Endogenous IL-6 From Bone Marrow Mesenchymal Stem Cells Suppresses Hippocampal Neurons Autophagy Of The Hypoxic-ischemic Brain Damage Rats And Its Possible Mechanism2

PART ? BONE MARROW MESENCHYMAL STEM CELLS SUPPRESSES HIPPOCAMPAL NEURONS AUTOPHAGY IN THE HYPOXIC-ISCHEMIC BRAIN DAMAGE RATSObjective: To investigate the effect of mesenchymal stem cells(MSCs)on autophagy in hippocampal neurons of neonatal rats with hypoxic-ischemic brain damage(HIBD).Methods: Seven-days-postnatal SD rats were permanently ligated the left carotid artery and treated with hypoxia 2.5h to establish HIBD model,and the hippocampus tissues were collected at 0h?12h?24h?48h?72h?5d and 7d following HIBD.The 5 ? L PBS with 2×105 MSCs was intracerebroventricularly injected in the HIBD neonatal rats named as the HIBD+MSCs group,while injected by the same volume of sterile PBS as its control(HIBD group).The hippocampus tissues of HIBD rats were collected at 12 h and 24 h following transplantation.The therapeutic effects of MSCs transplantation on learning and memory function of the neonatal rats subjected to HIBD were assessed by morris water maze test for 4w.The primary hippocampal neurons cultured for 5 days in vitro were subjected by OGD treatment,and then the hippocampal neurons were collected at 0h ? 12 h ? 24 h ? 48 h ? 72 h following OGD injury.The OGD-injured neurons were cocultured with MSCs.Meanwhile,the negative control groups were not subjected to OGD treatment.The OGD neurons were collected at 12 h and 24 h following coculture.Western blotting detected the changes of autophagy related proteins Beclin 1,LC3 II and p62 expression levels in the hippocampal neurons and OGD-injured neurons at different time points after HIBD and OGD injury.Transmission electron microscopy was also used to observe the number of autophagosomes in the OGD damaged neurons following coculture with MSCs.Results:(1)The results of western blotting displayed that levels of Beclin 1 and LC3 II protein expressions were obviously increased in the rat hippocampus at 12-24 h following HIBD(P<0.001).The MSCs transplantation statistically suppressed the increased levels of Beclin 1 and LC3 II,upregulated p62 protein expression levels in the hippocampus (P<0.001).(2)The rats of HIBD+MSCs groups required less time to find the platform than that in the HIBD group,and entering the platform region was significantly more than that of the HIBD rats in morris maze water test(P<0.01).(3)The OGD treatment also upregulated the levels of Beclin 1and LC3 II protein expressions in the primary neurons in vitro at 12h(P<0.001),while MSCs seperated coculture significantly downregulated the levels of Beclin 1 and LC3 II protein expressions in the primary hippocampal neurons by OGD injury(P<0.001),but induced p62 protein expression level(P<0.001).(4)Observation of transmission electron microscopy(TEM)found that the number of autophagosomes was significantly decreased at 12 h and 24 h after seperated coculture with MSCs(P < 0.001).Conclusion: Both HIBD and OGD injury in vitro can cause the upregulation of autophagy at the acute stage following damage,however,MSCs transplantation or seperated coculture could reduce the expression levels of Beclin 1,LC3 II and increase the level of p62 protein in the hippocampus of HIBD rats and OGD-injured neurons,in order to decrease the number of autophagosomes in the neurons by OGD injury at the acute stage and improve the learning-memory function of HIBD rats.PART ? INHIBITING ENDOGENOUS IL-6 IN BONE MARROW MESENCHYMAL STEM CELLS INCREASES AUTOPHAGY LEVEL IN HIPPOCAMPAL NEURONS OF THE HYPOXIC-ISCHEMIC BRAIN DAMAGE RATSObjective: To investigate the effect of endogenous IL-6 from bone marrow mesenchymal stem cells(MSCs)on autophagy in hippocampal neurons of neonatal rats with hypoxic-ischemic brain damage(HIBD)and its possible molecular mechanism.Methods: After the HIBD injury of 7-day-old postnatal rats,they were intracerebroventricularly injected by si IL-6 MSCs or GFP MSCs respectively.and then the hippocampus tissuses of HIBD rats were collected at 12 h and 24 h after transplantation.The primary hippocampal neurons in vitro subjected by OGD were separatedly cocultured with si IL-6MSCs or GFP MSCs,and the OGD-injured neurons were collected at 12 h and 24 h following coculture.Western blotting detected the expression levels of autophagy associated proteins Beclin 1,LC3 II,p62,IL-6,p-AMPK and p-m TOR in the hippocampus of HIBD rats following transplantation and the OGD-injured hippocampal neurons after seperated coculture.Transmission electron microscopy was also used to observe the number of autophagosomes in OGD damaged neurons following coculture.Results:(1)The results of western blotting showed that the levels of Beclin 1 and LC3 II protein expressions were obviously increased in the hippocampus of HIBD rats after si IL-6 MSCs transplantation at 12 h and24h,while the expression level of p62 was statistically decreased(P<0.001)compared with those of the GFP MSCs group.The changes of Beclin 1,LC3 II and p62 protein expressions levels in the OGD-injured primary neurons cocultured with si IL-6 MSCs were consistent with the detecting results in vivo(P<0.001).(2)The levels of IL-6 and phospho-m TOR(p-m TOR)protein expressions in the hippocampus of HIBD rats transplanted with si IL-6 MSCs and OGD-injured neurons after si IL-6MSCs coculture were significantly decreased than those in the GFP MSCs group,while the expressions of phospho-AMPK protein were obviously increased in 12 h and 24 h after the transplantion or coculture of si IL-6MSCs(P<0.001).(3)Observation of transmission electron microscopy(TEM)found that the number of autophagosomes were significantly increased in 12 h and 24 h after seperated coculture with si IL-6 MSCs(P <0.001).Conclusion: The above results in vivo and in vitro suggest that IL-6blocking in the MSCs significantly induces the levels of autophagy associated proteins Beclin 1,LC3 II in both the hippocampus of HIBD rats and OGD-injured neurons,suppresses p62 protein expression level,but also activate p-AMPK and inhibit phosphorylation of m TOR pathway in the hippocampal neurons injured by OGD,to increase the number of autophagosomes in the neurons by OGD injury at the acute stage.