| Introduction:Spinal cord ischemia reperfusion injury(SCIRI)is a phenomenon that spinal cord injury is more severe after reperfusion and is characterized by more obvious signs and morphological changes of nerve damage.Spinal cord injury includes primary injury and secondary injury.spinal cord neurons of SCIRI after a certain period of ischemia show no improvement in nerve function after blood reperfusion,but delayed spinal cord nerve dysfunction or even paralysis.Therefore,inhibition of neuronal apoptosis or reduction of Delayed neuronal death has become an important topic in the treatment of spinal cord injury.However,at present,most researches are still focused on protein,factor and other levels,and the molecular mechanism involved in transcription level is not yet clear.In particular,pathological molecular mechanism of SCIRI has not been clarified,which has become a huge obstacle to SCIRI drug and technology research and development.Therefore,it is urgent to reveal the pathological molecular mechanism of SCIRI.Micro RNA(mi RNA)is a short chain non-coding RNA molecule composed of 18-24 nucleotide molecules.mi RNA is a class of metabolic regulatory molecules with strong functions,which can specifically silence m RNA and regulate gene expression at the post-transcriptional level.mi RNA exists in the central nervous system(CNS)of mammals,including the brain and spinal cord,and plays an important regulatory role in the pathophysiological process of central nervous system injury,injury repair and degenerative diseases.Sevoflurane,an inhaled anesthetic,is widely used in clinical practice because of its characteristics such as rapid anesthesia,rapid resuscitation and stable hemodynamics.Recently,sevoflurane preconditioning regulates mi RNA,which has a protective effect on many organ injuries,including brain,heart,lung and liver,but whether sevoflurane affects spinal cord ischemia reperfusion injury by regulating mi RNA is rarely reported.Objective: this study aims to through mi RNAs chip technology,spinal cord ischemia/reperfusion injury in rats in the model and sevoflurane pretreatment in rat spinal cord ischemia-reperfusion injury model,the selection of screening differentially expressed mi RNAs,and further validation in rat,find out the target genes,explore the precise mechanism of spinal cord ischemia/reperfusion injury,through sevoflurane pretreatment adjusting the mi RNA.Methods:Part I Screening and validation of mi RNA expression profiles after SCIRI and sevoflurane pretreatment in rats 1.Male sprague-Dawley rats,weighing beween 280-320 g,were divided into three groups:Sham,I/R,SEVO+ I/R.Temporary aortic occlusion induces different extents of spinal cord ischemia.After 24 h of ischemia/reperfusion,rats were decapitated for pulp extraction(L4-6).2.Three groups of fresh rat spinal cord tissue were sent to Shanghai Jikai Gene Chemical Technology for mi RNA expression profiling of microarray chips.The obtained data were mapped to heat maps and analyzed by MEV software.we select the most significant mi RNAs.3.Verify the results of microarray screening.mi R-199a-5p decreased after spinal cord ischemia reperfusion injury in rats.3.1 qrt-pcr was used to verify the mi RNA content in the sham group and the model group.3.2 TUNEL staining was performed on the sham group and the model group for further verification.4.The time points of death at 0h,6h,12 h,24h,48 h and 72 h after spinal cord reperfusion injury in rats were selected.Specimens were collected and the content of mi R-199a-5p in spinal cord tissues was detected by qrt-pcr method to determine the optimal time point for sampling.5.Verification of chip screening results: sevoflurane preconditioning upregulates the protective effect of mi R-199a-5p on spinal cord ischemia/reperfusion injury.5.1 Grouping of experimental animals 18 healthy male SD rats(280-320g)were randomly divided into 3 groups: sham group(S group),model group(I/R group),and sevoflurane + model group(SEVO+I/R group).5.2 At 24 h after ischemia/reperfusion,the rats were sacrificed and spinal cord tissue(l4-6)was taken after the evaluation of neural motor function by BBB score.5.3 Apoptosis of cells in each group was observed by TUNEL staining.5.4 Evans blue(EB)determination of blood-spinal cord barrier(BSCB)permeability,5.5 qreal-time PCR was used to detect the mi R-199a-5p content in the spinal cord tissues of each group.5.6 Protein levels of caspase-9 and bcl-2 in spinal cord tissues were determined by Western blot.Part II Rats were intrathecally injected with lentivirus,and mi R-199a-5p was overexpressed or silenced to verify the protective effect of mir-199a-5p on spinal cord ischemia reperfusion injury The rat spinal cord I/R injury model was established,and the Basso Beattie Bresnahan scoring,Evans blue staining,HE staining,and TUNEL assay were used to assess the I/R-induced spinal cord injury.In addition,neuron expression was detected by immunostaining assay,while the expressions of p-ERK,ERK,p-JNK,JNK,caspase-9,Bcl-2,and ECE1 were evaluated by Western blot.The integrity of BSCB in spinal cord tissue was determined by Evans blue staining,and immunofluorescence histochemistry single-label staining was performed to confirm the expression of mi R-199a-5p in neuronal cells.Part III Verification of the targeted-regulating relationship between mi R-199a-5p and ECE1 to protect SCIRI in rat 1.The prediction bioinformatics software of the target genes of mi R-199a-5p and the construction of dual-luciferase reporter gene to identify the targeted-regulating relationship between mi R-199a-5p and ECE1.2.Lentivirus mediated ECE1 sh RNA silenced the target gene ECE1 in rats,to verifying that mi R-199a-5p protects rat SCIRI by regulating the target gene ECE1.the rat SCIRI model was established,and the Basso Beattie Bresnahan scoring,Evans blue staining,HE staining,and TUNEL assay were used to assess the I/R-induced spinal cord injury.In addition,while the expressions of p-ERK,ERK,p-JNK,JNK,caspase-9,Bcl-2,and ECE1 were evaluated by Western blot.Part IV Sevoflurane preconditioning protects against spinal cord ischemia/reperfusion injury by up-regulating micro R-199a-5p in rats.Twenty-fore SD rats were randomly divided into: group of sham(S group),group of ischemia-reperfusion(I/R group),group of sevoflurane and control(SEVO+I/R group).S group received the same operation,but did not inhale sevoflurane or close the aortic arch;The SCIRI model was established after 14 min of the aortic arch of the non-invasive arterial clip in I/R group.SEVO+I/R group,before setting up the SCIRI model,inhaled 2.4% of sevoflurane 3 h.Basso Beattie Bresnahan scoring method was used to evaluate neuromotor function and TUNEL staining to observe apoptosis.Evans blue(EB)determination of blood-spinal cord barrier(BSCB)permeability;real-time PCR detection of mi R-199a-5p content;Western blot determined the levels of Caspase-9 and Bcl-2 in spinal cord tissues.Results Part I Screening of mi RNA expression profiles after SCIRI and sevoflurane pretreatment in rats 1.Fold Change of mi R-199a-5p I/R group was 0.431 less than 0.5 compared with sham group.Fold Change of Sevoflurane pretreatment group was 2.39,greater than 2.0.mi R-199a-5p can significantly reverse spinal cord ischemia reperfusion injury after sevoflurane.2 To verify the chip screening results,mi R-199a-5p decreased after spinal cord ischemia reperfusion injury in rats.2.1 qrt-pcr verified that mi R-199a-5p in I/R group was significantly lower than that in sham group.2.2 TUNEL staining showed that after spinal cord/ischemia/reperfusion,the nuclei of neurons were larger,and some cells showed nuclear condensation 3.Hyperchromatic,on the side of the cell;Compared with group S,the apoptosis rate of I/R group was significantly increased.q RT-PCR method was used to detect the content of mi R-199a-5p in the spinal cord of the I/R group at each time point,and the expression of mi R-199a-5p at 24 h was the lowest.4.To verify the results of microarray screening,mi R-199a-5p decreased after spinal cord ischemia reperfusion injury in rats.4.1 compared with the S group,the motor function score of the I/R group was decreased,the structure of the spinal cord was disorganized with HE staining,and the nuclei were irregular.Sevoflurane pretreatment improved the neurological function score and histopathological changes.4.2 TUNEL staining showed that apoptosis rate increased after spinal cord ischemia/reperfusion injury,and the number of apoptosis decreased after sevoflurane pretreatment.4.3 BSCB permeability increased after spinal cord ischemia/reperfusion injury,sevoflurane pretreatment reduced BSCB permeability,and evansblue extravasation was significantly reduced.4.4 Western blot showed that after sevoflurane pretreatment,compared with spinal cord ischemia/reperfusion injury,bcl-2 was significantly increased and caspase-9 protein was significantly decreased.Part II Rats were intrathecally injected with lentivirus,and mi R-199a-5p was overexpressed or silenced to verify the protective effect of mir-199a-5p on spinal cord ischemia reperfusion injury 1.compared with sham group,BBB scores of rats in each I/R group were decreased at 6h,12 h,24h,48 h.After transfection with lv-mi R-199a-5p mimics or lv-mi R-199a-5p inhibitor,neurological function scores of rats were increased or decreased.2.Pathological conditions of spinal cord injury were detected by HE staining.Nuclear fragmentation was observed in the IR group.Transfection of LV-mi R-199a-5p mimics spinal cord tissue injury was significantly reduced,and transfection of LV-mi R-199a-5p inhibitor,spinal cord tissue damage was more serious 3.TUNEL staining results showed that the number of apoptotic neurons in I/R group increased significantly compared with Sham group.Compared with the I/R group,the number of apoptotic cells was significantly reduced after the transfection of lv-mi R-199a-5p mimics,and the number of apoptotic cells was significantly increased after the transfection of lv-mi R-199a-5p inhibitor.4.Evans blue staining was used to determine the integrity of spinal cord tissue BSCB.Evans blue infiltrated into spinal cord parenchyma in group I/R at 24 h,which broke BBSB.After transfection with lv-mi R-199a-5p mimics,EB infiltration was significantly reduced,and after transfection with lv-mi R-199a-5p inhibitor,evanth blue infiltration was significantly increased.5.q Real-time PCR was used to detect the expression of mi R-199a-5p in spinal cord tissues.Compared with Sham group,mi R-199a-5p expression was significantly decreased in I/R group and I/R+NC group.Compared with the I/R group,mi R-199a-5p expression level was significantly increased in the I/R+M group.However,mi R-199a-5p expression level was significantly reduced in the I/R+I group,indicating that lentivirus transfection to rats was successful.6.Compared with Sham group,the expression of Bcl-2 was decreased in the I/R group and I/R+NC group at 24 h after ischemia/reperfusion,while caspase-9,p-JNK,and p-ERK were significantly increased in the I/R group and I/R+NC group.Compared with the I/R group,Bcl-2 increased in the I/R+M group,while caspase-9,p-jnk and p-erk proteins decreased significantly.In the I/R+I group,Bcl-2 decreased and caspase-9,p-JNK and p-ERK protein expressions significantly increased.7.Immunofluorescence histochemical single-label staining was performed to determine the expression of mi R-199a-5p in neurons.Compared with Sham group,the density of neurons in IR group and I/R+NC group increased significantly.Compared with I/R+NC group,the density of neurons in I/R+M group increased significantly,while that in I/R+I group decreased significantly.Part III Verification of the targeted-regulating relationship between mi R-199a-5p and ECE1 to protect SCIRI in rat 1.The mi R-199a-5p had binding sites with wild-type ECE1.The mi R-199a-5p inhibited the luciferase activity of ECE1 wild type vector,which indicates ECE1 gene could be targeted regulated by mi R-199a-5p.2.mi R-199a-5p negatively regulated ECE1,and silencing the ECE1 gene also protected the rat spinal cord injury after I/R.Neurological function score were significantly increased at 6h,12 h,24h and 48 h after ECE1 was silenced.compared with the I/R group,HE staining in the ECE1-sh RNA group improved histopathological changes and brought them closer to normal tissues.TUNEL staining showed that the number of apoptotic cells decreased significantly.Evans blue staining showed that Evans blue extravasation decreased and BSCB permeability decreased.Western blot method showed compared with the I/R group,the expression of bcl-2,in the ECE1-sh RNA group increased,while the expression of Caspase-9,p-JNK,p-ERK protein and ECE1 protein decreased significantly.Conclusion : 1.mi RNA microarray analysis was performed to select significantly differentially expressed mi R-199a-5p to study its mechanism of action in spinal cord ischemia reperfusion injury for the first time.2.Sevoflurane pretreatment may reduce BSCB permeability and neuronal apoptosis by up-regulation of mi R-199a-5p and protect SCIRI of rats.3.mi R-199a-5p is abundantly expressed in neurons.Overexpression of mi R-199a-5p can reduce apoptosis of rat spinal cord tissue cells,while silence of mi R-199a-5p expression can promote apoptosis.4.ECE1 is the target gene of mi R-199a-5p.By down-regulating ECE1,mi R-199a-5p can reduce apoptosis of spinal cord cells in rats and protect spinal cord from ischemia reperfusion injury in the model of SCIRI. |
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