The Protective Mechanism Of Acaloric Ultrashort Wave On N2a Cells From Oxygen-glucose Deprivation And Reoxygenation Injury

Background and significance:Ischemic stroke has become one of the most life-threatening diseases in the world,accounting for about 70%to 80%of all stroke types.Perfusion will cause further irreversible damage,and the clinical treatment effect of cerebral ischemia-reperfusion injury is not ideal.No-calorie ultrashort wave therapy（USW）is a method in which ultra-high frequency alternating current acts on the human body to exert a therapeutic effect.It has been widely used in clinical practice.USW has the characteristics of strong penetration,comfort and non-invasiveness.After cerebral ischemia-reperfusion injury,brain tissue ischemia and edema are similar to joint and soft tissue ischemia injury.Therefore,we speculate that USW should be applied to nerve cells.Oxygen-glucose deprivation and reoxygenation（OGD/R）injury may also have a very good effect on reducing swelling and promoting repair.This research is mainly divided into two chapters:1.The protective effect of acaloric ultrashort wave on cellular oxygen and glucose deprivation and reoxygenation injury.2.The protective role of SPCA1 in USW treatment of cellular oxygen-glucose deprivation and reoxygenation injury.Objective:The purpose of this study was to investigate the effects of no-calorie ultrashort wave（USW）on cell morphology,cell activity,apoptosis and the expression of SPCA1 protein in N2a cells after oxygen-glucose deprivation and reoxygenation（OGD/R）injury,and to explore its protective mechanism.Correlation with SPCA1.Methods:The mouse neuroma blast cell line（N2a）was used as the research object,and the cells within three passages were taken.The first chapter verified the protective effect of acaloric ultrashort wave on cells after oxygen and glucose deprivation and reoxygenation.The patients were randomly divided into control group（CON group）,model group（OGD/R group）and ultrashort wave group（USW group）.The cells in the OGD/R group were modeled in a hypoxic incubator with a sugar-free balanced salt solution,95%N₂and 5%CO₂,underwent 4 hours of oxygen and sugar deprivation,and then placed in a 5%CO₂mild humidified incubator for reoxygenation;USW group cells were treated with USW after the same modeling procedure described above.The morphology of the three groups of cells was observed under the microscope,the cell proliferation activity was detected by CCK8,the apoptosis and calcium ion concentration were detected by flow cytometry,and caspase9（apoptotic protein）,GM130（Golgi membrane protein）,β-catenin（Wnt Pathway-related protein）were detected by WB analysis,SPCA1（Golgi membrane protein）expression changes.The second chapter verified the protective effect of SPCA1 on the injury of oxygen and glucose deprivation and reoxygenation of cells,and were randomly divided into OGD/R+sh CON group,OGD/R+sh RNA group,USW+sh CON group,USW+sh RNA group.In the OGD/R+sh CON vector group,the above modeling process was performed after adding empty lentivirus;in the OGD/R+sh RNA group,the above modeling process was performed after adding sh RNA that silences the gene encoding SPCA1;Modeling and ultrashort wave treatment were performed after virus;in USW+sh RNA group,sh RNA silencing SPCA1-encoding gene was added to cells for modeling and ultrashort wave treatment.The transfection rate of lentivirus was observed by fluorescence microscopy,the expression of SPCA1 was detected by WB analysis,apoptosis,calcium ion concentration,and ROS content were detected by flow cytometry,and SPCA1,i NOS（oxidative stress factor）,and Mn SOD were detected by RT-PCR analysis.and SOD（oxidative stress protective factor）expression changes.All data were analyzed and processed using Graphpad 9.0 statistical software.Result:（1）The N2a cell OGD/R model was constructed,and it was found that the morphology of the cells in the OGD/R group was damaged under an inverted optical microscope,while the cell morphology in the USW group was restored relative to the OGD/R group.The results of CCK8showed that the cell viability in the OGD/R group was lower than that in the CON group,while the cell viability in the USW group was higher than that in the OGD/R group（P<0.05）;（2）The results of flow cytometry apoptosis rate showed that compared with CON group,the apoptosis of OGD/R group increased,but after USW treatment,the cell survival rate increased significantly（P<0.05）.After the expression of the encoding gene SPCA1 was silenced by small hairpin RNA,the apoptosis rate in the USW+sh RNA group was higher than that in the USW+sh CON group（P<0.05）.（3）The results of flow cytometry calcium ion concentration showed that compared with the CON group,the Ca2+concentration of cells increased significantly after OGD/R,while the intracellular Ca2+concentration showed a downward trend after USW treatment.After sh SPCA1,the concentration of Ca2+in the USW+sh RNA group was lower than that in the USW+sh CON group（P<0.05）（4）The results of WB measurement of Golgi membrane proteins showed that after OGD/R,the protein expressions of Golgi proteins SPCA1 and GM130 were decreased.After USW treatment,the expressions of SPCA1 and GM130 were increased compared with those in the OGD/R group.slightly lower than the CON group.After sh SPCA1,the expression of SPCA1 protein was significantly decreased compared with the sh CON group,and the silencing of SPCA1 protein was successful（P<0.05）.After OGD/R,the expression of Wnt pathway-related proteinβ-catenin decreased,while the expression ofβ-catenin increased after USW compared with the OGD/R group,but was still slightly lower than that in the CON group（P<0.05）.WB detection of caspase9 protein showed that the expression of caspase9 protein in OGD/R group was significantly higher than that of CON group,and the apoptosis rate increased.The expression of caspase9 protein in USW group was lower than that in OGD/R group,but slightly higher than that in CON group（P<0.05）.（5）A large amount of green fluorescent protein after lentivirus transfection can be seen under the fluorescence microscope;（6）Flow measurement of ROS concentration showed that after sh SPCA1,the ROS concentration in the OGD/R+sh RNA group was higher than that in the OGD/R+empty group;the intracellular ROS concentration in the USW+sh RNA group was higher than that in the USW+empty group（P<0.05）.).（7）RT-PCR results showed that after the expression of SPCA1encoding gene was silenced by small hairpin RNA,the expressions of oxidative stress protective factors SOD and Mn SOD in the USW+sh RNA group were lower than those in the USW+empty group（P<0.05）;SPCA1expression results showed that,the USW+sh CON group had higher SPCA1 expression than OGD/R+sh CON group（P<0.05）.In conclusion:（1）USW can reduce the damage of N2a cells after OGD/R;（2）OGD/R inhibits the expression of Golgi membrane protein SPCA1;（3）USW can up-regulate Golgi-related proteins SPCA1 and GM130,Wnt signaling pathway proteinβ-catenin,and its neuroprotective mechanism may be as follows:by up-regulating SPCA1 expression,inhibiting over-activated Golgi stress and reducing oxidative stress injury,Wnt signaling pathway may be involved;（4）After sh SPCA1,OGD/R led to cell apoptosis,increased Ca2+concentration and ROS concentration,and OGD/R led to more obvious cellular oxidative stress;（5）The protective effect of USW on OGD/R injury may be achieved by inhibiting the down-regulation of SPCA1.23 Figures,16 Tables,184 References...