Effects Of Autophagy And The Relationship Between BMSCs Transplantation And Autophagy Following Cerebral Ischemia-Reperfusion Injury

[Background]Cerebral ischemia is a majorhealthhazard with acute onset and is associated with ahigh rate of disability.After Successful thrombolysis or recanalization,more severe secondary brain injury willhappen,known as cerebral ischemia/reperfusion(I/R)injury.Bone marrow mesenchymal stem cells(BMSCs)are a group of non-hematopoietic stem cells with self-renewal and multidirectional differentiation ability.They can alleviate the tissue damage and promote nerve function recovery after cerebral I/R injury by directly dividing or secreting paracrine processes,such as soluble factors.Recent studies show that the protective effect of BMSCs may be achieved partially through the regulation of autophagy.Autophagy is a stress-response pathway of lysosomal degradation that may play an essential role in the development and progression of cerebral ischemia.It leads to maintenance of cytoplasmichomeostasis by eliminating intracellular misfolded or aggregated proteins and damaged organelles.At the same time,autophagy is also a double-edged sword,the role of autophagy and the relationship between autophagy and BMSC transplantation in the cerebral I/R injury are still require the further study.[Objectgive]To investigate the role of autophagy in cerebral I/R injury and the effect of BMSCs transplantation on neurological function recovery in rats with cerebral I/R injury and to further explore the relationship between BMSCs transplantation and autophagy,and then to lead to a novel therapeutic strategy and theory basis of applying BMSCs transplantation in Ischemic stroke.[Methord]This experiment via establishing a rat model of cerebral ischemia-reperfusion injury(MCAO)according to the longa method.The successful modeling were randomly assigned to sham group,MCAO group,MCAO+BMSCs group,MCAO+3-MA group,MCAO+BMSCs+LY294002 group,and divided into 12h,24h,48h,3d,5d,and 7d subgroups according to the time point after reperfusion.The the mNSS nerve dysfunction rating methods(mNSS)and catwalk gait analysis was used to assess the neurologic evaluation of the rats in each groups.Besides,the morphological changes and the number of necrotic cells in thehippocampal tissue were observed byhE staining,the number of apoptotic cells was detected via using TUNEL,and TTC staining was used to estimate the cerebral infarction area in each group.At last,apply the transmission electron microscope to observe the ultrastructural changes and autophagosome formation in Ischemic tissue,Western blot to detect the expression of LC3Ⅱ/Ⅰ,Beclin-1,Akt/p-Akt,mTOR/p-mTOR,and the immunofluorescent staining was used to estimate the number of LC3 positive cells.[Statistical analysis]Statistical analysis was performed using SPSS 20.0 software.Descriptive statistic is showed by mean and standard deviation(x±S).Groups were compared with the methods of ANOVA.The significance level was set at P<0.05.Two further comparison with LSD-t method,P<0.05 for the difference has significant meaning.[Result]1.The role of autophagy in cerebral ischemia-reperfusion injury:The formation of autophagy in the MCAO group was clearly observed under microscope,and the ratio of LC3Ⅱ/Ⅰ and Beclin-Ⅰ expression in the MCAO groups were significantly increased(P<0.05)as compared with the sham group.Besides,comparing with the MCAO group,the mNSS scores of 3-MA group were significantly decreased 3,5 and 7d after surgery(P<0.05);HE staining indicated that the injury of neurons in the hippocampus of 3-MA group was alleviated,and the number of necrotic cells in the 3-MA group was significantly smaller than that in the MCAO group.2.Relationship between BMSCs transplantation and autophagy in ischemia/reperfusion injury① Transplantation of BMSCs can reduce the brain injury in I/R rats and promote their neurological recoveryTTC staining showed that the volume of cerebral infarction was significantly reduced in the MCAO+BMSCs group,when compared with the MCAO group,the difference was statistically significant(P<0.05);TUNEL positive neurons gradually increased from 12h to 72h after cerebral I/R injury.And the percentage of TUNEL-positive neurons in BMSCs+MCAO group was significantly lower than that in MCAO rats(P<0.05).The results of behavioral analysis showed that the scores of mNSS in BMSCs+MCAO group were lower than those in MCAO group,and the gait analysis was significantly improved,The difference was statistically significant(P<0.05).② The neuroprotective effect of BMSCs is related to the inhibition of autophagyUnder the transmission electron microscopy,we found that cerebral I/R injury activated autophagy,which was time-dependent.And the typical autophagosomes appeared within 24 hours after operation.At 72 hours after operation,the number of autophagosomes increased and autophagy lysosomes appeared.The expression of LC3Ⅱ/Ⅰ and Beclin-1 in the cerebral I/R injury brain tissue continued to increase,reaching the peak at 24h after transplantation.Moreover,compared with MCAO group,BMSCs transplantation significantly reduced the expression of LC3Ⅱ/Ⅰ and Beclin-1,the difference was statistically significant(P<0.05).Besides,the immunofluorescence results showed that the number of LC3 positive cells in the brain tissue after MCAO was significantly increased(P<0.05),while BMSCs transplantation significantly reduced the expression of LC3.③ The protective role of BMSCs in the cerebral I/R injury is related to the activation of PI3K/Akt/mTOR signaling pathwayCompared with sham group,cerebral I/R injury reduced the protein levels of p-Akt and p-mTOR,however,in the BMSCs + MCAO group,the expression levels of p-Akt and p-mTOR were significantly increased(P<0.05).The intervention of LY294002 significantly attenuated the expression of p-Akt and p-mTOR(P<0.05).In the BMSCs+MCAO+LY294002 group,the expression of LC3Ⅱ/Ⅰ was increased relative to the BMSCs+MCAO group,Statistical differences are statistically significant(P<0.05).[Consulsion]1.Ischemia-reperfusion injury can activate autophagy,and after the inhibition of autophagy,it can alleviate brain damage in rats with cerebral ischemia-reperfusion injury and improve its neurological dysfunction.2.BMSCs could inhibit the autophagy of neurons and promote the recovery of neurological function in rats after cerebral ischemia-reperfusion.3.BMSCs’ protective effect is achieved maybe through inhibiting autophagy via the activation of the PI3K/Akt/mTOR signaling pathway.