Based On RNAseq To Analyze The Effect Of Every-other-day Fasting At Different Times On Inflammatory Genes In Rats With Spinal Cord Injury

Objective Transcriptome sequencing(RNA-sequence,RNA-seq)detection methods were used to screen the differences between spinal cord injury rats that were involved in every-other-day fasting(EODF)at different times and normal diet spinal cord injury rats Express genes,biologically annotate differentially expressed genes through GO database and KEGG database,and select key differential genes related to inflammation for real-time fluorescence quantitative(Quantitative Real-time PCR,q PCR)verification,in order to explore different opportunities for intervention in EODF after spinal cord injury The influence of gene transcription,especially the influence of inflammation-related genes and pathways after spinal cord injury.Methods 1.Experimental grouping: A C5 unilateral clamp spinal cord injury model was used,and 100 male SD rats were included.First,they are randomly divided into pre-ad libitum(Pre-AL)(N=80)and pre-every-other-day fasting(Pre-EODF)(N=20),PreEODF The rats in the pre-AL group had food restriction and free water every other day before the operation;rats in the Pre-AL group had a normal diet and free water before the operation for 4 weeks,and then the model was established.After the Pre-EODF model was established,the diet continued to be restricted every other day;80 rats in the Pre-AL group were randomly divided into a normal diet group(ad libitum,AL)(N=20)and a sham operation group(Sham)(N =20),immediate every-other-day fasting,Immed-EODF(N=20),delayed every-other-day fasting,Delay-EODF(N=20)).The AL group continued to maintain a normal diet after modeling;the Immed-EODF group immediately began to eat every other day after modeling;the Delay-EODF group continued to eat normally for 6 days after modeling,and then began to eat every other day;Sham group only clamped during modeling Except for the lamina,the spinal cord was not injured,and the normal diet was continued after the operation,and all rats were free to drink water.After operation 1day(1 day post injury,1dpi),3 days(3 days post injury,3dpi),7 days(7 days post injury,7dpi),14 days(14 days post injury,14dpi),28 days(28 day post injury,28dpi)five time points,each group has a subgroup,that is,each of the five major groups has five post-operative time point subgroups(a total of 25 subgroups,each subgroup n=4),and the spinal cord tissue of the C5 segment was taken for transcriptome sequencing.The 1dpi and 3dpi subgroups of the Delay-EODF group were all normal diets(EODF intervention was started on the sixth day after surgery),which was consistent with the 1dpi and 3dpi subgroups of the AL group.The 1dpi and 3dpi subgroup samples of Delay-EODF were not tested during the test,so 23 subgroups were finally tested,a total of 92 samples.2.Analysis method: Compare the sequencing results of the three intervention groups(Pre-EODF group,Immed-EODF group,Delay-EODF group),sham operation group(Sham group)and normal diet group(AL group)respectively,according to P<0.05,the multiple of difference> 2 times the criteria to screen the differentially expressed genes(when the number of differential genes is small,it can be relaxed to 1.5 times),the differentially expressed genes are clustered,and then biologically annotated through the GO database and performed through the KEGG database Pathway notes.The annotation results of each subgroup were compared at 1dpi,3dpi,7dpi,14 dpi,28dpi,and key genes related to inflammation were selected for q PCR verification.Result 1.Changes in food intake and body weight of rats(1)Food intake: 1-4 weeks before surgery,the daily food intake of the Pre-EODF group was more than that of the Pre-AL group(P<0.05),and the average food intake of the Pre-EODF group was higher than that of the Pre-AL group The average daily food intake increased by 28%;the average daily food intake of Pre-EODF was reduced by 38% compared with the average daily food intake of Pre-AL.Within 1 week after the operation,the daily food intake of rats in all groups decreased first and then increased.The average daily food intake of the Pre-EODF group,Immed-EODF group,and Delay-EODF group was higher than that of the AL group for 1-4 weeks after surgery.Food intake decreased by 27%,25%,and 26%,respectively.(2)Body weight: There was no significant difference in body weight between the Pre-EODF group and the Pre-AL group at the time of entry and 4 weeks before the operation(P>0.05).After intervention,the weight of the rats in the Pre-AL group was gradually higher than that in the pre-EODF group.One week before the operation,the weight of the rats in the pre-AL group was significantly higher than that in the preEODF group(P<0.05).The weight of rats in the Pre-EODF,Immed-EODF,DelayEODF,and AL groups decreased after the operation,and then gradually increased after 1 week;the postoperative weight of the Sham group maintained an upward trend.2.The impact of SCI modeling on gene transcription(1)Overall analysis of differential genes: The Sham group and AL group were screened for differential genes with the screening conditions of P<0.05 and |log2(foldchange)|>1(fold change>2).A total of 4410 differentially expressed genes(DEGs)were screened at 1dpi,3dpi,7dpi,14 dpi,and 28 dpi,of which 516(12%)were up-regulated and 3,894(88%)were down-regulated.(2)GO enrichment analysis: GO enrichment analysis for DEGs from three levels: biological process(BP),cellular component(CC),and molecular function(MF).(1)Analyzed from the BP level,the DEGs of the Sham group and the AL group at each time point were mainly enriched in inflammatory response,immune response,granulocyte chemotaxis,T cell proliferation,response to lipopolysaccharide,phagocytosis,and active regulation of cell migration,etc.entry.(2)Analyzed from the CC level,it was found that the DEGs of the Sham group and the AL group were enriched into items such as NLRP3 inflammasome and AIM2 inflammasome at 3dpi,7dpi,and 14 dpi.(3)Analyzed from the MF level,the DEGs of the Sham group and the AL group at each time point were mainly enriched in items such as Toll-like receptor binding,immunoglobulin receptor binding,and superoxide-producing NADPH oxidase activity.(3)KEGG pathway analysis: DEGs at each time point are mainly enriched in NF- κB signaling pathway,NOD-like receptor signaling pathway,Toll-like receptor signaling pathway,IL-17 signaling pathway,TNF signaling pathway,JAK-STAT signaling Pathways,PI3K-AKT signaling pathways and other pathways.3.The influence of EODF intervention on gene transcription(1)Overall analysis of differential genes: Pre-EODF group and AL group,ImmedEODF group and AL group,Delay-EODF screening conditions of P<0.05 and |log2(foldchange)|>0.58(fold change>1.5)Differential gene screening was performed between the AL group and the AL group.Pre-EODF group and AL group were screened at 1dpi,3dpi,7dpi,14 dpi,28dpi to a total of 1636 DEGs,of which 742(45%)were increased and 894(55%)were decreased;Immed-EODF and AL were at 1dpi,3dpi,7dpi,14 dpi,28dpi screened a total of 1402 DEGs,of which 774(55%)increased and 628(45%)decreased;Delay-EODF group and AL group were screened to 1413 at 7dpi,14 dpi,28dpi DEGs,of which 722(51%)were raised and 691(49%)were lowered.(2)GO enrichment analysis: GO enrichment analysis on DEGs from three levels of BP,CC and MF.1)Analysis from the BP level:(1)DEGs in the EODF group and the AL group at 1dpi and 3dpi are mainly enriched in inflammation,immune response,active regulation of interleukin-4,6,and 12,response to TNF,and NF-κB pathway Actively regulate biological processes such as the process of apoptosis and the differentiation of brown adipocytes.(2)At 7dpi,14 dpi,and 28 dpi,biological processes such as inflammation,immune response,interleukin-1 active regulation,TNF active regulation,response to lipopolysaccharide,and active regulation of apoptosis are still enriched;2)Analysis from the CC level: each EODF The items enriched in DEGs of group and AL group include protein extracellular matrix,extracellular area,base outer plasma membrane,plasma membrane components,etc.;3)Analysis from the MF level: DEGs enrichment of each EODF group and AL group The entries include: heparin binding,protein kinase binding,histone deacetylase binding,cell adhesion molecule binding,carbohydrate binding,etc.(3)KEGG pathway analysis: 1)The main enrichment pathways of DEGs in EODF and AL groups at 1dpi and 3dpi are: IL-17 signaling pathway,Toll-like receptor signaling pathway,TNF signaling pathway,PI3K-Akt signaling pathway.2)At 1dpi,Pre-EODF is also enriched in items such as m TOR signaling pathway,FOXO signaling pathway,HIF-1 signaling pathway,and RIG-1-like receptor signaling pathway.3)At 3dpi,the enriched KEGG pathway entries increased,including NF-κB signaling pathway,TNF signaling pathway,insulin resistance and other entries.4)The main enrichment pathways at 7dpi,14 dpi and 28 dpi are: IL-17 signal pathway,TNF signal pathway,PI3K-Akt signal pathway,etc.4.The effect of EODF intervention on inflammatory gene transcription Under the intervention of EODF,there are both differentially up-regulated and differentially down-regulated inflammatory genes.At 1dpi,the transcription of inflammatory genes is down-regulated;at 3dpi,inflammatory genes are up-regulated;at 7dpi,inflammatory genes are down-regulated;14dpi and 28 dpi,inflammatory gene transcription is still different,but the difference is more The details are reduced.At 1dpi,the pro-inflammatory genes were mainly down-regulated.At 3dpi and 7dpi,some proinflammatory genes were up-regulated and some pro-inflammatory genes were downregulated;anti-inflammatory genes were mainly up-regulated at 3dpi and gradually down-regulated at 7dpi.Conclusion 1.Gene transcription caused by EODF is mainly related to biological events such as inflammation.The main pathways for transcription changes include signaling pathways such as NF-κB.2.One day after SCI,EODF reduces the transcription of pro-inflammatory genes and has a certain effect on reducing inflammation in SCI.3.One day after SCI,pre-EODF and immediate EODF intervention have different effects on gene transcription.Compared with EODF started after SCI,pre-EODF 4 weeks before injury promotes the most significant down-regulation of proinflammatory genes.