Gene Regulation Study Of Spinal Ganglion Neurons In Colorectal Hypersensitive Mice By Electroacupuncture

BackgroundVisceral hypersensitivity occurs in deep organs and be hard to located,accompanied by complex clinical manifestations and characteristics such as referred pain and autonomic nerve reflex.Therefore,people’s understanding of visceral hypersensitivity lags far behind that of somatic pain,which also makes the treatment of chronic visceral hypersensitivity lack specific measures.Previous studies on the mechanism of visceral hypersensitivity in mice have found that TRPV1 channel are related to visceral hypersensitivity.And a series of molecules or pathways such as substance P and nerve growth factor have regulatory effects on TRPV1 channel.However,the specific regulatory mechanism has not yet been clarified.TRPV1 channel is a non-selective ion channel with six trans-membrane structures,which can be activated by capsaicin,various inflammatory mediators(bradykinin,protease,etc.)and endogenous cannabinoid and arachidonic acid.Because of its ability to respond to various stimuli,it is also known as the multisensory receptor,which mainly distributes in the primary sensory nerve fibers and non-nervous tissues such as gastrointestinal tract,vascular smooth muscle cells and T cells.It plays an important role in pain transmission,modulation and integration of multiple pain information.At present,most of the stimulants found to activate TRPV1 channel proteins are exogenous chemical or physical stimuli,while the reports of endogenous molecules and pathways regulating TRPV1 channel participation in visceral hypersensitivity are limited.The mechanism of interaction between molecules and pathways has not been clarified yet.Transcriptome sequencing can quickly lock in genes related to phenotypic changes and their significant enriched biological function and metabolic pathway which is a new way to study visceral hypersensitivity,but the transcriptome characteristics related to electro-acupuncture alleviating visceral hypersensitivity are scarce.We have studied the regulation of Electro-acupuncture on TRPV1 and substance P in spinal ganglion neurons of Zymosan-induced rectal colon hypersensitivity model mice in the past.It was found that:(1)The alleviation of rectal colon hypersensitivity by Electroacupuncture was related to the down-regulation of TRPV1 and SP in spinal ganglion neurons of hypersensitive model mice.(2)Small non-peptidergic neurons are the main nerve fibers of TRPV1 channel involved in electro-acupuncture to alleviate the high sensitivity of the rectal colon in the spinal ganglion of mice.Based on this,we speculate that:(1)TRPV1 activated in primary sensory neuronsplays an important role in the formation of visceral hypersensitivity.Electro-acupuncture alleviates visceral hypersensitivity by inhibiting the activation of TRPV1 channel,reducing tissue permeability,reducing the release of pain-causing substances,and inhibiting the secretion of excitatory neurotransmitters,thus reducing the exaggerated transmission of noxious information to the senior central nervous system;(2).TRPV1 channel participating in electro-acupuncture alleviating visceral hypersensitivity is accomplished by a series of cascaded signaling pathways.ObjectiveTo obtain the molecular pathways involved in the regulation of TRPV1 channel by Electro-acupuncture in the process of rectal hypersensitivity in mice,to explore potential regulatory genes,and to elucidate the regulatory mechanism of TRPV1 in rectal hypersensitive at the level of transcripts in mice.To provide more complete scientific evidence for TRPV1 pathway as a target for electro-acupuncture to regulate the release of peripheral pain-causing substances,the plasticity of spinal ganglion neurons and the secretion of neurotransmitters and provide new ideas and targets for clinical treatment of IBS patients.Materials and methodsPart One:Twenty-four normal male C57BL/6 mice(20-30g)were randomly divided into four groups:saline anal injection group(6 mice),saline anal injection electro-acupuncture group(6 mice),zymosan anal injection group(6 mice),Zymosan anal injection electro-acupuncture group(6 mice):experimental mice were given straight colon balloon dilatation test(CRDO)on the first day.For 3,4 and 5 consecutive days.Yeast polysaccharide suspension(Zymosan,30 mg/mL,0.2 ml/rat)or saline were injected into the anus;CRD tests(CRDl,CRD2,CRD3)were performed on all experimental mice on day 7,9 and 11,and the rectal colon sensitivity of mice was assessed by abdominal wall withdrawal reflex score(AWR score);CRD tests(CRD4.CRD5,CRD6)were given on day 13,15 and 17,respectively.On the 13th,14th,15th.16th and 17th days,electro-acupuncture intervention was given in Z+A,S+A groups,(bilateral Housanli and Shangjuxu acupoints,1mA,2/15Hz,once a day,30 minutes each time).On the 17th day,after CRD test,The left side L5-S2 spinal ganglia of mice in each group were taken out.The contraction degree of abdominal muscles of mice was evaluated by AWR score in each CRD test to detect the changes of rectal colon hypersensitivity in mice after electro-acupuncture intervention.Part Two:The left side L5-S2 spinal ganglia were taken out form the mice after finished intervention in the first part and the high-throughput gene sequencing was carried out on the spinal ganglia.According to the analysis of gene expression and differential expression,the related genes could alleviating the high sensitivity of rectal colon in mice after electro-acupuncture were screened out.The genes obtained were enriched and analyzed based on gene ontology(GO)database and Kyoto gene and genome Encyclopedia(KEGG)metabolic pathway database to screen out the significant enriched functional and metabolic pathways.Finally,gene co-expression network analysis was carried out to screen out the genes with strong regulatory ability,and annotation analysis of their functional or metabolic pathways was carried out.Results1.Behavioral test of Electro-Acupuncture Relieving Hypersensitivity Symptoms of the rectal colon in Mice.The abdominal muscle contraction induced by CRD recorded by AWR score show ed that the abdominal muscle contraction recorded by CRD3 at 60 mmHg pressure wa s 150%higher than that recorded by CRDO at 60 mmHg pressure,which confirmed t he formation of rectal colon hypersensitivity in the hypersensitive model group.After t he third electro-acupuncture intervention,the abdominal muscle contraction AWR score induced by CRD5 showed a downward trend in Z+A group,and after the fifth electr o-acupuncture intervention,the AWR score of abdominal muscle contraction induced b y CRD6 showed that the order of abdominal muscle contraction from large to small was:Z+C group>Z+A group>S+A.S+C group.The AWR score of Z+C group wa s significantly higher than that of Z+A and Z+C group(P<0.05).There was no signifi cant difference between Z+A group with S+C group and S+A group,indicating that el ectroacupuncture effectively inhibited Zymosan-induced rectal hypersensitivity in mice.2.Electroacupuncture can reverse down-regulated genes of spinal ganglion neurons in hypersensitive model mice.56 target genes such as Car3,Hp and Gm29216 were screened out by crossover of different groups by venn diagram.The expression trend of 56 genes in the hypersensitive model group was lower than that in the saline control group(P<0.05),while the expression in the hypersensitivity model elctro-acupuncture group was higher than that in the hypersensitivity model group(P<0.05),and the differential expression in the saline control group and the saline electro-acupuncture group was not significant.And there was no statistical difference in the expression of hypersensitive model electro-acupuncture group compared with saline control group(P>0.05).3.Analysis of Differential Expression of 56 GenesBased on the expression of 56 differentially expressed genes in saline control group,hypersensitivity model group and hypersensitivity model electro-acupuncture group,23 of the first 24 genes(P=5 X 10-5)in saline control group were significantly different from those in high sensitive model group,and the first 39 genes(P=5 X 10-5)in high sensitive model group and high sensitive model electro-acupuncture group which including the 23 genes referred before were significantly different(P=5 X 10-5).Overlapping shows that the expression of these 23 genes(Car3,Hp.Gm29216,Hist2h2c,Thbsl,Elane,Klf2,Pglyrp1 Anxal,Ly6c2,etc.)in saline control group,hypersensitive model group and hypersensitive model electro-acupuncture group showed the most obvious change trend.The expression of Car3,Hp,Gm29216,Hist2h2ac in each group was significantly higher than that of other genes.4.GO enrichment analysis of 56 genesAmong the 290 terms annotated by GO database,the first 20 terms were significantly enriched as follows:positive regulation of immune response,activation of adenylate cyclase,signal pathway of cell surface receptor,positive regulation of transforming growth factor beta receptor,negative regulation of cGMP signal pathway,protein phosphorylation,inflammatory response,positive regulation of p38MAPK pathway,biosynthesis of cAMP,electrical stimulation response,hosphatidylserine binding.Activation of acid lipid A2 inhibitor,adenylate cyclase binding activity,cAMP binding protein activity,calcium-dependent protein kinase activity,mitogen-activated protein kinase,adenylate cyclase activation,etc.The corresponding differential genes mainly include Thbsl(6 times),Adcy4(3 times),Anxa 1(3 times),Mapkapk3(3 times),Adrb2(2 times),Ctsg(2 times),Elane(2 times),Xdh(2 times).5.KEGG Metabolic Pathway Analysis of 56 GenesAccording to KEGG annotation,56 genes were significantly located in 20 specific metabolic pathway branches,including calcium signaling pathway,insulin secretion.cGMP-dependent protein kinase,p53 signaling pathway,lipid metabolism,gastrointestinal gland secretion,apoptosis,thyroid hormone synthesis,adrenergic signaling pathway of cardiomyocyte,positive immunoregulation and so on.The corresponding differential genes mainly include:Adcy4(11 times),Plcb2(9 times),Adrb2(6 times),Casp8(3 times),H2-Q6(2 times),Thbs1(2 times),Hk2(2 times),etc.6.Co-expression Network analysis of 56 GenesThe results showed that 15 genes including Ly6c2,Lrmp,Plcb2,Anxal,Pglyrp1,Ncf4,Sikl,Elf4,Alox5,Stkl7b,Slc16a10,Fcnb,Vsir,Rin3 etc.had more than 5 regulatory genes,of which F630028010Rik had no significant enrichment function or pathway.The remaining genes were mainly concentrated in cell membrane,cytoplasmic vesicles,endoplasmic reticulum,intracellular and extracellular signal transduction,protein phosphorylation.phosphorus.Negative regulation of acid lipase A2,calcium signaling pathway,lipid metabolism and other functions.ConclusionsIn this experiment,we used balloon dilatation stimulation and electro-acupuncture to treat the chronic rectal colon hypersensitive mice,and evaluated the effect of electro-acupuncture by abdominal withdrawal reflex score.We observed and analyzed the visceral pain sensation and pain behavior after electro-acupuncture stimulation on the Housanli(ST36)and Shangjuxu(ST37)acupoints on the rectal colon hypersensitive mice.We also discussed and analyzed the transcriptome regulation on spinal ganglion neurons of the rectal colon hypersensitive mice by electro-acupuncture based on TRPV1.The results showed that:1.Electro-acupuncture at ST36 and ST37 acupoints can significantly alleviate Zymosan-induced rectal colon hypersensitivity in mice,and the effect is positively correlated with the frequency of electro-acupuncture intervention.2.The alleviation of hypersensitive symptoms in mice by Electro-acupuncture may be related to the up-regulation of 56 genes abnormally expressed in spinal ganglion neurons of hypersensitive model mice;56 differentially expressed genes are significantly enriched in tissue structures(cell membranes,membrane surface receptors,cytoplasmic vesicles,etc.)and bioactive substances(proteins,lipid metabolic intermediates,intracellular and extracellular signal transduction pathways)that closely related to the function of TRPV1 channel.The expression values of Car3,Gm29216,Hist2h2ac and Hp genes in 56 genes were significantly higher than those of other genes in each group;5 genes,including Ly6c2,F630028010Rik,Anxal,Pglyrpl and sikl had the most obvious change trend among saline control group,hypersensitive model group and hypersensitive model electro-acupuncture group,and at the same time,they have the most strong regulation ability in the co-expression networks of 56 genes.Adcy4,Plcb2,Adrb2 and Thbsl appeared most frequently in the genes corresponding to the significant enrichment function and metabolic pathway.3.Electro-acupuncture therapy may regulate the function of TRPV1 by interfering with Adcy4,Adrb2,Plcb2 and metabolic pathways such as cGMP,cAMP-PKA and MAPK,so as to alleviate the symptom of hypersensitivity of rectal colon in mice.