| BackgroundSpinal cord injury is mainly divided into traumatic spinal cord injury and non-traumatic spinal cord injury,and traumatic spinal cord injury is the most common,which often leads to serious dysfunction of the limbs below the injured segment.Spinal cord injury will not only bring serious physical and psychological harm to the patients,but also cause a huge economic burden to the whole society.The prevention,treatment and rehabilitation of spinal cord injury has become a major topic in the medical field.As a medical intervention,anesthesia is closely related to the life,health and safety of surgical patients.With the improvement of people's educational background and the change of health concept,the safety of operation and the risk of anesthesia have been widely concerned by patients and their families.Intraspinal anesthesia is one of the commonly used anesthetic methods,mainly through the puncture technique to inject anesthetic drugs into the subarachnoid space or epidural space of the spinal canal,that is,the nerve root is blocked,so that the corresponding area innervated by the nerve root produces anesthetic effect,collectively referred to as intraspinal anesthesia.The effect of intraspinal anesthesia is accurate and has low requirements for instruments and equipment.it can provide sufficient analgesia for clinical surgery and significantly avoid respiratory complications caused by airway-related operations.and can maintain communication with conscious patients during the whole operation,which has been widely used in surgery.Improper intraspinal puncture may cause motor dysfunction,local vascular damage and local microcirculation disturbance in a certain period of time,and the condition of spinal cord injury is also aggravated by the above-mentioned factors.Electroacupuncture isan effective acupuncture method,which has been successfully used to treat a variety of diseases related to spinal cord injury.it can effectively restore the damaged central nervous system function,inhibit nerve cell apoptosis,improve the microenvironment of the injured area and so on.MicroRNA(miRNA)is a type of small non-coding RNA with a length of about22 nt.In most cancers,tumor proto-oncogene expression is affected by miRNA inhibition to participate in various biological processes of tumor cells,including cancer cell metabolism,proliferation,invasion and migration,and induction of cell autophagy and apoptosis.One of the pathologies after spinal cord injury is the apoptosis and inflammatory response of neurons and axon cells.Studies have shown that the pathophysiological changes caused by spinal cord injury are closely regulated by the expression of specific miRNAs.After spinal cord injury,multiple miRNAs regulate the inflammatory response of spinal cord injury by targeting the regulation of mRNAs of anti-inflammatory cells and pro-inflammatory cytokines.However,it is unclear whether electroacupuncture therapy repairs spinal cord injury by regulating miRNA.Therefore,this study constructs a model of spinal cord injury caused by acupuncture in rats and uses RNA sequencing technology to analyze whether electroacupuncture repairs spinal cord injury caused by acupuncture by regulating miRNA and explore its related mechanisms.Part ? Observation on the Behavior and Morphology of Spinal Cord in Acupuncture Rat Models Treated with ElectroacupunctureObjective: To detect the effect of electroacupuncture on spinal acupuncture injury.Methods: Eighteen SD rats were divided into Sham group,puncture injurymodel(SCI)group and puncture injury + electroacupuncture(SCI + EA)group(6rats in each group).The Sham group only needs to perform surgical procedures on the lower back,without other operations;SCI group: spinal puncture,but no electroacupuncture;SCI + EA group: daily electroacupuncture stimulation of Dazhui point three days after acupuncture injury,Mingmen,Jiaji points for treatment.The motor function of rats in each group was evaluated by BBB scoring,and the pathological morphology was observed in combination with HE staining.The expression of IL-1? and TNF-? in spinal cord tissue was detected by enzyme-linked immunosorbent assay(ELISA)method.Results:(1)BBB scores showed that after 7 days of acupuncture in the spinal cord of rats,the BBB score(20.50 ± 0.55)of the SCI + EA group was significantly higher than that of the SCI group(19.17 ± 0.75).After 21 days of electroacupuncture,the SCI + The BBB score(20.67 ± 0.52)of the EA group was not significantly different from that of the Sham group(21.00 ± 0.00),and was significantly higher than that of the SCI group(19.83 ± 0.75).Electroacupuncture can obviously improve the motor function of rats with spinal acupuncture injury.(2)Observing the results of HE staining of pathological morphology of spinal cord,it was found that after electroacupuncture,spinal cord tissue was more complete,the outline of cell morphology and structure was clearer,the number of inflammation-related cells decreased,and the number of normal nerve cells increased.(3)ELISA and qRT-PCR were used to detect the inflammatory factors IL-1? and TNF-?.After 21 days of acupuncture,the expression levels of TNF-? and IL-1? in the SCI + EA group were 616.25 and 699.16 pg / mg,respectively.It was significantly lower than the SCI group(718.01,1366.62 pg / mg).It is shown that the expression of inflammatory factors in spinal cord tissues is down-regulated afterelectroacupuncture treatment,and electroacupuncture treatment may improve spinal cord injury by reducing the release of inflammatory factors.Part ? Differential Expression Analysis and Functional Annotation of Electroacupuncture for Repairing Spinal Needle InjuryObjective:To screen the differentially expressed miRNAs in the model of spinal cord injury by electroacupuncture using small RNA sequencing technology.Methods:The small RNA sequencing technology was used to determine the miRNA expression profile of SCI group and SCI + EA group rats.Differentially expressed miRNAs were analyzed between GO groups and KEGG metabolic pathway enrichment methods.Differentially expressed miRNAs were analyzed.The detection of random miRNA expression levels determines the reliability of sequencing results.Results :(1)Using small RNA sequencing technology,a total of 168 differentially expressed miRNAs were screened.Among the SCI + EA group,29 were significantly up-regulated and 139 were significantly down-regulated.(2)Multi-database analysis of target genes that differentially expressed miRNA in sequencing results,predicting a total of 36,336 target genes.These target genes are significantly enriched in GO entries such as phosphorylation,transcription regulation,DNA-templated,and protein phosphorylation,and are significantly enriched in signal pathways such as adhesion,insulin and axon guidance.(3)qRT-PCR verification results show that compared with the SCI group,in the SCI + EA group,rno-miR-219a-5p,rno-miR-128-3p,rno-miR-136-5p,rno-miR-7b The expression was down-regulated,and the expression of rno-miR-486 was up-regulated,and the expressions of rno-miR-486 and rno-miR-7b were not significant.The expression trend of qRT-PCR results was similar to that of sequencing results,indicating that the sequencing results are accurate and reliable.Part ? Effect of miR-136-5p on microglial-related functions in ratsObjective : To further explore the function of miR-136-5p in regulating microglia during the repair of acupuncture spinal cord injury by electroacupuncture.Methods: qRT-PCR was used to detect the expression of three target genes after transfection of miR-136-5p mimics in microglial cells.The microgels were overexpressed by CCK-8 and flow cytometry.Proliferation and Apoptosis of Plasma Cells.Results:(1)Three target genes verified by qRT-PCR showed that miR-136-5p mimics significantly inhibited the expression of Sema3 a,Vangl2,and Gng3,indicating that the expression of Sema3 a,Vangl2,and Gng3 was related to miR-136-5p expression.(2)Compared with the control group,after microglial cells interfered with miR-136-5p expression,the cell proliferation ability decreased and the apoptosis increased.Conclusion: In this study,small RNA sequencing was used to study the differential expression profiles of electroacupuncture in repairing spinal cord acupuncture and non-electroacupuncture.The results showed that miR-136-5p expression was down-regulated after electroacupuncture treatment.It was found in in vitro microglial studies that miR-136-5p promotes microglial apoptosis and inhibits its cell proliferation ability;overexpression of miR-136-5p significantly inhibits Sema3 a,Vangl2,Gng3 mRNA expression,and Vangl2 protein expression.Experimental studies have shown that electroacupuncture can reduce the expression of miR-136-5p,inhibit the proliferation of microglia and promote its apoptosis,and reduce the expression of Vangl2 protein,thereby promoting the recovery of spinal cord injury. |
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