A Study Of Low-frequency Pulsed Electromagnetic Fields Inhibiting Oxidative Stress To Repair Spinal Cord Injury In Rats

Objective:Spinal cord injury(SCI)which has high incidence rate,high disability,low mortality and difficulties in repairment,is one of the most serious traumatic diseases.The purpose of this study is to clarify the anti oxidative stress effect and the best stimulation parameters of low-frequency pulsed electromagnetic fields(LPEMFs)through the experimental study of oxidative stress injury model of SH-SY5Y cells in vitro and spinal cord contusion model of rats in vivo.In this study whether the mechanism of LPEMFs'anti oxidative stress effect is related to PI3K/Akt/HSP70signal pathway is also be verified,so as to provide a non-invasive alternative treatment for the early intervention of SCI in the future.Method:1.Cell survival and active oxygen generation:The oxidative stress injury model of SH-SY5Y cells was constructed in vitro with different concentration of H₂O₂,and the standard model was constructed with semi inhibitory concentration(IC50).LPEMFs with different intensity(0.5m T-3m T)and different duration(0.5h-2.5h/d)were selected;The morphological changes and activities of cells were observed in order to determine the best parameters of LPEMFs(50Hz,2.5m T,1h/d).ROS assay kit was used to detect the production of ROS in SH-SY5Y cells;2.Spinal cord injury model:T10 spinal cord contusion model of rats was made by Impactor Model-III percussion instrument.LPEMFs with best parameters were selected to intervene the rats with SCI.BBB score was given to the experimental animals for 2 weeks to study the effect of LPEMFs on behavioral changes of rats.Somatosensory evoked potential(SEP)and motor evoked potential(MEP)of rats was measured by electrophysiological instrument 2 weeks after injury;3.Inflammatory factors:Enzyme-linked immunosorbent assay(ELISA)was used to measure the expression of TNF-?and IL-1?after 2 weeks;immunohistochemistry was used to evaluated the expression of NF-?B in the spinal cord tissues;4.Oxidative stress:Western blot was used to measure the expression of i NOS in the injured spinal cord;ELISA was used to measure the expression of SOD and CAT;ROS assay kit was used to detect the production of ROS in spinal cord;The expression of HSP70 was evaluated by immunohistochemistry;5.PI3K/Akt signaling pathway:Western blot was used to detect the expression changes of phosphorylated and non phosphorylated Akt and HSP70 in SH-SY5Y cells before and after intervention with LY294002;6.Statistical analysis:Graphpad prism 8 was used for statistical analysis,results were expressed in the form of Mean±SD.Use one-way ANOVA as statistical method,and use Student-Newman-Keuls method to analyze the differences between groups afterwards.P<0.05 indicates that the differences are statistically significant.Result:1.With the increase of H₂O₂ concentration,the mortality of SH-SY5Y cells increased while the viability decreased.The standard model was constructed with IC50concentration(50?M).The cell viability(77.80±6.22%)of the optimal LPEMFs group(50Hz,2.5m T,1h/d)was significantly higher than that of oxidative stress group(54.60±7.37%)(P<0.05).Also the optimal LPEMFs could reduce the production of ROS in SH-SY5Y cells(5.87±0.62:2.53±0.96)(P<0.05);2.The optimal LPEMFs stimulation(50Hz,2.5mt,1h/d)was selected to intervene moderate SCI rats.BBB scores of experimental animals in each group were 21 before injury and 0 after injury.There was no significant difference in BBB score between the control group and the LPEMFs group in the first 7 days after the injury.However after7 days,the LPEMFs group showed better motor function recovery than the control group(P<0.05).The amplitude of MEP and SEP in LPEMFs group was significantly higher than that in the control group(P<0.05),but the latency of MEP and SEP in LPEMFs group was not significant different from that in control group and sham group;3.The expression of TNF-?and IL-1?in the spinal cord tissue of rats was increased 2 weeks after injury.But he expression of TNF-?and IL-1?was significantly decreased by LPEMFs intervention(P<0.05);The expression of NF-?B at the anterior horn of the injured spinal cord was significantly reduced after the intervention of LPEMFs(P<0.05);4.The expression of i NOS and ROS in the injured spinal cord was significantly decreased After LPEMFs intervention(P<0.05);While the expression of SOD,CAT and HSP70 in the injured spinal cord was significantly increased(P<0.05);5.The PI3K/Akt signaling pathway of SH-SY5Y cells could be activated by LPEMFs,and the expression of HSP70 in SH-SY5Y cells in vitro could also be increased by LPEMFs(P<0.05);the intervention of LY294002,an inhibitor of PI3K/Akt signaling pathway,could reduce the expression of HSP70 in SH-SY5Y cells and antagonize the anti oxidative stress injury of LPEMFs.Conclusion:1.LPEMFs can inhibit oxidative stress injury of SH-SY5Y cells in vitro.The optimal stimulation parameters of LPEMFs are 50Hz,2.5m T,1h/d;2.LPEMFs can improve the recovery of motor function and increase the amplitude of SEP and MEP in SCI rats.LPEMFs can reduce inflammatory response,and reduce oxidative stress by upregulating CAT and SOD.Its potential mechanism may be related to the high expression of HSP70;3.The mechanism of LPEMFs inhibiting oxidative stress injury is closely related to the activation of PI3K/Akt/HSP70 signaling pathway.