| Objective: 1.Randomized controlled trial was conducted to observe the clinical effect of mild moxibustion at different temperatures on Diarrhea-predominant irritable bowel syndrome(IBS-D)patients and its influence on abdominal symptoms,quality of life,TCM syndrome,emotions;2.To observe the changes of Trp-Kyn(tryptophankynurenine)/5-HT(5-hydroxytryptamine)metabolites before and after treatment in IBS-D patients and explore the pathogenesis of IBS-D and Trp-Kyn/5-HT mechanism of moxibustion treatment by comparing the differences of Trp-Kyn/5-HT metabolites in serum between IBS-D patients and healthy volunteers;3.To explain Trp-Kyn/5-HT metabolism mechanism of moxibustion treatment.To determine the expression of TrpKyn/5-HT rate-limiting enzyme and the content of related metabolites in colonic tissue of IBS mice model by using IDO inhibitor 1-MT in Trp-Kyn pathway and rate-limiting enzyme TPH inhibitor PCPA in Trp-5-HT pathway.Methods: Part 1 Clinical Effect of Moxibustion on IBS and Trp-Kyn/5-HT Metabolism 108 IBS-D patients were randomly divided into moxibustion group 1(54 cases,43±1℃)and moxibustion group 2(54 cases,37±1℃),and control group(20 healthy volunteers).Tianshu(ST25,30min)and Zusanli(ST36,30min)were chosen for treatment.All the patients were treated 18 times in all(3 times a week).At week 0(before treatment),the 3rd week,the 6th week and the 8th week,abdominal pain score and Bristol are assessed as the primary clinical indicators.The secondary clinical indicators are IBS-SSS,TCM syndrome score,IBS-QOL and SAS and SDS.Trprelated metabolites were detected by LC-MS/MS were assessed to compare the difference of Trp metabolites between IBS-D patients and healthy volunteers and the effects of moxibustion at different temperatures on them.Part 2 Regulation Mechanism of Moxibustion on IDO and TPH in IBS Model Mice C57BL/6 mice were randomly divided into normal group,model group,moxibustion group,1-MT group and PCPA group.IBS model was established by TNBS enema,and intervened by moxibustion,inhibitor 1-MT and PCPA.The visceral hypersensitivity of mice was assessed by AWR.TRP-related metabolites in colonic tissue of mice were detected by LC-MS/ Ms.The protein and m RNA expressions of IDO1 and TPH1 in colonic tissue were detected by immunohistochemistry and RTPCR.Results: Part 1.Clinical Effect of Moxibustion on IBS and Trp-Kyn/5-HT Metabolism 1.Abdominal Pain Score,Bristol The total effective rate of moxibustion group 1 was 87.76%,and that of moxibustion group 2 was 51.11%.The difference between the two groups was statistically significant(P<0.05).Compared with the week 0,it shows that abdominal pain score and Bristol of moxibustion group 1 and 2 at the 3rd,6th and 8th week were significantly lower(P<0.05);compared with the 3rd weeks,moxibustion group 1 and 2 at the 6th and 8th week were significantly lower(P<0.05).At the 3rd,6th and 8th week,that of moxibustion group 1 was significantly lower than moxibustion group 2(P<0.05).2.IBS-SSS Compared with the week 0,the IBS-SSS scores of two moxibustion groups were significantly lower at all the week points(P<0.05);compared with the 3rd week,moxibustion group 1 and 2 at the 6th and 8th week were significantly lower(P<0.05),compared with the 6th week,moxibustion group 1 and 2 at the 8th week were significantly decreased(P<0.05).At the 3rd,6th and 8th week,the IBS-SSS score of moxibustion group 1 was significantly lower than that of moxibustion group 2(P<0.05).Comparison of sub-items: the scores of IBS-SSS in moxibustion group 1 were significantly lower than those in moxibustion group 2 at the 6th week(P<0.05).And IBS-SSS scores in both moxibustion groups were significantly decreased(P<0.05).3.TCM Syndrome Score The total effective rate of moxibustion group 1 was 87.76%.while the total effective rate of moxibustion group 2 was 37.78% after 6 weeks’ treatment.And the difference between the two groups was statistically significant(P<0.05);Compared with the week 0,it shows that TCM Syndrome Score of both moxibustion groups were significantly decreased at the 3rd,6th and 8th week(P<0.05);compared with the 3rd week,moxibustion group 1 and 2 were significantly lower at the 6th and 8th week(P<0.05);moxibustion group 1 was significantly decreased at the 8th week(P<0.05),and moxibustion group 2 had no significant difference at the 6th week(P>0.05).At the 6th and 8th week,the TCM syndrome scores of moxibustion group 1 were significantly lower than that of moxibustion group 2(P<0.05).Comparison of sub-items: the sub items as diarrhea,abdominal distension,abdominal pain,bowel sounds,abdominal fullness,mental fatigue and lazy state of speaking in moxibustion group 1 were lower than those in moxibustion group 2 at the 6th weeks(P<0.05).Compared with the scores before treatment,two moxibustion groups were significantly lower at the 6th week(P<0.05).4.IBS-QOL Compared with the week 0,it shows that IBS-QOL of moxibustion group 1 and 2 at each week point were significantly lower(P<0.05);compared with the 3rd week,the total score of IBS-QOL in moxibustion group 1 and 2 were significantly lower at the 6th and 8th week(P<0.05);compared with the 6rd week,the total score of IBS-QOL in moxibustion group 1 and 2 were significantly lower at the 8th week(P<0.05).At the 6th and 8th week,the IBS-QOL total score of moxibustion group 1 was significantly lower than that of moxibustion group 2(P<0.05).5.SAS,SDS Compared with the week 0,it shows that SAS and SDS of moxibustion group 1 and 2 were significantly lower at the 3rd,6th and 8th week(P<0.05);compared with the 3rd week,SAS and SDS scores of moxibustion group 1 at the 6th and 8th week were significantly lower(P<0.05);compared with the 6th week,SAS and SDSscores of moxibustion group 1 at the 8th week was significantly lower(P<0.05)and there was no significant difference in moxibustion group 2 at the 8th week(P>0.05).At the 6th and 8th week,SDS score of moxibustion group 1 was significantly lower than that of moxibustion group 2(P<0.05).At the 8th week,SDS score of moxibustion group 1 was significantly lower than that of moxibustion group 2(P<0.05).6.Trp-Kyn/5-HT Metabolism Compared with healthy volunteers,significant increasing was found in the content of tryptophan in serum,5-HT pathway motabolites(5-HT,5-HTP,5-HIAA),kynurenine pathway motabolites(KYN,KYNA,Xa,3-OH-AA and KYN/Trp in serum of IBS-D patients(P<0.05).7.Change of Trp-Kyn/5-HT Metabolism in Moxibustion Groups after Treatment The 5-HT and 5-HT/Trp in serum of patients were decreased by moxibustion at different temperatures(P<0.05).After treatment,KYN,KYNA and XA in serum were significantly decreased.The content of 3-HK was significantly increased.The ratio of kyn/trp was decreased(P<0.05).Part 2.Regulation Mechanism of Moxibustion on IDO and TPH in IBS Model Mice 1.General State of Mice and Histological Observation of Colon The normal group mice have glossy skin color,normal food intake,increased body weight and the stool color was brown and yellow;after enema,the skin and hair of the model mice were slightly dry.Mice looked tired.And the weight gain was slightly slowed down.And the stool was getting soft.After the intervention,the body weight of mice in 1-MT group and PCPA group increased slowly,while the skin color and mental state of mice in moxibustion group were improved in varying degrees.And the food intake increased gradually.The results showed that the colon of mice in each group was similar in general,and the bowel was uniform in thickness with no dilation and no adhesion to surrounding tissues.The intestinal mucosal surface was smooth and neat,the mucosal folds were complete,the direction was regular,and there was no congestion,edema,erosion and ulcer.2.Rectocele Stimulation Score Compared with the normal group,the AWR scores of the model group and moxibustion group under 30 mm Hg were higher than those of the normal group before treatment(P<0.05);The AWR score of moxibustion group under 30 mm Hg was significantly lower than that before treatment(P<0.05).3.Expression of IDO,TPH Protein and m RNA in Colon Tissue of Mice Compared with the normal group,the Expression of TPH1 and ido1 protein in colon tissue of model group was significantly higher than that in the normal group(P<0.05);compared with the model group,after mild moxibustion,AOD of TPH1 and IDO1 protein in mice colon tissue was significantly decreased(P<0.05),the expressions of AOD and m RNA in PCPA group was significantly lower than that in the model group(P<0.05),and the AOD and m RNA expression of IDO1 protein in the colon tissue of 1-MT group were significantly lower than that in the model group(P<0.05);Compared with moxibustion group,IDO activity of moxibustion group was similar to that of 1-MT group after treatment.There was no significant difference in KYN,Kyna,3-HK,XA,3-OH-AA,QUIN and Kyn/Trp between the two groups(P>0.05).4.Changes of Trp-Kyn/5-HT Metabolites in Colon Tissue of Mice The content of 5-HT and Trp-Kyn in IBS model mice were higher than those in normal group(P<0.05);compared with the model group,the Trp/Kyn moxibustion group was lower(P<0.05);Kyn content and Kyn/Trp in IDO inhibitor 1-MT group were significantly lower than those in model group(P<0.05);The ratio of 5-HT/Trp was significantly decreased in the TPH inhibitor PCPA group(P<0.05).Conclusion 1.Moxibustion(43℃ and 37℃)on Tianshu(ST25)and Zusanli(ST36)can improve the symptoms of IBS-D(abdominal pain,diarrhea),TCM syndrome score and IBS-QOL score.syndromes like abdominal pain and diarrhea started to be released,and those effects were increased by time.The effect of 43℃ moxibustion is better than 37℃.2.Compared with healthy volunteers,the abnormal Trp metabolism in IBS-D patients may be caused by the increased activity of the rate-limiting enzyme IDO through kynurenine pathway in TRP metabolism.Moxibustion may reduce Kyn/Trp and 5-HT/Trp in serum,the activities of IDO and TPH and contents.That may be one of the mechanisms of moxibustion effect in the treatment of IBS-D.3.Moxibustion can reduce the abnormally increased Kyn/Trp to inhibit the over expressed IDO activity and down regulate IDO1 protein expression in colon tissue of IBS model mice,which may be one of the mechanisms of moxibustion improving visceral hyperalgesia of IBS model mice. |
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