To Explore The Effect Mechanism Of Acupuncture And Moxibustion At Biao-Ben Acupoint Combination In The Prevention And Treatment Of Diarrhea Irritable Bowel Syndrome Based On Metabonomics Technology

ObjectiveBased on the idea of "treating diseases and seeking the essence"and the Biao-Ben theory of traditional Chinese medicine,the rat model of diarrhea predominant irritable bowel syndrome（IBS-D）with emotional disorder was prepared by CAS modeling method.The method of acupuncture and moxibustion at Biao-Ben acupoint combination based on the principle of "regulating the mind and protecting the intestines" was selected to treat the disease.To observe the effects of acupuncture and moxibustion at Biao-Ben acupoint combination on intestinal function,heart rate variability and cardiac endocrine peptides in IBS-D model rats.On this basis,metabonomics technology was used to analyze the effects of acupuncture and moxibustion at Biao-Ben acupoint combination on differential metabolites and metabolic pathways in serum,heart and colon of IBS-D model rats,in order to structure the metabolic model of acupuncture and moxibustion at Biao-Ben acupoint combination in the prevention and treatment of IBS-D serum,heart and colon,and explore the biological effect mechanism of acupuncture and moxibustion at Biao-Ben acupoint combination in the prevention and treatment of irritable bowel syndrome,providing experimental basis for improving the clinical efficacy of irritable bowel syndrome with acupuncture and moxibustion at Biao-Ben acupoint combination.MethodsSixty SD rats were randomly divided into three groups:control group（CT）,model group（IBS）,BB group（BB）and CG group（CG）.The IBS-D model was prepared by CAS method in CT group,BB group and CG group.The visceral pain threshold of the model group was measured before and after modeling.If the visceral pain threshold of the model group was higher than that of the control group（P<0.05）,which indicated that the modeling was successful.Before modeling,rats in BB group was treated by acupuncture and moxibustion at the acupoints of "Neiguan","Zusanli" and "Guanyuan",while rats in CG group was treated by acupuncture and moxibustion at the acupoints of "Zusanli","Shangjuxu" and "Tianshu".The CT group and IBS group were not given any intervention.The rats were weighed on day 1,day 2,day 5,day 14,day 23 and day 29,and calculated the growth rate of body mass.On the 28th day,the sugar water preference experiment was measured,and the feces were collected for Bristol fecal character score.On the 29th day,open field test,diarrhea index related test,fecal water content and heart rate variability were carried out.Then the rats were anesthetized with 2%Pentobarbital Sodium（0.2ml/100g）.The blood was taken from the abdominal aorta.After centrifugation,one part was put into the refrigerator at-4℃ for ELISA to detect ANP and BNP,and the other part was put into the refrigerator at-80℃ after liquid nitrogen quenching for metabolomics detection.Take out the heart and colon segments,clean them with PBS,and carefully absorb the water with filter paper.Part of the colon tissue is fixed in 4%paraformaldehyde solution for HE staining detection.After quenching with liquid nitrogen,part is stored in refrigerator at-80℃ for metabolomics detection.All heart tissues were quenched with liquid nitrogen and stored in-80℃ refrigerator for ELISA detection of ANP,BNP and metabolomics.All results were statistically analyzed.Metabonomics detection is to screen metabolites and metabolic pathways by using ultra-high performance liquid chromatography-mass spectrometry（UPLC-MS/MS）detection platform and diversified analysis methods.Result1.Evaluation of the curative effect of acupuncture and moxibustion at Biao-Ben acupoint combination on IBS-D model rats:（1）Comparison of body weight and body mass growth rate of rats:on the first day of modeling,there was a very significant difference in body weight between the IBS group and the CT group（P<0.01）.There was significant difference in body weight between the CG group and the IBS group on the 23rd and 29th days（P<0.05）.There was no significant difference in body weight between the BB group and the CG group（P>0.05）.There was a significant difference in the weight growth rate of rats in the IBS group and the CT group（P<0.01）.There was a significant difference in the weight growth rate measured on the 14th and 29th days between the BB group and the IBS group（P<0.05）.There was significant difference in body weight growth rate measured on the 29th day between the CG group and the IBS group（P<0.05）.There was no significant difference in body weight between the BB group and the CG group（P>0.05）.（2）Comparison of visceral pain threshold of rats:before modeling,there was no significant difference among the CT group,IBS group and BB groupthe（P>0.05）.After modeling,there was significant difference in visceral pain threshold between the CT group and IBS group（P<0.01）.Compared the IBS group with the BB group and the CG group in the visceral pain threshold,the difference was statistically significant（P<0.01）.There was no significant difference in visceral pain threshold weight between the BB group and the CG group（P>0.05）.（3）Behavioral observation of rats:the sugar water preference rate of rats in the IBS group was significantly higher than that in the CT group（P<0.01）.The sugar water preference rate of rats in the BB group and CG group was significantly lower than that in the IBS group（P<0.01）,which was close to that in the CT group.The rats in the CT group had free trajectories and a long total distance,while the rats in the IBS group had more trajectories in the periphery and a short total distance.Compared with the IBS group,the range of motion trajectories of the rats in the BB group and CG group was wider and the total distance of the rats in the two groups was increased.PCA analysis showed that the intra group aggregation of the four groups of data was good and stable,and the difference among groups was significant.There was no significant difference between the BB group and CG group.There were significant differences in distance,move duration,rest duration,central time,central dist and zone crossing between the IBS group and the CT group（P<0.01）.The distance,move duration,rest duration,central time,central dist and zone crossing of the rats in the BB group and CG group were significantly different compared with those in the BB group（P<0.01）.There was significant difference in the speed between the BB group and the IBS group（P<0.01）.There was no significant difference in distance,move duration,rest duration,central time,central dist and zone crossing between the BB group and CG group（P>0.05）.（4）Diarrhea in rats:there was a significant difference in fecal water content between IBS group and CT group（P<0.01）.Compared the IBS group with the BB group,the difference of the fecal water content was significant（P<0.01）.Compared the IBS group with the CG group,the difference of the fecal water content was significant（P<0.01）.Bristol fecal trait score in the IBS group was higher than that in the CT group,and the difference was highly statistically significant（P<0.01）.The Bristol fecal character score of rats in the BB group was lower than that in the IBS group,and the difference was highly statistically significant（P<0.01）.There was no significant difference in Bristol fecal character score between the BB group and the CG group（P>0.05）.（5）Histopathological observation of rats’ colon:no significant pathological changes were observed in the colon tissue of the CT group.The mucosal layer of colon tissue was discontinuous,the arrangement of glands was slightly disordered,and there were more inflammatory cell infiltration in submucosa and muscle layer in the IBS group.The structure of colonic mucosal epithelium in the BB group and the CG group was relatively complete without inflammatory cell infiltration.（6）Heart rate variability measured in rats:there was a significant difference in HF value between IBS group and CT group（P<0.05）.There were significant differences in SDNN,RMSSD,RR mean,TP,VLF,LF,HF and LF/HF between BB group and IBS group（P<0.01,P<0.05）.The difference of the TP,VLF and HF values of the rats between the CG group and the IBS group were sigificantly（P<0.05）.There was no significant difference in SDNN,RMSSD,RR mean,TP,VLF,LF,HF and LF/HF between the BB group and the CG group（P>0.05）.（7）The rat serum,heart tissue ANP,BNP content changes:the contents of ANP and BNP in serum of the IBS group were significantly higher than those of the CT group（P<0.01）.The contents of ANP and BNP in serum of rats in the BB group were significantly lower than those in the IBS group（P<0.01）.The levels of ANP and BNP in serum of rats in CG group were significantly different from those in IBS group（P<0.01）.The contents of ANP and BNP in the heart tissue of the IBS group were higher than those of the CT group（P<0.05）.The contents of ANP and BNP in the heart tissue of rats in the CG group were higher than those in the BB group（P<0.05）.2.2.Effects of acupuncture and moxibustion at Biao-Ben acupoint combination on serum metabolomics of IBS-D model rats（1）Comparison of different metabolites in each group:compared with the control group,the contents of serum metabolites TXB2,（±）15 HETE,PGE2,PGJ2,12-oxoETE,12,13-EpOME,FFA（16:1）,FFA（18:3）,FFA（14:0）and 9,10-DiHOME in the IBS group decreased.Compared with the IBS group,the contents of blood metabolites TXB2,（±）15-HETE,PGE2,PGJ2,12-oxoETE,12,13-EpOME and FFA（16:1）in the BB group increased.Compared with the IBS group,the contents of FFA（16:1）,FFA（18:3）,FFA（14:0）,9,10-DiHOME and 12,13-EpOME decreased in the CG group.Compared with the BB group,the contents of Kynurenic acid,uric acid and Thr-Leu decreased in the CG group.（2）Compare different metabolic pathways in each group:compare the IBS group with the CT group,different metabolic pathways included arachidonic acid metabolism,linoleic acid metabolism,serotonergic synapse,amoebiasis,fatty acid biosynthesis,unsaturated fatty acid biosynthesis,and the regulation of TRP channel metabolism by inflammatory mediators.Compared with the model group,the differential metabolic pathways in the BB group included arachidonic acid metabolism,serotonergic synapse,TRP channel regulation metabolism by inflammatory mediators,linoleic acid metabolism,human papillomavirus infection,oxytocin signaling pathway,human cytomegalovirus infection,leishmaniasis and rheumatoid arthritis.Compared with the IBS group,the differential metabolic pathways included linoleic acid metabolism and fatty acid biosynthesis.There was no difference in metabolic pathway between the BB group and the CG group.It showed that there is little difference in the metabolites and metabolic pathway of the two methods of acupuncture and moxibustion on serum.3.Effects of acupuncture and moxibustion at Biao-Ben acupoint combination on cardiac metabolomics in IBS-D model rats（1）Comparison of different metabolites in each group:compared with the CT group,the contents of cardiac metabolites adenosine 5’-diphosphate,TXB2,PGE2,PGJ2,2-deoxyribose 1-phosphate,deoxyguanosine diphosphate（dGDP）,5’-adenylyl sulfate（APS）,2-deoxyribose 1-phosphate and kinurenine in the IBS group increased,and the contents of N-acylornithine,inosine diphosphate The content of kynurenic acid decreased;Compared with the IBS group,the contents of cardiac metabolites adenosine 5’-diphosphate,2-deoxyribose 1-phosphate,deoxyguanosine diphosphate（dGDP）,5’-adenylyl sulfate（APS）,inosine diphosphate,2-deoxyribose 1-phosphate,kinurenine and kynurenic acid decreased,while the contents of N-acetylcholine increased;Compared with the IBS group,the contents of cardiac metabolites TXB2,PGE2,PGJ2,2-deoxyribose and 1-phosphate in the CG group decreased,while the contents of N-acetylcholine and increased.Compared with the BB group,the contents of cardiac metabolites adenosine 5’-diphosphate,deoxyguanosine diphosphate（dGDP）,5’-adenylyl sulfate（APS）and kinurenine in the CG group increased,while the contents of TXB2,PGJ2 and 2-deoxyribose 1-phosphate decreased.（2）Compare different metabolic pathways in each group:compare the IBS group with the CT group,different metabolic pathways included steroid hormone biosynthesis,African trypanosomiasis,metabolism of starch and sucrose and serotonergic synapse.Compared with the model group,the differential metabolic pathways in the BB group included purine metabolism,olfactory conduction,light conduction,cGMP PKG signal pathway,African trypanosomiasis,FOXO signal pathway and morphine addiction.Compared with the IBS group,the differential metabolic pathways included arachidonic acid metabolism,bile secretion,serotonergic synapse,inf lammatory mediator regulation of TRP channel and linoleic acid metabolism.Compared with the CG group,the differential metabolic pathways include arachidonic acid metabolism,African trypanosomiasis,renin secretion,inflammatory mediator regulation of TRP channel,FOXO signal pathway and platelet activation.4.Effect of acupuncture and moxibustion at Biao-Ben acupoint combination on colon metabolomics of IBS-D model rats（1）Comparison of different metabolites in each group:compared with the CT group,the contents of 13 oxoode and 9-oxoode in the IBS group increased,while the contents of cytidine,deoxycytidine,cytarabine,anserine and 2,4-dihydroxypyridine decreased.Compared with the IBS group,the contents of 13 oxode and 9-oxode decreased,while the contents of cytidine,deoxycytidine,cytarabine,anserine and 2,4-dihydroxypyridine increased in BB group.Compared with the IBS group,the contents of colonic metabolites cytidine,deoxycytidine,cytarabine,anserine and 2,4-dihydroxypyridine in the CG group increased.The contents of cytarabine and 2,4-dihydroxypyridine increased in the CG group compared with the BB group.（2）Compare different metabolic pathways in each group:compare the IBS group with the CT group,different metabolic pathways included steroid hormone biosynthesis,lysine degradation,aldosterone synthesis and secretion.Compared with the model group,the differential metabolic pathways in the BB group included thiamine metabolism,porphyrin and chlorophyll metabolism and linoleic acid metabolism.Compared with the IBS group,the differential metabolic pathways included insect hormone biosynthesis,thyroid hormone signal pathway and autoimmune thyroid diseases.Compared with the CG group,the differential metabolic pathways include insect hormone biosynthesis.Conclusion1.The IBS-D model of this experiment was successfully prepared.The method of acupuncture and moxibustion at Biao-Ben acupoint combination better improved the gastrointestinal symptoms of rats,regulate heart rate variability and cardiac endocrine peptides,regulate sympathetic nerve and vagus nerve,improve the anxiety and depression mental state of rats,and achieve the purpose of regulating mind.2.The method of acupuncture and moxibustion at Biao-Ben acupoint combination can up regulate the contents of metabolites TXB2,（±）15 hete,PGE2,PGJ2,12-oxoete and 12,13-EpOME in IBS-D model rats,and participate in arachidonic acid metabolism,serotonin synapse,regulation of TRP channel and linoleic acid metabolism by inflammatory mediators,indicating that this therapy can improve gastrointestinal symptoms and regulate mind by regulating these metabolites.These metabolites may be potential serum biomarkers for the diagnosis,treatment and prognosis of irritable bowel syndrome.3.Acupuncture and moxibustion at Biao-Ben acupoint combination can change the contents of metabolites adenosine 5’-diphosphate,2-deoxyribose 1-phosphate,deoxyguanosine diphosphate（dGDP）,5’-adenylyl sulfate（APS）and kinurenine in the heart of IBS-D model rats,and participate in African trypanosomiasis and FOXO signaling pathways,indicating that this therapy can alleviate stress and anxiety by regulating these metabolites and metabolic pathways,which suggested that these metabolites may be potential cardiac biomarkers to distinguish whether irritable bowel syndrome with emotional disorders and the degree of mental disorders.4.Acupuncture and moxibustion at Biao-Ben acupoint combination can up regulate the metabolites cytarabine and 2,4-dihydroxypyridine in the colon tissue of IBS-D model rats,and involve three metabolic pathways:thiamine metabolism,porphyrin and chlorophyll metabolism and linoleic acid metabolism,indicating that this therapy can reduce oxidative stress and inflammatory response through these metabolites and metabolic pathway.It is suggested that these metabolites and metabolic pathways may be the metabolic model of the colon in the prevention and treatment of irritable bowel syndrome.5.The change of serotonergic synaptic metabolic pathway is an important factor in the occurrence of IBS.The therapy of acupuncture and moxibustion at Biao-Ben acupoint combination can regulate serotonergic synaptic metabolic pathway in serum,but no regulate serotonergic synaptic metabolic pathway in heart,suggesting that this therapy have different effects on serotonergic synaptic metabolic pathway in different tissue samples.How to prevent and treat irritable bowel syndrome through 5-HT related pathway needs further research and verification.6.The therapy of acupuncture and moxibustion at Biao-Ben acupoint combination can regulate the linoleic acid metabolism of serum,heart and intestine at the same time,and participate in the regulation of immune response and nerve center,so as to prevent and treat IBS.It is suggested that the relationship between serum,heart and intestine may be related to linoleic acid metabolism.How to prevent and treat irritable bowel syndrome through linoleic acid metabolism may be a direction of the next research on the effect mechanism.