Study On Protective Mechanism Of Troxerutin In Spinal Cord Injury

Background : Spinal cord injury(SCI)is a common and highly destructive neurological disease worldwide,causing changes in sensory,motor dysfunction,muscle strength,and reflexes in the corresponding segments.It mainly includes primary damage and secondary damage.Primary damage occurs suddenly,causing damage to neurons and vasculature.It is irreversible damage that cannot be treated clinically;secondary damage It appears after a few hours of primary spinal cord injury,and its pathological changes can be reversed.The damage mechanism is related to the interaction of spinal cord hemorrhage,edema,inflammatory cell infiltration,microcirculation disorder,and apoptosis.At present,spinal cord injury is mainly treated by surgical decompression and fixation,nutritional nerve drugs and hormones,stem cell transplantation and hyperbaric oxygen to protect the spinal cord tissue at the injured site and promote the recovery of nerve function.However,these treatments have not achieved satisfactory results.Troxerutin is a semi-synthetic derivative of natural bioflavonoid rutin,with a wide range of pharmacological effects,such as antioxidant,anti-inflammatory,inhibiting red blood cell platelet aggregation,improving microcirculation,promoting neovascularization and protecting nerves.At present,it is mainly used for the treatment of diseases such as edema,hemorrhoids,diabetic complications,venous thrombosis,and cardiovascular and cerebrovascular diseases.However,its potential therapeutic efficacy in spinal cord injury has never been found.In order to explore the effect of troxerutin on spinal cord tissue,this study treated SCI rat models with troxerutin,and performed bioinformatics analysis of differential gene expression in the whole genome of the spinal cord tissue,and found that the differential gene m RNA in the troxerutin group Levels of expression increased significantly.Finally,Western Blot was used to verify the expression of differential genes,and the results showed that the protein expression results were consistent with the sequencing results;the findings of this study provided a certain scientific basis for the clinical treatment of spinal rutin in spinal cord injury.1 : Effect of troxerutin on spinal motor function and morphological functionObjectiveInvestigate whether troxerutin has a recovery effect on hindlimb motor function_and nerves in spinal cord injured rats,and analyze the effect of troxerutin on spinal cord injured rats,compared with normal rats andcontrol rats at different time periods Differences in hindlimb motor function and local morphological function of spinal cord injury.Methods1.Establishment of spinal cord injury model in SD rats,Sham group:Laminectomy only,no spinal cord injury.SCI group and troxerutin group:Spinal Cord Injury Model Based on Allen ’s Strike.2.Troxerutin group: troxerutin injection via tail vein 30 minutes after spinal cord injury,6mg/kg,injection per day for 4 weeks.SCI group and Sham group were given the same amount of 0.9% sodium chloride injection via tail vein once a day for 4 weeks.3.Hind limb motor function assessment by using Basso,Beattie and Bresnahan(BBB)exercise rating scale at 1,2,and 4 weeks after surgery.4.Spinal cord tissue sections were stained with HE,and the local microcirculation of spinal cord tissue was observed with an optical microscope.Result1.The BBB scores of the SD rats in the three groups before surgery were all 21 points;the BBB scores in the Sham group after surgery were the same as the BBB scores before the operation,At any observation time,the BBB score was significantly higher than that of troxerutin group and SCI control group.2.At the first week of postoperative medication,the BBB score of theSCI control group and the troxerutin group was not statistically significant.3.The BBB scores at the 2nd and 4th week postoperative medication showed that the lower limb motor function of the troxerutin group was significantly better than that of the SCI group(P<0.05).4.The spinal cord morphology and structure of the Sham group were complete,and the cell morphology was complete and uniformly distributed.5.In the SCI group,there were a large number of vacuoles in the spinal cord,some neurons were dissolved,a small number of inflammatory cells infiltrated around the spinal cord,the spinal cord structure was disordered,and glial scars were generated.6.The vaccinations in the spinal cord tissue of the troxerutin group were reduced,and at the same time,vascular hyperplasia was seen at the injury site.ConclusionBased on the BBB score of the lower limb motor function of the rats,we found that after 2 weeks of troxerutin treatment,the BBB score of the troxerutin group was higher than that of the SCI group.After 4 weeks of treatment,the difference was more obvious.By HE staining,we found that the vacuoles in the spinal cord tissue of the troxerutin group were reduced.At the same time,vascular hyperplasia at the injury site can be seen.The recovery of motor function in spinal cord injured rats may be related to troxerutin in the chronic phase after spinal cord injury,which promotesvascular proliferation in the injured area.It is related to improving the microcirculation of the spinal cord injury site and reducing the expression of apoptotic factors,so as to protect the nerve.the second part2 : Effect of troxerutin on gene expression of rat spinal cord injury tissueObjectiveIn order to detect the molecular mechanism of the protective effect of troxerutin on spinal cord injury,spinal cord tissues from the SCI control group and the troxerutin group were collected for transcriptome sequencing.Western blot was used to verify the expression of the differential gene caspase-6 in the spinal cord tissue of each group.Methods1.After 4 weeks of treatment of spinal cord injured rats with troxerutin,the spinal cord tissues from the troxerutin and SCI control groups were collected and sent to the company for sequencing.2.Sequencing the transcriptome on the Hiseq X Ten sequencing platform.3.To ensure the repeatability and reliability of the results,targetedquality control checks are applied at different stages of the analysis.4.Differentially expressed genes were screened by edge_R software,and GO function and KEGG pathway enrichment of differentially expressed genes were analyzed by go_enrichment software.5.Verify the expression of the differentially expressed gene caspase 6,collect the spinal cord tissues from each group,and take them out into a mixture of lysate and protease inhibitor.6.After grinding,cryogenic centrifugation,and taking the supernatant,etc.,the final BCA kit measures the protein concentration,and then performs electrophoresis,transfer,and blocking.7.Incubate the primary antibody for 12 h at 4℃and incubate the secondary antibody for 2 h at room temperature;8.The exposure meter exposes and analyzes the gray value of each group of bands using Image J.Result1.Data quality control The library has a uniform base distribution and N% is within a reasonable range;it meets the subsequent analysis requirements.2.Troxerutin significantly alters gene expression in injured spinal cord After 4 weeks of troxerutin treatment,the m RNA expression of differential genes in the troxerutin group increased significantly(P<0.05).Compared with the SCI group,there were 261 differentiallyexpressed genes in the troxerutin group,of which 217 were upregulated and 44 were downregulated.3.Western blot results showed that the expression of caspase 6 in the SCI group was the highest.The expression of caspase 6 protein in the troxerutin group was significantly lower than that in the SCI group(P<0.05),but higher than that in the Sham group.4.Enrichment analysis of differentially expressed genes GO and KEGG pathway This experiment uses the GO and KEGG databases to annotate 261 DEGs.This experiment uses the GO and KEGG databases to annotate 261 DEGs.There are several signaling pathways such as troxerutin in the treatment of spinal cord injury and enrichment of KEGG.Conclusion1.Through differential gene analysis of m RNA sequencing,we found that compared with the SCI group,there were 261 differentially expressed genes in the troxerutin group,of which 217 were up-regulated and 44down-regulated.2.GO and KEGG enrichment analysis showed that troxerutin was mainly concentrated in signal pathways such as Wnt,Hippo and TGF-β,and Apoptosis,suggesting that troxerutin has a broader protection mechanism for SCI.3.Western blot results confirmed that the expression of caspase 6protein in the troxerutin group was significantly lower than that in the SCIgroup after 4 weeks of troxerutin treatment.The results suggest that troxerutin can promote the recovery of spinal cord injury by inhibiting the apoptotic pathway and reducing the expression of apoptotic protease caspase 6.Apoptosis plays an important role in the process of spinal cord injury.After spinal cord injury,spinal cord ischemia and hypoxia can cause local internal environment imbalance,cell necrosis,and induce apoptosis,which leads to the expansion of the scope of the injury and the severity of the injury.Therefore,by inhibiting the apoptotic pathway of nerve cells,it can have a protective effect on motor function injury caused by spinal cord injury.