The Mechanism Of BMSCs Exosomes Regulating ALP Through MTOR/TEFB To Improve Cerebral Ischemia-Reperfusion Injury

Research Background:The pathophysiological process after Cerebral Ischemia Reperfusion Injury(CIRI)is complex,in which the accumulation of misfolded proteins and damaged organelles is an important cause of neuronal death,and the Autophagy-Lysosomal Pathway(ALP)is an important pathway for neurons to clear long-term misfolded proteins and damaged organelles to maintain cellular homeostasis.Extensive studies have demonstrated that exosomes can improve various diseases by regulating autophagy,but no studies have reported the regulatory role of Bone Mesenchymal Stem Cells-Exosomes(BMSC-Exos)in ALP dysfunction after CIRI.Our group has previously demonstrated in vitro experiments that BMSC-Exos can inhibit mTOR expression in PC 12 cells after OGD/R.It is known that when mTOR expression is inhibited,TFEB can be induced to dephosphorylate and thus promote TFEB translocation nucleus and activate the ALP pathway.In this study,we intend to investigate the alteration of ALP at different time points after CIRI,clarify whether BMSC-Exos intervention can regulate ALP dysfunction after CIRI and its specific mechanism of action,and provide a theoretical basis for the clinical translation of BMSC-Exos.Objectives:To investigate the changes of ALP at different time points after tMCAO rat modeling;to verify that BMSC-Exos can improve ALP dysfunction after CIRI,and to investigate its specific mechanism of action.Methods:1.Exosomes were extracted and identified using ultracentrifugation method.2.The effects of ALP alteration at different time points after tMCAO rat modeling and BMSC-Exos intervention were detected by Western blot and immunofluorescence,respectively.3.The effect of BMSC-Exos on neurobehavioral deficits in tMCAO rats was verified using mNSS score,corner test and stick-turning test.4.The effects of BMSC-Exos on the expression of TFEB and mTOR in cytoplasmic nuclei were detected by western blotting and immunofluorescence5.CCK8 was used to explore the cell survival rate at different time points of OGD/R,determine the appropriate modeling time,and further verify the effect of BMSC-Exos intervention on the survival rate of PC 12 cells after OGD/R.And the effect of MHY1485(mTOR agonist),Rapamycin(mTOR inhibitor)on the survival rate and the admission of TFEB into the nucleus;The immunofluorescence intensity of CTSB in different groups was further verified by immunofluorescence staining.Results:1.BMSCs as well as BMSC-Exos were successfully extracted and identified as compliant.2.BMSC-Exos can reduce the area of brain infarction in tMCAO rats and improve the short-term behavior of mNSS.3.BMSC-Exos can improve the behavioral performance of tMCAO rats in the corner test as well as the stick-turning test.4.tMCAO rats showed ALP dysfunction 72h after modeling.5.BMSC-Exos improved ALP dysfunction in tMCAO rats at 72h post-modeling.6.BMSC-Exos inhibited mTOR expression and promoted TFEB expression in the nucleus7.BMSC-Exos improved the survival rate of PC 12 cells after OGD/R and promoted the nucleation of TFEB.This effect was reversed by MHY1485,and was similar to that of Rapamycin,suggesting that BMSC-Exos improved ALP dysfunction mainly by inhibiting mTOR.Conclusion:BMSC-Exos may reduce cerebral ischemia-reperfusion injury by inhibiting mTOR,promoting TFEB entry into the nucleus,and improving ALP.