| Objective:Observe the changes of behavior and spinal cord morphology after spinal cord injury in rats.Investigate the effects of curcumin on the microbial diversity of cecal and the influence of metabolic pathway by high-throughput sequencing.Explore the effect and pathway of curcumin on gene expression after spinal cord injury in rats to providing theoretical basis for clinical treatmentMethods:1.SD Rat spinal cord injury(SCI)_models were prepared using HI-0400 spinal cord beater,grouped into:(1)Sham control group(Sham),model group(SCI),model+curcumin group(CUR 50 mg/kg),model+curcumin group(CUR 100 mg/kg),model+curcumin group(CUR 200 mg/kg).Every group has 10 animals.The neurological BBB score and Rivlin test were used at 1d,3d,5d,7d and 14 d after operation.Rats were evaluated for behavior and HE staining was used to observe the morphological changes of rat spinal cord tissue.2.After aortic perfusion on the 14 th day after operation,the cecal tissue of sham,SCI and CUR group were taken,the total DNA of the microorganisms in the tissue was extracted,and the V3+V4 variable region of 16 S rDNA was subjected to high-throughput sequencing.The changes of microbial flora diversity after spinal cord injury treated curcuminin in rats by analyzing the diversity of α and β,the relative abundance of the flora,the LEfSE method to find the difference speciesand the prediction of KEGG function.3.After 14 days of post-operative through aortic perfusion,the rat spinal cord was sequenced by Illumina sequencing platform.The expression of the target was statistically analyzed.The differences were analyzedin gene expression in the sham group,the SCI group and the CUR group.The differentially expressed genes were screened.The related genes were annotated according to the KEGG function analysis to obtain the core enriched genesso that the mechanism of curcumin treatment of spinal cord injury in rats was further studied.Results:1.Rat behavior: After 5 days of spinal cord injury,the BBB score and the oblique plate experiment angle were significantly increased by BBB score and oblique plate test.The CUR group was significantly higher than the model group.The effect of the concentration of 100 mg/kg was obvious than the lower dose of curcumin at 50 mg/kg.The two group have significantly difference(p<0.05).There was no significant difference between the high dose(200 mg/kg)and the media dose(100 mg/kg).Through HE staining,it was found that the degenerative necrosis and inflammatory response of the damaged spinal cord neurons in curcumin were significantly reduced,and the cavity in the tissue decreased significantly.2.Microbial diversity:(1)Differences in bacterial flora: The bacteria that contribute the most to spinal cord injury are: Firmicutes,Bacteroidetes,Proteobacteria,Enterobacteriaceae,Erysipelotrichaceae,Shigella and Bacillus,etc.The most important bacteria for curcumin treatment of spinal cord injury are: Epsilonbacteraeota,Helicobacteraceae,Lactobacillaceae,Helicobacter,Lactobacillus etc.(2)KEGG pathway analysis: The treatment of spinal cord injury in rats by Curcumin may be achieved through metabolic pathways such as carbohydrate metabolism,membrane transport,cell movement,energy metabolism,and signal transduction.The most obvious difference of pathway was carbohydrate metabolism.3.Transcriptome analysis: Compared with the SCI group,(1)using DEseq software for differential screening,the number of differentially expressed genes was 6,394 of which 3148 were up-regulated genes and 3246 were down-regulated.(2)Through GO function analysis,differentially expressed genes were enriched in 24 GO items,including metabolic processes,biological regulation,cell processes,developmental processes,monomers,stimuli,cells,cell components,binding,catalytic activity,molecular sensor activity etc.(3)Differential expression was significantly enriched in the pathways of neuroactive ligand-receptor interaction,osteoblast differentiation,MAPK signaling pathway,axon guidance,cell growth and apoptosis,and cell adhesion through KEGG pathway analysis.Compared with the CUR group,the model group:(1)the number of differentially expressed genes was 3,500 of which 1876 were up-regulated,and 1624 were down-regulated.(2)3028 differentially expressed genes can be annotated into 25 GO entries,including cellular processes,biological regulation,metabolic processes,multicellular biological processes,signaling pathways,organelles,macromolecular complexes,cellular components,binding,catalysis activity,molecular sensor activity etc.(3)Differentially expressed genes were significantly enriched in the TNF signaling pathway,phagosome,MAPK signaling pathway,Osteoclast differentiation,Cell adhesion molecules pathway through KEGG pathway analysis.Conclusions:1.Medium Curcumin through intra-abdominal injection obviously improve the limbs of spinal cord injury to protect and repair the injured spinal cord.2.The diversity of intestinal flora after spinal cord injury increased.The curcumin of moderate dose after peritoneal injection affect the diversity of intestinal flora and the abundance of intestinal flora,which may be related to the increase of the abundance of Epsilonbacteraeota and Helicobacteraceae,the decrease of the abundance of Proteobacteria and actinobacteria,and achieved through carbohydrate metabolic pathway.3.There were 3148 up-regulated genes and 3246 down-regulated genes in the sham group and the SCI group.There were 1876 up-regulated genes in the CUR group and the SCI group,which were down-regulated mainly including inflammation and immune-related genes.Curcumin treatment of spinal cord injury may be achieved by down-regulating TNF signaling pathway,phagosome,PI3K-Akt signaling pathway,Toll-like receptor signaling pathway and calcium signaling pathway. |
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