Research Of Autophagy Involved In The Protection Of Minocycline Against Cerebral Ischemia-reperfusion Injury In Rats

Existing studies have shown that autophagy plays an important role in the process of cerebral ischemia-reperfusion injury.The oxidative stress and endoplasmic reticulum stress after cerebral ischemia reperfusion may be helpful in inducing autophagy.Many reports proved that there following autophagy activation after cerebral ischemia reperfusion,and which have proved to be a protective role in this processMinocycline is a semisynthetic tetracycline antibiotic with the strongest antimicrobial activity among the tetracycline family.In addition to its own antimicrobacterial properties,minocycline has been reported to exert neuroprotective effects over various experimental models especially in traumatic brain injury and brain ischemia diseases.Minocycline has been focused as a neuroprotective agent over neurodegenerative disease since it has been first reported by Yrjanheikki J that minocycline has neuroprotective effects in animal models of ischemic injury.However,the mechanism remains unclear.This study aims to explore the mechanism from the protection of minocycline against cerebral ischemia-reperfusion injury both in vitro(oxygen-glucose deprivation and reperfusion in PC12 cells)and vivo(middle cerebral artery occlusion in rats)modelsPart 1 Research of the relationship between autophagy and protection ofminocycline in PC12 cells after oxygen-glucose deprivation and reperfusionObjective:To investigate the effects of autophagy on the protection of minocycline in PC 12 cells after oxygen-glucose deprivation and reperfusion,exploring the mechanisms of minocycline against cerebral ischemia-reperfusion injuryMethods Cultured PC 12 cells were exposed to oxygen-glucose deprivation and then reperfusion,and treating with minocycline(1μM,10μM,100μM)or Trimethyl adenine(3-MA,5mM)during reperfusion.And cell viability was determined by MTT assay after 6h of reperfusion.We also stain cells with monodansylcadaverin to observe autophagic vacuole The expression of protein LC3 Ⅱ,Beclinl and p62/SQSTM 1 were determined by western blot.Results The cell viability of PC12 cells decreased significantly after oxygen-glucose deprivation and reperfusion.Low dose(1,10 μM)minocycline significantly attenuates cell death cascades after oxygen-glucose deprivation,while the high dose(100μM)exacerbates the cell injury.The expression levels of LC3 Ⅱ and Beclinl increased more pronouncedly when PC 12 cells were treated minocycline at the higher concentration(1-100μM).Whereas the autophagy inhibitor 3-MA reversed the protection of minocycline by reducing the expression of LC3 Ⅱ and Beclin1 and increasing p62/SQSTM 1.Conclusion Low dose(1,10μ M)minocycline promotes PC12 cells survival after oxygen-glucose deprivation and reperfusion,which might be mediated by up-regulating the protein LC3 Ⅱ and Beclinl expression and promoting the degradation of p62/SQSTM 1 and then inducing autophagyPart 2 Research of the relationship between autophagy and protection ofminocycline against cerebral ischemia-reperfusion injury in ratsObjective To investigate the effects of autophagy on the protection of minocycline against cerebral ischemia-reperfusion injury in rats,exploring the mechanisms of minocycline against cerebral ischemia-reperfusion injuryMethods The middle cerebral artery occlusion(MCAO)model was established by intraluminal suture method,reperfusion was allowed after 1.5 hours occlusion.Intraperitoneal injection of minocycline(22.5,45,90 mg/kg)or intracerebroventricular injection of Trimethyl adenine was operated at the onset of reperfusion.Score on rat neural function and determine infarct volume by TTC staining method at 24h after reperfusion.The ultrastructure of neurone and the quantity changes of autophagic vacuoles were observed by using transmission electron microscopy(TEM).The expression of protein LC3 Ⅱ,Beclinl and p62/SQSTM1 were determined by western blotResults Minocycline(22.5mg/kg,45mg/kg)group significantly reduces rat nerve function scores,reducing cerebral infarction volume after cerebral ischemia-reperfusion compared with model group,improving the expression of autophagy related proteins LC3 Ⅱand Beclinl,increasing the number of autophagosome and degradation of p62/SQSTM 1.Whereas the autophagy inhibitor 3-MA reverses the protection of minocycline by reducing the expression of LC3 Ⅱ and Beclin1 and increasing p62/SQSTM 1Conclusion Low dose(22.5mg/kg,45mg/kg)minocycline attenuates cerebral ischemia reperfusion injury in rats,which might be mediated by up-regulating the protein LC3 Ⅱ and Beclinl expression and promoting the degradation of p62/SQSTM 1 and then inducing autophagy.