staining.The expression of apoptotic proteins(cleaved caspase-3,Bcl-2 and Bax),autophagy marker proteins(LC3B,p62 and Beclinl)and autophagy pathway proteins(PI3K,Akt and mTOR)were detected by western blot assay.Results:1.Compared with the model group,intracerebroventricular administration of Apelin-13 could significantly reduce the infarct sizes(observed in TTC staining).And Apelin-13 significantly improved the neurological score.The level of LDH in rat hippocampus was also decreased by Apelin-13 treatment.Together,these results indicate that Apelin-13 is neuroprotective against cerebral I/R injury.2.Apelin-13 intervention effectively inhibited the increase of cleaved caspase-3 and LC3B-Ⅱ and the decrease of p62 in the MCAO rat model,which was determined by immunofluorescence double staining and western blot assay.It shows that Apelin-13 intervention significantly inhibited apoptosis and autophagy in cerebral I/R injury.3.The Bcl-2/Bax ratio was obviously higher due to Apelin-13 treatment.In addition,Beclinl expression was increased in the MCAO rat model,whereas Bcl-2 expression was decreased.Apelin-13 treatment was able to reverse these effects.These findings indicate that Bcl-2 upregulation participates in the apoptotic and autophagic inhibition of Apelin-13.4.In the MCAO rat model,the PI3K/Akt/mTOR pathway was significantly inhibited.However,Apelin-13 treatment reversed this inhibition by upregulating p-PI3K/PI3K,p-Akt/Akt,and p-mTOR/mTOR.These results suggest that the PI3K/Akt/mTOR pathway participates in the inhibitory effects of apelin-13 on apoptosis and autophagy.Conclusion:Apelin-13-induced neuroprotection against cerebral I/R injury involved the inhibition of apoptosis and autophagy by Bcl-2 and the PI3K/Akt/mTOR pathway.