To Study The Sensitization Mechanism Of Heat-Sensitive Moxibustion In Intervention Of IBS-D Based On BDNF-TrKB Signaling Pathway

Part I Evidence-based researchObjective: this study aimed to comprehensively analyze the effectiveness and safety of heat-sensitive moxibustion in the treatment of diarrhea-predominant irritable bowel syndrome(IBS-D)and evaluate its clinical application value.Methods: Relevant research articles were collected and eligible RCTs were screened by retrieving Chinese Biomedical Database,China National Knowledge Infrastructure Database,Wang Data,Chinese Medicine Database System,Google Scholar,Pub Med,and Embase databases in both Chinese and English.Meta were extracted and quality was assessed by two independent reviewers using Rev Man 5.3 for meta-analysis of included study results and funnel plot for publication.Results: A total of 14 RCTs involving 928 patients were included,including 466 cases in the treatment group and 462 cases in the control group.The treatment group was treated with heat-sensitive moxibustion,and the control group was treated with sham treatment,conventional treatment or other treatment methods.The main evaluation indicators included the severity of diarrhea,abdominal pain and quality of life after treatment.The difference in combined effect factor RR = 1.15,95% CL [1.07,1.23],and Z = 3.99(P < 0.0001)was statistically significant.Conclusion: The efficacy of heat-sensitive moxibustion in the treatment of IBS-D patients is superior to that of the control group,and the diarrhea severity and quality of life have been significantly improved after the treatment.In terms of safety,the heat-sensitive moxibustion treatment group had no obvious adverse reactions and side effects as compared with the control group.Heat-sensitive moxibustion is an effective and safe treatment for IBS-D patients,and the mechanism can be explored through animal experiments.Part ii experimental studyObjective: Male SD rats were used as the experimental subjects to model IBS-D rats.Starting from the BDNF-TrKB signaling pathway,which is closely related to the visceral hypersensitivity of IBS-D,the relationship between the reduction of visceral hypersensitivity by heat-sensitive moxibustion and BDNF-TrKB signaling pathway was studied,and the peripheral sensitization mechanism of acupoint heat sensitization was explored,in order to provide experimental basis for the clinical treatment of IBS-D by heat-sensitive moxibustion.Method: After adaptive feeding for 7 days,42 healthy male SD rats were randomly assigned using the RAND function,and the experimental subjects were divided into a blank group(6 rats)and a model group(36 rats).The control group will be fed normally without any intervention.The IBS-D model was established in the model group by the combination of intragastric administration of Folium sennae decoction and chronic unpredictable stimulation,for 21 days.After the successful establishment of the model,the model groups were randomly divided into the model group,the 7,8-DHF group,the ANA-12 group and the moxibustion group using the RAND function again.Each group received the corresponding intervention measures.The moxibustion group was divided into the heat-sensitive moxibustion group and the non-heat-sensitive moxibustion group according to the tail temperature changes of rats during the moxibustion at "Mingmen"(BL 17).The changes of general condition,body weight and visceral sensitivity of rats in each group before and after treatment were observed.The samples taken after the treatment were used to detect the expression of BDNF and TrkB in the colon by immunohistochemistry and WB,respectively.The mRNA and protein expressions of BDNF and TRKb in the frontal lobe and hippocampus were detected by real-time quantitative PCR and WB.The downstream factors of the pathway were determined by biochemical detection of Ca2+ concentration in the hippocampus and quantitative fluorescent PCR of CREB and BCL-2 contents in the hippocampus.The immunofluorescence was used to detect the contents of SP,CGRP and TRPV1 in the skin tissue,and the toluidine blue staining was used to observe the number of mast cells in Mingmen(BL 25)region,so as to explore the peripheral sensitization mechanism of acupoint heat sensitivity.Results:(1)The rats in the model group showed mental fatigue,slow reaction,decreased spontaneous activity,more curled-up,messy and withered hair,dry and dilute feces,soft feces,and the body weight showed a trend of loss;After intervention,the states of rats in the 7,8-DHF group were aggravated,and the states of rats in ANA-12 group,non-heat-sensitive moxibustion group and heat-sensitive moxibustion group were improved to different degrees.The state of rats in the heat-sensitive moxibustion group was more significant.(2)The colonic tissue of rats was observed under the microscope,and it was found that the tissues and mucosa of the blank control group were structurally intact and showed no obvious damage,proliferation of fibrous connective tissue,interstitial edema,inflammatory reaction,etc.;In the model group,the tissue structure was disordered but there was no obvious damage,and mild inflammatory cell infiltration,no proliferation and edema were observed.(3)After intervention,compared with the blank group,the intestinal water injection volumes of the model group and the 7-8-DHF group were decreased(P < 0.01).Compared with the model group,the intestinal water injection volumes in the ANA-12 group,the heat-sensitive moxibustion group,and the non-heat-sensitive group were significantly increased(P < 0.01).Compared with the ANA-12 group,the expression of intestinal water injection in the 7-8-DHF group was significantly reduced(P < 0.001).Compared with the 7-8-DHF group,the intestinal water injection volumes in the heat-sensitive moxibustion group and the non-heat-sensitive group were increased(P< 0.001).(4)Compared with the blank group,the protein expression levels of BDNF and TrkB in the colon tissue of the model group were increased.Compared with the model group,the protein expression levels of BDNF and TrkB in the colon tissue of the 7,8-DHF group were increased(p<0.05).The protein expression levels of BDNF and TrkB in ANA-12 group,the non-heat-sensitive moxibustion group and the heat-sensitive moxibustion group were decreased(p<0.05).The decrease was most significant in the heat-sensitive moxibustion group.Compared with the 7-8-DHF group,the intestinal water injection volumes in the heat-sensitive moxibustion group and the non-heat-sensitive group were increased(P < 0.001).(5)Compared with the blank group,the mRNA expression levels of BDNF and TRKb in the frontal lobe and hippocampus of the model group were increased.Compared with the model group,the mRNA expression levels of BDNF and TRKb in the frontal lobe and hippocampus of the 7,8-DHF group were increased(p<0.05).The mRNA expression levels of BDNF and TRKb in ANA-12 group,non-heat-sensitive moxibustion group and heat-sensitive moxibustion group were decreased(p<0.05).The reduction was most significant in the heat-sensitive moxibustion group.(6)Compared with the blank group,the expression levels of Ca2+ concentration in the model group,ANA-12 group,7-8-DHF group and non-heat-sensitive moxibustion group were increased(P <0.05).Compared with the model group,the Ca2+ concentration in the 7-8-DHF group was increased(P < 0.01),and the expression levels of ANA-12,heat-sensitive moxibustion and non-heat-sensitive groups were significantly decreased(P < 0.01).(7)Compared with the blank group,the CREB expressions in the model group,ANA-12 group,7-8-DHF group,and non-heat-sensitive moxibustion group were increased(P <0.05).Compared with the model group,the CREB expression levels in the ANA-12 group and the heat-sensitive moxibustion group were significantly decreased(P <0.01).Compared with the blank group,the expressions of BCL-2 in the model group and the 7-8-DHF group were increased(P < 0.05).Compared with the model group,the expressions of BCL-2 in the ANA-12 group,the heat-sensitive moxibustion group,and the non-heat-sensitive moxibustion group were decreased significantly(P < 0.01).(8)After intervention,compared with the blank control group,the expression levels of SP,CGRP and TRPV1 in the model group and 7-8-DHF group were significantly increased in immunofluorescence detection(P < 0.01).Compared with the model group,the expression levels of SP,CGRP,and TRPV1 in the ANA-12 group and the heat-sensitive moxibustion group were significantly decreased(P < 0.01).Compared with the ANA-12 group,the expressions of SP,CGRP and TRPV1 in the 7-8-DHF group were significantly increased(P < 0.01),and the expressions of heat-sensitive moxibustion group and non-heat-sensitive group in immunofluorescence were significantly decreased(P < 0.01).(9)Compared with the blank group,the numbers of mast cells in ANA-12 group,heat-sensitive moxibustion group and non-heat-sensitive moxibustion group were increased.Compared with the non-heat-sensitive moxibustion group,the degranulation of mast cells was noticed in the heat-sensitive moxibustion group and ANA-12 group.Conclusion: Heat-sensitive moxibustion can effectively improve the visceral hypersensitivity in IBS-D rats.Heat-sensitive moxibustion could effectively reduce the expression of BDNF,TrkB and downstream factors CREB,BCL-2 and Ca2+ in the hippocampus,frontal cortex and colon of IBS-D model rats,suggesting that heat-sensitive moxibustion could effectively treat IBS-D and might play a role by regulating the BDNF/TrkB pathway.SP,CGRP,TRPV1 and mast cells are one of the material bases for acupoint heat sensitization in Mingmen(GV 14).