| Functional constipation(FC)is a clinical common Functional gastrointestinal disease,but its pathophysiological mechanism is still unclear.Since 1988,the study of correlation between constipation and emotional problem has been reported.A large number of clinical observation data show that constipation patients often company with anxiety,depression and other emotional disorders,and emotion abnormal are often associated with constipation presenting comorbidity status.But the relationship between constipation and the emotional problems are still unclear.Functional constipation is the advantage of acupuncture clinical diseases,Back-shu point and front-mu point in large intestine channel are the classical acupoints in the acupuncture treatment of gastrointestinal diseases.A number of relevant RCTs carried out in our country showed the two acupoints used alone or together can increase the stool frequency,improving stool property,increase the colon bowel electric-amplitude and frequency,and relieve the patient's mental state.Acupoint compatibility is not only an important part of acupuncture and moxibustion prescription,and also an important factor in the curative effect of acupuncture and moxibustion.How's different acupoints compatibility mechanism(cooperation or antagonism)?Effect targeted have or not overlap?Work site similarities or differences?Explore the mechanism of the acupuncture theories and clinical research has the important value.Objective:1.From the transcriptome level to explore the molecular pathogenesis of functional constipation.2.From the transcriptome level reveal the molecular mechanism of acupoints compatibility effect for functional constipation.Methods:1.Pathological morphology observation of FC rat hippocampal and colon:26 male SD rats were randomly divided into 2 groups:12 in blank control group,14 in model group.Adopting compound diphenoxylate suspension liquid lavage method the rat model with functional constipation,subsequent model to determine success.Blank control group and determine the successful model rats by HE staining,immunohistochemical and image collection and analysis,observe two groups of colon of rats,the hippocampus morphology,and BDNF protein expression.Feeding compound diphenoxylate mixed suspension to established the FC rats model.HE staining,immunohistochemical and image collection and analysis were used in blank control group and successful model rats.Colon and hippocampus morphology,BDNF protein expression were observed in two groups.2.Shu-mu compatibility for bowel movement effect in FC rats:36 SD rats were randomly divided into 2 groups:blank control group 6,model making group 30.Successful model rats were randomly divided into model group,shu-mu group,shu group and mu-group.Unilateral Tianshu and/or ta chang shu were choose for shu-mu group,shu-group and mu-group,alternate acupuncture per day,followed by electric-acupuncture 15-20min,qd,continuous treatment of 10d.Blank control group and model group rats with the same method of bondage,didn't accpet acupuncture intervention.After the 10d acupuncture intervention,next day took the material,Observed two groups rats general situation,24h defecation number,24h feces water content.3.Difference gene expression analysis of colon tissue in model rats and Shu-mu compatibility groups:50 SD rats were randomly divided into 2 groups:blank control group 12,model making group 38.Successful model rats were randomly divided into model group,shu-mu group,shu group and mu-group.Intervention are same to"Experiment two".Randomly selected 6 cases of colon tissue in each group,extracting total RNA,interrupt amplification,DNA library construction and used BGI-500 sequencer to filter gene expression profiling of each sample.The significant difference gene were examed in clustering analysis,GO funtioanl enrichment,pathway enrichment analysis.From the transcriptome level explore the FC colon molecular pathogenesis and shu-mu compatibility effect mechanism.4.Difference gene expression analysis of hippocampus tissue in model rats and Shu-mu compatibility groups:synchronous extraction "experiment three" rats hippocampus total RNA,interrupt amplification,DNA library construction and used BGI-500 sequencer to filter gene expression profiling of each sample.The significant difference gene were examed in clustering analysis,GO funtioanl enrichment,pathway enrichment analysis.From the transcriptome level explore the FC hippocampus tissue molecular pathogenesis and shu-mu compatibility effect mechanism.5.Hippocampal tissue gene quantitative analysis of shu-mu points treated with FC rats:genes with further research value in "experiment four" were examined with qRT-PCR to verify sequencing results.Results:1.HE staining results:blank control group vs model group in colon tissue,mucous membrane epithelial cell removal(2 vs 1),submucosa lymphatic edema(1 vs 1),submucosa lymphangiectasia(0 vs 4),the incidence of each pathological change was no statistically significant difference(P>0.05);two groups of rats hippocampal structure stratification clear,neurons and vertebral body cell structure complete,did not see the abnormal tissue pathological changes.2.BDNF immunohistochemical results:compared with blank control group,BDNF positive accumulated in each hippocampus district of model group reduced inordinately,differences in CA3 and DG area has statistically significant(P<0.05).3.Shu-mu compatibility for bowel movement effects:?24h defecation number:compared with model group,three EA groups were all increased in 24h defecation number,and difference of mu-points group was statistically significant(P<0.05),no statistically significant difference between three EA groups(P>0.05).?24h feces water content:compared with model group,three EA groups were all increased in 24h feces water content but difference has no statistically significant(P>0.05),no statistically significant difference between three EA groups(P>0.05).4.Difference gene expression analysis of colon tissue in model rats and Shu-mu compatibility groups:?compared with the blank group,model group has 61 significant difference genes(27 down,34 up),GO function enrichment and pathway enrichment results consistently show that the FC in colon site is given priority to with lipid metabolism,Secondly affect cell process,moreover SDGs enrichment in PI3K-Akt signaling pathway,P53 stress signaling pathways,tumor necrosis factor signaling pathways and immune response,etc.;?compared with model group,shu-mu group has 12 SDGs,GO enrichment mainly around the immune response and lipoprotein regulating function,and no significant pathway enrichment.?mu-group has 43 SDGs,GO function enrichment and pathway enrichment results consistently show that Tianshu acupoint mainly by promoting the intestinal smooth muscle movement to realize the effect.Related genes including myosin light chain gene,actin,calcium binding protein gene;?shu-group has 18 SDGs,GO function and pathway both has no enrichment(P>0.05).5.Difference gene expression analysis of hippocampus tissue in model rats and Shu-mu compatibility groups:?compared with the blank group,model group has 36 significant difference genes(14 down,22 up),GO function enrichment mainly in oxidative stress response,cholinergic synapses pathways has been significantly enrichment;?compared with model group,shu-mu group has 48 SDGs,GO enrichment mainly for regulating oxidative stress response,antioxidant,etc.,secretion pathway has been significantly enrichment.?mu-group has 9 SDGs,serotonin synaptic pathway has been significantly enrichment.?shu-group has 19 SDGs,pentose and glucuronic acid conversion pathway has been significantly enri chm ent(P>0.05).6.qRT-PCR gene quantitative results:mRNA expression of Igf2,Enpp2,Igfbp2,KI and Ptgds in hippocampus of model group and shu-mu group are consistent with the sequencing results.Difference of Enpp2 mRNA,Igfbp2 mRNA and KI mRNA are statistically significant(P<0.05).Conclusion:1.Local pathological changes(colon)in FC,mainly affects lipid metabolism,cell process,relevent gene expression abnormal in body pressure signal pathways.In addition,hippocampus presents athological changes in FC rats mainly in the function of blood vessels and blood carry oxygen and cholinergic system abnormalities.And the changes may cause abnormal emotional state that is one of the reasons,directly or indirectly.2.The effect mechanism of shu-mu compatibility in colon site treating with FC:?local effects of shu-acupoint is uncertain,although SDGs are mainly present in regulating immune function,but the overall function distribution is scattered and untargeted.?Mu-acupoint through the promotion of multiple gene expression in smooth muscle cells,play a catalytic function of intestinal contractions;?local effect of shu-mu acupoints is between the back-Shu and front-Mu group,mainly in immune and lipoprotein regulation.3.Back-Shu and front-Mu compatible therapeutic mechanisms in FC hippocampal:?back-Shu acupoint therapy on hippocampal function mainly for improvement in oxygen-carrying capabilities;?front-Mu acupoint function on hippocampal included the protected physiological function of vascular and blood,and affect serotonin neurotransmitter levels and releases,but enrichment to less number of genes,the strength of evidence remained to be tested.?Shu-mu acupoint function in hippocampal is most significant in three EA groups,mainly reflected improved oxygen-carrying function,vascular calcification in vitro resistance and improved cognitive function. |
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