| Spinal cord injury is a traumatic disease that occurs in the central nervous system.It can lead to motor function deficits,limb sensory disturbances,and autonomic dysfunction,which seriously affects the patient’s physical,psychological,and social health status.So far,there is no ideal clinical treatment methods.As a combination of traditional Chinese medicine acupuncture and modern medical technology,electro-acupuncture has been widely used in clinical practice,and its good clinical effect on spinal cord injury has been widely recognized by the medical community.Previous studies have found that electroacupuncture promotes the repair of spinal cord injury by inhibiting the Notch signaling pathway,but the molecular mechanism remains unclear.Therefore,this study explored the molecular mechanism of electroacupuncture inhibiting Notch signaling pathway in spinal cord injury to repair spinal cord injury.This study is divided into three parts.The first part used electroacupuncture to treat a rat clamp-type spinal cord injury model,to study the factors H19 and EZH2 that may play a role in the Notch signaling pathway in spinal cord injury,and to study the effect of electroacupuncture on H19,EZH2 and endogenous neural stem cells.In the second part,a PC-12 cell oxygen and glucose deprivation model was constructed in vitro to simulate the internal environment of spinal cord injury in rats,and the H19,EZH2 and Notch signaling pathways were studied by silencing H19,overexpressing EZH2 and using EZH2 inhibitors.Furthermore,the effects of H19,EZH2 and Notch signaling pathways on apoptosis and viability of PC-12 cells in an oxygen-glucose deprivation model were investigated.In the third part,in vivo experiments were conducted to treat rats(injected with H19 overexpressing lentivirus)with spinal cord injury by electroacupuncture to explore that electroacupuncture promoted the proliferation of endogenous neural stem cells and inhibited their differentiation into astrocytes by inhibiting the H19/EZH2/Notch signaling axis,thereby promoting the Mechanisms of recovery from spinal cord injury inrats.This study deepens our understanding of the mechanism of electroacupuncture inhibiting the Notch signaling pathway in the treatment of spinal cord injury,and proves that the H19/EZH2/Notch signaling axis is expected to become a new important potential target for the treatment of spinal cord injury,and it provides important enlightenment for a new way of clinical electroacupuncture treatment of spinal cord injury.Part 1:Effects of electroacupuncture on the expression of endogenous neural stem cells,EZH2 and H19 in rats with spinal cord injuryObjective:To study.the effect of electroacupuncture on endogenous neural stem cells,EZH2 and H19 in rats with spinal cord injury.Methods:A total of 3 groups were set up in this experiment.They are:sham operation group(Sham),model group(SCI)and model+electroacupuncture group(SCI+EA).After successful modeling,the SCI+EA group received electroacupuncture every day.After 28 days,spinal cord tissue samples from all groups were used for the following experiments:1.Immunofluorescence staining to detect the expression of Nestin protein and GFAP protein;2.Western blot to detect the expression of EZH2;3.RT-qPCR to detect the expression of H19.Experimental data were compared between groups using Tukey’s comparison method with one-way ANOVAs.Results:1.Immunofluorescence results showed:Compared with the Sham group,the expression of Nestin in the SCI group was decreased,and the expression of GFAP was increased;compared with the SCI group,the expression of Nestin in the SCI+EA group was increased,and the expression of GFAP was decreased.2.Western blot results showed:Compared with the Sham group,the expression of EZH2 in the SCI group was increased(P<0.01);compared with the SCI group,the expression of EZH2 in the SCI+EA group was decreased(P<0.01).3.RT-qPCR results showed:Compared with the Sham group,the expression of H19 in the SCI group was increased(P<0.01);compared with the SCI group,the expression of H19 in the SCI+EA group was decreased(P<0.01).Conclusion:Electroacupuncture treatment can inhibit the expression of H19 and EZH2,promote the proliferation of endogenous neural stem cells and inhibit their differentiation into astrocytes.Part 2:Study on the relationship between H19,EZH2 and Notch signaling pathway based on PC-12 cell oxygen and glucose deprivation modelObjective:To study the relationship between H19,EZH2 and Notch signaling pathways,and to explore the effects of H19,EZH2 and Notch signaling pathways on apoptosis and viability of PC-12 cells in an oxygen-glucose deprivation model.Methods:This experiment includes 4 stages,the research objects of stage 1 are H19 and Notch signaling pathway,the research objects of stage 2 are H19 and EZH2,the research objects of stage 3 are EZH2 and Notch signaling pathway,and the research objects of stage 4 are H19,EZH2 and Notch signaling pathway.Stage 1:Four groups were set up by constructing a PC-12 cell oxygen-glucose deprivation model and silencing H19.They are:negative control group(NC),oxygen-glucose deprivation group(OGD),oxygen-glucose deprivation+unregulated H19 group(OGD+si-NC)and oxygen-glucose deprivation+down-regulated H19 group(OGD+si-H19).The following experiments were performed on the above groups:1.RT-qPCR to detect the expression of H19;2.Western blot to detect the expression of Notch signaling pathway-related proteins;3.Flow cytometry to analyze cell apoptosis;4.CCK-8 to detect cell viability.Stage 2:Six groups were set up by constructing a PC-12 cell oxygen-glucose deprivation model,silencing H19 and overexpressing EZH2.They are:NC group,OGD group,OGD+si-NC group,OGD+si-H19 group,OGD+down-regulated H19+EZH2 not overexpressed group(OGD+si-H19+oe-NC)and OGD+down-regulated H19+o verexpressing EZH2 group(OGD+si-H 19+oe-EZH2).The following experiments were performed on the above groups:1.RT-qPCR to detect the expression of H19;2.Western blot to detect the expression of EZH2;3.Flow cytometry to analyze cell apoptosis;4.CCK-8 to detect cell viability.Stage 3:Three groups were set up by constructing a PC-12 cell oxygen-glucose deprivation model and using EZH2 inhibitors.They are:NC group,OGD group and OGD+GSK126 group(OGD+GSK126).The following experiments were performed on the above groups:1.Western blot to detect the expression of Notch signaling pathway-related proteins;3.Flow cytometry to analyze cell apoptosis;4.CCK-8 to detect cell viability.Stage 4:Seven groups were set up by building a PC-12 cell oxygen-glucose deprivation model,silencing H19,overexpressing EZH2,and using EZH2 inhibitors.They are:NC group,OGD group,OGD+si-NC group,OGD+si-H19 group,OGD+si-H 19+oe-NC group,OGD+si-H 19+oe-EZH2 group and OGD+GSK126 group.The following experiments were performed on the above groups:1.RT-qPCR to detect the expression of H19;2.Western blot to detect the expression of EZH2 and Notch signaling pathway-related proteins;3.Flow cytometry to analyze cell apoptosis;4.CCK-8 to detect cell viability.Experimental data were compared between groups using Tukey’s comparison method with one-way ANOVAs.Results:1.Results of Stage 1:RT-qPCR results showed:Compared with the NC group,the expression of H19 in the OGD group was increased(P<0.01);compared with the OGD group,the expression of H19 in the OGD+si-H19 group was decreased(P<0.01).Western blot results showed:Compared with the NC group,the expression of Notch1、Notch3、Notch4、Hes1、PS-1 in the OGD group was increased(P<0.01);compared with the OGD group,the expression of Notchl、Notch3、Notch4、Hesl、PS-1 in the OGD+si-H19 group was decreased(P<0.01).Flow cytometry results showed:Compared with NC group,the apoptosis rate of PC-12 in OGD group was increased(P<0.01);compared with the OGD group,the apoptosis rate of PC-12 in the OGD+si-H 19 group was decreased(P<0.05).The results of CCK-8 showed:Compared with the NC group,the viability of PC-12 in the OGD group was decreased(P<0.01);compared with the OGD group,the viability of PC-12 in the OGD+si-H19 group was increased(P<0.01).2.Results of Stage 2:RT-qPCR results showed:Compared with the NC group,the expression of H19 in the OGD group was increased(P<0.01);compared with the OGD group,the expression of H19 in the OGD+si-H19 group was decreased(P<0.01);compared with the OGD+si-H19 group,there was no difference in the expression of H19 in the OGD+si-H19+oe-EZH2 group.Western blot results showed:Compared with the NC group,the expression of EZH2 in the OGD group was increased(P<0.01);compared with the OGD group,the expression of EZH2 in the OGD+si-H19 group was decreased(P<0.01);compared with the OGD+si-H19 group,the expression of EZH2 in the OGD+si-H19+oe-EZH2 group was increased(P<0.01).Flow cytometry results showed:Compared with NC group,the apoptosis rate of PC-12 in OGD group was increased(P<0.01);compared with the OGD group,the apoptosis rate of PC-12 in the OGD+si-H19 group was decreased(P<0.01);compared with OGD+si-H19 group,the apoptosis rate of PC-12 in OGD+si-H19+oe-EZH2 group was increased(P<0.01).The results of CCK-8 showed:Compared with the NC group,the viability of PC-12 in the OGD group was decreased(P<0.01);compared with the OGD group,the viability of PC-12 in the OGD+si-H19 group was increased(P<0.01);compared with the OGD+si-H19 group,the viability of PC-12 in the OGD+si-H19+oe-EZH2 group was decreased(P<0.01).3.Results of Stage 3:Western blot results showed:Compared with the NC group,the expression of Notchl、Notch3、Notch4、Hes1、PS-1 in the OGD group was increased(P<0.01);compared with the OGD group,the expression of Notch 1、Notch3、Notch4、Hes1、PS-1 in the OGD+GSK126 group was decreased(P<0.01).Flow cytometry results showed:Compared with NC group,the apoptosis rate of PC-12 in OGD group was increased(P<0.01);compared with the OGD group,the apoptosis rate of PC-12 in the OGD+GSK126 group was decreased(P<0.01).The results of CCK-8 showed:Compared with the NC group,the viability of PC-12 in the OGD group was decreased(P<0.01);compared with the OGD group,the viability of PC-12 in the OGD+GSK126 group was increased(P<0.01).4.Results of Stage 4:RT-qPCR results showed:Compared with the NC group,the expression of H19 in the OGD group was increased(P<0.01);compared with the OGD group,the expression of H19 in the OGD+si-H19 group was decreased(P<0.01);compared with the OGD+si-H19 group,there was no difference in the expression of H19 in the OGD+si-H19+oe-EZH2 group;compared with the OGD group,there was no difference in the expression of H19 in the OGD+GSK126 group.The results of Western blot detection of EZH2 showed:Compared with the NC group,the expression of EZH2 in the OGD group was increased(P<0.01);compared with the OGD group,the expression of EZH2 in the OGD+si-H19 group was decreased(P<0.01);compared with the OGD+si-H19 group,the expression of EZH2 in the OGD+si-H19+oe-EZH2 group was increased(P<0.01).The results of Western blot detection of Notch signaling pathway-related proteins showed:Compared with the NC group,the expression of Notch1、Notch3、Notch4、Hes1、PS-1 in the OGD group was increased(P<0.01);compared with the OGD group,the expression of Notchl、Notch3、Notch4、Hes1、PS-1 in the OGD+si-H19 group was decreased(P<0.01);compared with the OGD+si-H 19 group,the expression of Notchl、Notch3、Notch4、Hes1、PS-1 in the OGD+si-H19+oe-EZH2 group was increased(P<0.01);compared with the OGD group,the expression of Notch1、Notch3、Notch4、Hes1、PS-1 in the OGD+GSK126 group was decreased(P<0.01).Flow cytometry results showed:Compared with NC group,the apoptosis rate of PC-12 in OGD group was increased(P<0.01);compared with the OGD group,the apoptosis rate of PC-12 in the OGD+si-H19 group was decreased(P<0.01);compared with OGD+si-H19 group,the apoptosis rate of PC-12 in OGD+si-H19+oe-EZH2 group was increased(P<0.05);compared with the OGD group,the apoptosis rate of PC-12 in the OGD+GSK126 group was decreased(P<0.01).The results of CCK-8 showed:Compared with the NC group,the viability of PC-12 in the OGD group was decreased(P<0.01);compared with the OGD group,the viability of PC-12 in the OGD+si-H19 group was increased(P<0.01);compared with the OGD+si-H19 group,the viability of PC-12 in the OGD+si-H19+oe-EZH2 group was decreased(P<0.01);compared with the OGD group,the viability of PC-12 in the OGD+GSK126 group was increased(P<0.01).Conclusion:Inhibition of H19 can inhibit the EZH2/Notch signaling axis,thereby attenuating PC-12 cell damage induced by oxygen and glucose deprivation.Part 3:Mechanism of electroacupuncture inhibiting Notch signaling pathway via H19/EZH2 axis to promote recovery from spinal cord injury in ratsObjective:To study the mechanism of electroacupuncture promoting the recovery of spinal cord injury rats by inhibiting the H19/EZH2/Notch signaling axis to promote the proliferation of endogenous neural stem cells and inhibit the differentiation of endogenous neural stem cells to astrocytes.Methods:A total of 5 groups were set up in this experiment.They are:Sham group,SCI group,SCI+EA group,model+electroacupuncture+H 19 overexpression group(SCI+EA+Lenti-H19)and model+electroacupuncture+H 19 non-overexpression group(SCI+EA+Lenti-NC)).After successful modeling,the SCI+EA+Lenti-H19 group and the SCI+EA+Lenti-NC group were injected with H19 overexpressing lentivirus and blank lentivirus,respectively.SCI+EA group,SCI+EA+Lenti-H 19 group and SCI+EA+Lenti-NC group received electroacupuncture every day.During the experiment,the survival rate,body weight and BBB score of the rats were observed.After 28 days,spinal cord tissue samples were taken from all groups for the following experiments:1.RT-qPCR to detect the expression of H19;2.Western blot to detect the expression of EZH2 and Notch signaling pathway-related proteins;3.Immunofluorescence staining to detect Nestin and GFAP expression.Experimental data were compared between groups using Tukey’s comparison method with one-way ANOVAs.Results:1.RT-qPCR results showed:Compared with the Sham group,the expression of H19 in the SCI group was increased(P<0.01);compared with the SCI group,the expression of H19 in the SCI+EA group was decreased(P<0.01);compared with the SCI+EA group,the expression of H19 in the SCI+EA+Lenti-H 19 group was increased(P<0.01).2.The results of Western blot detection of EZH2 expression showed:Compared with the Sham group,the expression of EZH2 in the SCI group was increased(P<0.01);compared with the SCI group,the expression of EZH2 in the SCI+EA group was decreased(P<0.05);Compared with SCI+EA group,the expression of EZH2 in SCI+EA+Lenti-H 19 group was increased(P<0.01).3.The results of Western blot detection of Notch signaling pathway-related proteins showed:Compared with the Sham group,the expression of Notch1、Notch3、Notch4、Hes1、PS-1 in the SCI group was increased(P<0.01);compared with the SCI group,the expression of Notch1、Notch3、Notch4、Hes1、PS-1 in the SCI+EA group was decreased(P<0.05);Compared with SCI+EA group,the expression of Notchl、Notch3、Notch4、Hes1、PS-1 in SCI+EA+Lenti-H19 group was increased(P<0.01).4.Immunofluorescence results showed:Compared with the Sham group,the expression of Nestin in the SCI group was decreased,and the expression of GFAP was increased;compared with the SCI group,the expression of Nestin in the SCI+EA group was increased,and the expression of GFAP was decreased;compared with the SCI+EA group,the expression of Nestin in the SCI+EA+Lenti-H19 group was decreased,and the expression of GFAP was increased.5.The results of survival rate,body weight,and BBB score showed:The average survival rate,average body weight,and average BBB score of SCI group,SCI+EA group,SCI+EA+Lenti-H19 group,and SCI+EA+Lenti-NC group were all lower than the Sham group;the average survival rate,average body weight and average BBB score of SCI+EA group and SCI+EA+Lenti-NC group were higher than those of SCI group and SCI+EA+Lenti-H19 group.Conclusion:Electroacupuncture promoted the proliferation of endogenous neural stem cells and inhibited the differentiation of endogenous neural stem cells into astrocytes by inhibiting the H19/EZH2/Notch signaling axis,thereby promoting the recovery of spinal cord injury rats. |
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