Neuroprotective Effect And Mechanism Of Exogenous Spermidine On Spinal Cord Ischemia-reperfusion Injury In Rats

Objective:To establish a rat model of spinal cord ischemia-reperfusion injury,to observe whether spermidine has neuroprotective effects on rats with spinal cord ischemia-reperfusion injury by treating them with spermidine,and to explore its possible mechanism of action initially,and to verify it in in vitro experiments.Methods:1.Construction of a rat model of spinal cord ischemia-reperfusion injury in rats.2.The recovery of the limb was assessed by the motor function score of the rat hind limb,and the size of the ischemic infarct area was detected by TTC staining.3.The neuronal survival in rat spinal cord tissues were detected by NeuN staining.4.mRNA and protein expression levels of apoptosis,autophagy and polyamine metabolizing enzymes were detected by qPCR and immunoblotting assay.5.The neuroprotective effect of spermidine was verified by constructing a glutamate-induced PC 12 cell injury model,CCK-8.6.The levels of oxidative stress and autophagy in PC 12 cells treated with spermidine were detected by flow cytometry.Results:1.A rat spinal cord ischemia-reperfusion injury model was successfully constructed.2.motor scores showed higher scores in the SCIRI+SPD group than in the SCIRI group at 72 h after injury(P<0.05).3.TTC staining showed that the area of spinal cord ischemic infarction was smaller in the SCIRI+SPD group than in the SCIRI group(P<0.05).4.NeuN IHC results showed that the number of NeuN-positive cells in both the SCIRI group and the SCIRI+SPD group was very less than that in the Sham group,The number of NeuN positive cells in SCIRI+SPD group was remarkably higher than that in SCIRI group(P<0.05).5.Western Blot results for detection of autophagy and apoptosis Bax was remarkably higher and Bcl2 was remarkably lower in the SCIRI group and SCIRI+SPD group compared with the Sham group,while Bax protein expression level was clearly lower in the SCIRI+SPD group compared with the SCIRI group,while Bcl2 protein expression level was clearly higher in the SCIRI+SPD group compared with the SCIRI group.The expression levels of LC3 and p62,The protein level of LC3 and p62,the key proteins of autophagy,were significantly increased in the SCIRI and SCIRI+SPD groups,and the LC3 level was further increased in the SCIRI+SPD group compared with SCIRI,and the p62 level was decreased in the SCIRI+SPD group compared with SCIRI.The level of Beclinl protein expression in the SCIRI group was not significantly different from that in the Sham group(p>0.05),while the level of Beclinl protein expression in the SCIRI+SPD group was upregulated more than in the SCIRI group.Compared to the Sham group,the P300 protein expression level was significantly lower in the SPD group,The expression levels of ATG13 and ULK1 were significantly increased in the SCIRI group compared to the sham group,whereas FIP200 did not change markedly in the SCIRI group compared to the sham group.The protein level of ATG13 and FIP200 was significantly increased in the SCIRI+SPD group compared to the SCIRI group,while ULK1 was not significantly altered(p>0.05).6.Changes in enzymes related to spermidine metabolism after spinal cord injury:The immunohistochemistry data showed that SRM protein levels The qPCR results showed that the mRNA expression levels of the Satl gene in spinal cord tissues were markedly increased in the SCIRI and SCIRI+SPD groups compared with the sham group,while the mRNA levels of Satl were significantly decreased in the SCIRI+SPD group when compared with the SCIRI group(P<0.05).7.In vitro experiments,CCK-8 results showed that 10 μL SPD had a marked effect on promoting PC12 cell proliferation compared to other concentrations.Compared to the control group,PC 12 cell viability was greatly inhibited in the Glu and Glu+SPD groups,while PC 12 cell viability was significantly higher in the Glu+SPD group with statistical significance.The flow ROS analysis results showed that the ROS levels in the Glu and Glu+SPD groups were significantly higher than those in the control group,and the ROS levels in the Glu+SPD group were significantly lower than those in the Glu group.The data from the flow MDC autophagic assay showed that the autophagy levels in the Glu group and Glu+SPD group were significantly higher than those in the control group,and the autophagy levels in the Glu+SPD group were at a significantly higher level than those in the Glu group(P<0.05).Conclusion:1.spermidine can significantly reduce the number of apoptotic cells,increase the number of surviving neurons,and play a neuroprotective role.2.While reducing apoptosis,spermidine may promote the expression of autophagy-related proteins by inhibiting P300,promote the formation of ATG13-FIP200-ULK1 complex,and also significantly reduce iNOS and eNOS content to alleviate oxidative stress,ultimately promoting autophagy and improving autophagic flux.3.Exogenous spermidine supplementation can improve the abnormalities of polyamine metabolizing enzymes SRM and Satl after spinal cord ischemia-reperfusion injury and repair the imbalanced homeostasis of polyamine metabolism.4.Spermidine can alleviate glutamate-induced excitatory neurotoxic injury in PC 12 cells by improving oxidative stress and promoting autophagy.