| Objective: a common internal mechanism of neuropathic pain is inflammation of injured or affected nerves.This inflammatory response causes a series of events that cause innate immune cells to concentrate and be activated near the damaged tissue.The release of immunoactive substances,including cytokines,neurotrophic factors and chemokines,causes local reactions and further leads to broader immune responses.Sensitization of peripheral and central nervous system is the main reason for the occurrence and maintenance of neuropathic pain,and inflammatory immune mechanism is the key to regulate these links.Neuropathic pain belongs to the category of "Arthromyodynia" in TCM syndrome differentiation.The compound decoction of ShenTongZhuYu has the effect of promoting Qi,promoting blood circulation,removing wind,removing dampness and relieving pain.It is the representative prescription of treating arthromyodyni,especially blood stasis Bi.Modern pharmacological studies have proved that the whole prescription hassignificant anti-inflammatory,analgesic,microcirculation improvement and anti allergic effects.High throughput sequencing(RNA-seq)based on second-generation sequencing technology is the most widely used transcriptome sequencing technology,which can quickly obtain all genetic information of test samples.RNA-seq has the advantages of low cost and high throughput.It can be used to study the structure and function of genes,identify the changes of gene expression,and explore the alternative splicing mode of regulation.In the field of life science,this technology has been used to explore the pathogenesis of disease,clinical diagnosis and pharmacopharmacology research.Therefore,this study will explore the potential target of ShenTongZhuYu Decoction in the treatment of neuropathic pain based on RNA-seq technology,and provide new clues and theoretical basis for the treatment of the disease.Methods: 1)Through literature search,to explore the mechanism of ShenTongZhuYu Decoction in the treatment of neuropathic pain,search for RBP gene related to neuropathic pain in Genecard database with search results and RNA binding as key words,and determine the RBP gene because of our research object;2)to construct stau1;3)to transfect HeLa cells,and to detect RBP after transfection by rt-q-pcr The expression of stau1 gene and Western blot were used to detect the expression of stau1 protein.4)MTT method was used to detect the effect of stau1 gene overexpression on cell proliferation.5)RNA-seq was used to analyze the transcriptional regulation of stau1 overexpression.Establishment of control cells and stau1 Four sample groups of OE cells(two biological repeat samples)were used to prepare cDNA libraries of total RNA from four groups of samples respectively,which were sequenced and analyzed after construction;6)to explore the differential expression genes regulated by stau1 and the biological processes or signal pathways enriched in go or KEGG databases;7)to explore the variable shear events regulated by stau1 and the signal pathways enriched in go or KEGG databases by related genes;8)fluorescent quantitative Q-PCR was used to verify the differentially expressed genes and differentially variable splicing genes detected in RNA SEQ.Results: 1)ShenTongZhuYu Decoction plays an important role in the development of neuralgia through p38 MAPK pathway,and stau1 is related to neuralgia,which is a double stranded RNA binding protein and p38 MAPK pathway.2.;2)stau1 vector with GFP fluorescent label and flag label(fused with the target gene)was constructed successfully in this experiment;3)GFP fluorescent expression effect was observed under fluorescence microscope after the vector was transfected to confirm the success of cell transfection.Rt-q-pcr showed that stau1 gene was over expressed 48 hours after transfection,Western blot showed that stau1 gene was successfully transfected and over expressed between 50-70KD;4)MTT assay showed that stau1 gene over expression could promote cell proliferation.5)The average value of base quality q30 was 93.93%.The correlation analysis between samples showed that stau1 OE cells and control cells were highly correlated,and two biological replication samples were highly correlated;6)MA plot showed that there were 801 significantly different expression genes caused by stau1 overexpression,including 423 up-regulated genes and 378 down regulated genes,which indicated that stau1 widely regulated gene transcription in HeLa cells.Overexpression of stau1 positively regulated the expression of IFIT2,oasl,IFIT3 and negatively regulated the expression of CCL2 genes in HeLa cells,all of which were related to inflammation and immune response;7)stau1 could globally regulate the variable splicing events in HeLa cells,and part of the regulated differential variable splicing factors were enriched in nerve growth factor receptor signaling pathway(p-value was about 0.01).By detecting the variable splicing events of six selected genes in the NGF receptor signaling pathway,we can see the obvious difference of variable splicing,which indicates that stau1 is of great significance to the survival,differentiation,growth,repair and regeneration of neurons after injury.8)The results of Q-PCR showed that the differentially expressed genes and alternative splicing events were consistent with the results of RNA sequencing,which further confirmed the reliability ofour results.Conclusion: we know that ShenTongZhuYu Decoction plays an important role in the development of neuralgia through p38 MAPK pathway.The overexpression of p38 MAPK and staul is confirmed by studying p38 MAPK and staul Staul can regulate gene transcription and alternative splicing of HeLa cells globally.It is also found that staul has a positive regulatory effect on gene transcription involved in inflammation and immune response related functions.At the same time,staul can also regulate and enrich the pathways involved in regulating nerve growth and repair related genes,such as nerve growth factor receptor signal pathway,reverse transport,transcription regulation and myocyte differentiation Because of the variable splicing.This study provides new clues for the treatment of neuropathic pain in the future,and suggests that stau1 is the potential target of Shentongzhuyu Decoction in neuropathic pain treatment. |
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