| Part 1 Impact of Direct Peritoneal Resuscitation with Pyruvate on Spinal Cord Ischemia Reperfusion Injury in RatsObjective To investigate the effects of pyruvate-based peritoneal dialysis solution(PyrPDS)on the recovery of neurological function of(SCII)after spinal cord ischemiareperfusion injury in rats.Methods Twenty-four rats were allocated evenly to four groups.In sham group,rats were treated withoutligation or reuscitation except for open surgery followed by abdominal aorta exposure.In Group SCII,animals were treated with aortic occlusion above right renal artery for 60 min without reuscitation.In the Groups SCII+Saline and SCII+Pyr-PDS,after surgery,rats were performed of DPR with normal saline(NS)pyruvate-based PDS(Pyr-PDS),respectively.BBB score was used to evaluate neurologic function during 0/6/12/24/36/48 hours after injury.Serum and spinal cord tissue were collected 48 hours after surgery,renal function was detected by serum creatinine and spinal cord injury was observed by H&E staining and Nissl staining.Results BBB scores showed neurologic function was damaged after surgery,whereas there was no injury of renal function which means success of model establishment.In the meantime,BBB score showed that Pyr-PDS could promote recover of neurologic function which is close to normal 48 h post-injuy.Besides,neuron swollen was observed in both SCII and SCII+Saline group according to H&E staining,as well as vague boundary between grey matter and white matter,which were alleaviated in Pyr-PDS group.The result of Nissl staining showed accumulation of nissl body in cytoplasm after surgey which was improved by Pyr-PDS.Conclusion SCII model was successfully established by aortic occlusion below right ranal aortic for 60 min without renal injury.Pyr-PDS could effectively recover neurologic function injuried by surgery,alleaviate infiltration of inflammation cells and production of nissl body.Part 2 The Change of Autophagy in the Protective Effect of Direct Peritoneal Resuscitation with Pyruvate on Spinal Cord IschemiaReperfusion Injury in RatsObjective We observed the alteration of autophagy during SCII and investigated whether autophagy participated into the protection of pyruvate-based peritoneal dialysis solution(Pyr-PDS)to SCII in rats.Methods Forty-eight rats were allocated evenly into three groups.In sham group,rats were treated without ligation or reuscitation except for open surgery followed by abdominal aorta exposure.In SCII Group,rats were only treated with aortic occlusion for 60 min withoutfluidreuscitation.In the Group SCII+Pyr-PDS,rats were performed of DPR with pyruvate-based PDS(Pyr-PDS)after surgery.48 h after surgery,Spinal cord tissues were collected at 48 hours after surgery.Q-PCR and western blot were applied to observe the level of autophagy related genes and proteins,and transmission electron microscope(TEM)was used to observe ultrastructure of cells and autolysosome.Results Results of q-PCR and Western blot showed that the level of autophagy was higher compared to sham group,and Pyr-PDS could further promote the degree of autophage compared to SCII group.Furthermore,numerous lysosomes and disordered structure of mitochondria and parts of cytoplasmic organelles andnuclear pyknosis were observed by TEM,while after treated with Pyr-PDS,the injury of cells was alleviated and more autophagosome was exhibited compared with SCII group.Besides,the level of PHD2 was downregulated in SCII group and Pyr-PDS could further reduce the expression of PHD2.Downregulation expression of PHD2 resulted inincreased expression of HIF-1?/BNIP3,which resulted upregulated expression of downstream gene that inhibit autophay.Conclusion Autophagy was activated after SCII,Pyr-PDS could effectively recover neuron ultrastructure destroy and further promote autophagy,the underline mechanism might be related to regulating expression of PHD2/HIF-1 and downstream autophagy related genes.Part 3 Protective Mechanism against Oxygen-glucose deprivation and reoxygenation by Pyruvate in SH-SY5 Y CellsObjective We ought to elucidate the mechanism protective effect of pyruvate to neuron cells in vitro,and the underline mechanism that pyruvate regulates autophagy during injury.Methods SH-SY5 Y cells were treated with oxygen-glucose deprivation and reoxygenation to mimic SCII in vitro.Cells were devided into 5 groups:(1)Control group;(2)OGD group: cells were treated with oxygen-free without reoxygenation;(3)OGD/R group: cells were treated with OGD and harvested during reoxygenation 0/6/12/24 hours;(4)Pyruvate group: cells were treated with OGD and pyruvate were applied to cells during reoxygenation;(5)IOX2 group: cells were treated with OGDand PHD2 inhibitor IOX2 were applied to cells during reoxygenation;To explore whether pyruvate could exert protective effects in vitro,we apply CCK-8 and flow cytometry to detect drug toxicity and cell viability;Additionally,q-PCR and western blot were applied to observe the levelexpression of PHD/HIF-1 and autophagy related genes.Results The results of CCK-8 andflow cytometry showed protective effect of pyruvate to neurons,and the expression of autophagy-related genes,PHD2,and its downstream HIF-1?/BNIP3 pathway showed the same trend as the results in vivo.Besides,results of IOX2 groupare as same as the results in pyruvate group.Then,we observed the expression of autophagy-related genes and the HIF-1? signal pathway in the process of reoxygenation;the results showed that as the reoxygenation goes,the expression of the HIF-1? signal pathway and degree ofautophagy came to decrease gradually,while treated with pyruvate could maintain autophagy high and stable through keeping PHD2 at a lower level during reoxygenation,and the latter was observed downregulated during reoxygenation process from 0 to 24 hours in a time-effect way.Conclusion Pyruvate is protective to neurons in vitro through activating autophagy via acting on PHD2 and its downstream HIF-1?/BNIP3 pathway... |
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