| Objective:RNA-seq technique was used to detect differentially expressed genes in jejunum and hippocampus of rats treated with functional diarrhea by Back-Shu and Front-Mu compatibility,and qRT-PCR was used to verify differentially expressed genes,so as to investigate the mechanism of treating functional diarrhea rats with electroacupuncture Back-Shu and Front-Mu compatibility.Methods:1.Establishment of functional diarrhea rat model and observation of acupoint effect: 30 SPF grade male SD rats aged 8 weeks were randomly divided into 5 groups: blank group,model group,shu-mu point group,shu point group and mu point group.Rats in the acupuncture group and the model group were given the liquid of senxie leaf by gavage to make the rat model of functional diarrhea.Rats in the blank group were given the same amount of 0.9% sodium chloride solution by gavage.After one hour’s gavage,on the same day,electroacupuncture shu point group,mu point group,and shumu point group,each time for 20 minutes.The model group and the blank group were bound in the same way without acupuncture and fixed for 20 minutes.Once a day,ten times in all.After the end of the intervention,the general condition,fecal number and fecal water content of each group were observed.2.Analysis of differentially expressed genes in the treatment of functional diarrhea with the combination of shu mu point: 30 SPF grade male SD rats aged 8 weeks were randomly divided into 5 groups: blank group,model group,shu-mu point group,shu point group and mu point group.The intervention measures were the same as "experiment 1".Subsequently,total RNA was extracted from jejunum and hippocampal tissues of rats in each group,amplification was interrupted,DNA library was constructed,and gene expression profile was screened by bgi-500 sequencing machine.Cluster analysis,GO functional enrichment analysis and pathway enrichment analysis were carried out on the selected significantly different genes.3.Quantitative Real-time PCR(qRT-PCR)detection: according to the gene sequencing results,differentially expressed genes were screened for PCR verification.Results:1.Compared with the blank group,the number of defecation grains in the model group increased significantly,showing a statistical difference(P < 0.05).Compared with the model group,the number of fecal grains in the rats in the shu-mu point group was significantly reduced,with a statistical difference(P < 0.05).The number of fecal grains in the rats in the mu point group and the shu point group in the 24-hour period was not significantly different from that in the model group.Comparison between groups of rat feces moisture content concluded: 24 hours water content model group significantly greater than the blank group,draw a statistical difference at P < 0.05,the water content of model group compared with.Comparison between groups of rat feces moisture content concluded: 24 hours water content model group significantly greater than the blank group,draw a statistical difference at P < 0.05,the water content of model group compared with shu-mu and mu point group significantly increased water content,it is concluded that the statistical difference at P < 0.05,and the model group compared with moisture content shu point group did not change significantly,there is no statistically significant difference.2.Analysis of gene expression differences in jejunum tissues of rats treated with functional diarrhea by shu-mu point:(1)Compared with the blank group,the model group had 78 significantly different genes,27 down-regulated genes and 51 up-regulated genes.The enrichment results of GO and Pathway showed that the differentially expressed genes in jejunum of rats with functional diarrhea were mainly reflected in the effects on the synthesis and metabolism of the digestive system.Mineral binding,transport and absorption;Protein synthesis and metabolism;Affecting bile secretion,complement system and other signaling pathways(P<0.05).(2)Compared with the model group,there were 8 genes with significant differences in shu-mu point group,1 gene was down-regulated,7 genes were up-regulated,the GO function was enriched mainly in the REDOX enzyme activity in terms of molecular function,and the biological processes involved were significantly enriched in the drug reaction and the rhythm process.Pathway significantly enriched in rhythmically equivalent pathways(P<0.05).(3)Compared with the model group,there were 57 significantly different genes,33 down-regulated genes and 24 up-regulated genes in the mu point group.The enrichment results of GO function showed that the differentially expressed genes were significantly enriched in the extracellular,mainly in the growth factor binding in the molecular function,and the biological processes were significantly enriched in the rhythm process.In pathway analysis,differential gene enrichment was found in vascular smooth muscle contractions(P<0.05).(4)Compared with model group,the group with significant difference of shu point gene 49,lowered 25 genes,raised gene 24,GO enrichment results show that the enrichment of differentially expressed genes in significant function in the ribosome in cells,main enrichment in iron ion on molecular function combination,combined with hemoglobin,antioxidant activity and transition metal ions,to participate in the biological process of significant concentration in oxygen metabolism,protein synthesis and metabolism pathway(P<0.05).3.Differentially expressed genes in hippocampal tissues of rats treated with functional diarrhea by shu mu point:(1)There were 13 significantly different genes,9 down-regulated genes and 4 upregulated genes between model group and blank group,and no significant enrichment of GO function and pathway(P<0.05).(2)Compared with the model group,there were 5 significantly different genes,4 down-regulated genes and 1 up-regulated gene in the shu-mu point group.The enrichment of GO function mainly centered on the regulation of immune response and lipoprotein,but only 2 enriched genes(P<0.05);(3)Compared with the model group,there were 22 significantly different genes in the mu point group,including 2 down-regulated genes and 20 up-regulated genes.The differentially expressed genes were mainly located outside the cell and involved in molecular functions,including G protein coupling receptor binding and receptor binding,and involved in biological processes,including nervous system processes(P<0.05).(4)Compared with the model group,there were 5 significantly different genes,1 down-regulated gene and 4 up-regulated genes in the shu point group,and there was no significant enrichment of GO function and pathway(P<0.05).4.qRT-PCR gene quantitative results: The results of RNA-seq and qRT-PCR of the four target genes of AQP8,Htra2,NOD2 were compared between the model group and the shu-mu point group.The ratio of expression levels of the shu-mu point group and the model group was taken as a multiple of the difference.It was found that the variation trend of the three genes of AQP8,Htra2 and NOD2 was consistent,indicating that the RNA-seq results were of good quality.Conclusions:1.Acupuncture shu mu point of the large intestine can improve the intestinal dynamic hyperactivity of rats with functional diarrhea,and reduce the amount of defecation and fecal water content of rats.2.(1)The local effect of shu point treatment for FD is not clear,the overall function distribution is scattered,the targeting is not clear.(2)The local effect of mu point recruitment on FD was positive.By down-regulating the expression of myosin and actin related genes,the dynamic hyperactivity state of the intestinal tract of functional diarrhea rats was adjusted.(3)The effect of FD in the shu-mu point group may be due to the influence of dopaminergic synaptic pathway on ion channel proteins to change the synaptic transmission function,thereby increasing the content of intestinal BDNF,and finally regulating the state of intestinal dynamic hyperactivity in rats with functional diarrhea.3.The mechanism of the hippocampal effect of the treatment of FD by the combination of electroacupuncture shu mu point may be related to the pressure signal pathway,but it still needs to be further verified. |
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