Regulation Of The Gut Flora And Mucosal TLRs/TRAF6 Signaling Pathway Of Obese Mice Based On The Jian-pi Yi-qi Acupuncture

Objective:Obesity as a chronic disease is intensively related to mucosal immunity,chronic inflammatory reaction and intestinal flora.In the current thesis,the obesity mice associated with Spleen deficiency syndrome were established based on the high-fat food.The regulational pattern of Jian-pi Yi-qi acupuncture against the disordered gut flora and abnormal TLRs/TRAF6 signaling pathway was then explored.Methods:In the first part,establishment of spleen deficiency syndrome obesity model,grouping and electroacupuncture treatment.Based on the replication of the nutritional obesity model,according to the main syndromes of spleen deficiency syndrome of Chinese Traditional Medicine（TCM）,screened of eligible mice into obese model of spleen deficiency syndrome from the aspect of daily food intake,open field test and fecal water content,and evaluated though weight,Lee’s index,total cholesterol（CHO）,triacylglyceride（TG）,blood glucose（Glu）,leptin（LP）,adiponectin（ADPN）,CD₄⁺,CD₈⁺,CD₄⁺/CD₈⁺ratio and CD⁺_11cof T lymphocytes and inflammatory factors（IL-6,IL-10,TNF-α）,and obtained the obese mice of spleen deficiency syndrome,and dived those randomly into model group,Eletroacupuncture 7^th（A7）group,Eletroacupuncture 14^th（A14）group,Eletroacupuncture 21^st（A21）group,Eletroacupuncture 28^th（A28）group,respectively.Eletroacupuncture group was pinprick Tianshu,GuanYuan,Zusanli,Sanyinjiao points,and connected to electroacupuncture treatment to compared with the difference of weight,Lee’s index,fat mass around the epididymis,CHO,TG,LP,ADPN,CCK,FFA,apo-A1,apo-B,T lymphocytes and inflammatory factors and morphology of epididymal fat.In the second part,detection of TLRs/TRAF6 signaling pathway in intestinal mucosa.The immunohistochemical method was used to compare the difference five proteins distribution of TLR₁,TLR₂,TLR₄,TLR₇ and TLR₉ in peyer’s patches,andreverse transcription polymerase chain reaction was used to compare the gene relative expression difference of TLR₁,TLR₂,TLR₄,TLR₇,TLR₉,MyD88,TRAF6.In the third part,Analysis of structure and function of intestinal flora.16S rDNA sequencing was used to observe the difference of relative abundance,species classification tree,sample complexity and diversity of different species level（Phylum,Class,Order,Family,Genus）in intestinal flora.Metagenomic sequencing was used to observed the differences of gene prediction and abundance analysis,species annotation analysis and function annotation analysis.Results:The body weight,Lee’s index,fecal water content,and and the parameter content of CHO,TG,LP,ADPN,CD₈⁺and CD⁺_11cof T lymphocytes,IL-6,IL-10,TNF-αin model group of spleen deficiency syndrome obese mice were significantly higher than normal group,and the exercise time and distance,CD₄⁺/CD₈⁺ratio,CD₄⁺of T lymphocytes in model group was significantly lower than normal group（P<0.01）.After acupuncture treatment,the body weight,Lee’s index,and the parameter content of CHO,TG,LP,ADPN,CCK,FFA,apo-B,IL-6,IL-10 and TNF-α,CD₈⁺and CD⁺_11cof T lymphocytes,the diameter of adipocytes were significantly lower than model group,and the content of apo-A1 and the apo-A1/apo-B ratio,CD₄⁺/CD₈⁺ratio,CD₄⁺of T lymphocytes were was significantly increased than model group.Compared with A7 group,the A21 and A28 group were significantly lower（P<0.05）.The protein distribution density of TLR₁,TLR₂,TLR₄ and TLR₉ was higher than normal group（P<0.01）,TLR₇ protein distribution density lower than normal group.The distribution of TLR₁,TLR₂,TLR₄,TLR₇ and TLR₉ was significantly improved after acupuncture intervention,and the improvement of A21 group was the most significant（P<0.05）.The relative expression of TLR₁,TLR₄,TLR₄,TLR₉ and TRAF6 was significantly increased（P<0.01）,and the relative expression of TLR₇and MyD88 gene in PP was significantly decreased（P<0.01）;and after acupuncture intervention,the relative expression of TLR₁,TLR₄,TLR₄,TLR₉ and TRAF6 were down-regulated,and the relative expression of TLR₇,MyD88 were up-regulated.Compared with A7 group,the A21 and A28 was improved the change significantly（P<0.05）.Analysis of relative abundance of species and cluster thermal graphs displayed that the Firmicutes,Tenericutes and synergistetes in the model group were significantly higher than normal group,the Bacteroides,Caldiserica and proteobacteria lower than the normal group.After the intervention of acupuncture,the flora imbalance could be gradually improved,especially the Caldithrix,Elusimicrobia,Crenarchaeota of A7group were close to normal group.The NMDS analysis,sample clustering analysis,T-test analysis and the LEfSe analysis showed that there was significant difference between the normal group and the model group（P<0.01）,and the acupuncture intervention was rapid recovery and the effect was better in A21 group.From the KEGG database,the K01915,K03057,K03081,K10117,K10440,K10118,K10439,K1019 and other functional genes in model group were significantly higher than normal group,and the K01601 and K02110 functional gene expression was significantly lower than normal group（P<0.01）.From the eggNOG database,the expression of COG1129,COG4753,COG2972,COG0395,COG1663,COG1175,ENOG410XNMH and others homologous relevant gene in model group were significantly higher than normal group（P<0.01）,and the COG1850,COG2801,COG3293 of homologous gene were expressed lower than normal group（P<0.01）.From the CAZy database,the expression of GH65,GH25,GH18,GH32,GH1,GT35,CE4 and other functional genes in model group was significantly higher than normal group,and the functional gene expression of GTS and GTS1 was significantly lower than normal group（P<0.01）.The functional gene was gradually improved after acupuncture intervention,and the change of A21 group was obvious significantly.The acupuncture treatment of obesity in the first 7 days gained the most obvious effect,in21 days to reach the most stable state.Conclusion:（1）Successful establishment of spleen deficiency syndrome obesity model based on dirt-induced obese mice.And acupuncture was significant effect to loose weight.（2）Acupuncture could regulate distribution density of TLR₁,TLR₂,TLR₄,TLR₇,TLR₉ and other five TLRs protein,and down-regulation expression of TLR7,TLR4,TLR9,TRAF6 and other genes,and up-regulation expression of TLR7,MyD88genes,and adjust the TLRs/TRAF6 signaling pathway to improve intestinal mucosal immune dysfunction,reduce intestinal mucosal chronic inflammatory response,and intervene 21 Days to achieve the best therapeutic effect.（3）Acupuncture improves the structure and quantity of intestinal flora,and benign adjustment of Tenericutes,synergistetes,Caldiserica,proteobacteria,Caldithrix,Elusimicrobia,Crenarchaeota and other strains of bacteria,and regulation of functional genes,such as K01915,K03057,K0307,K101140,K101140,K101439,K1019,CO101H9,COG1759,COG2972,COG0395,COG1663,COG1175,ENOG410XNMH,GH65,GH25,GH18,GH32,GH1,GT35,CE4.And promotion the the establishment of a new healthy balance of ecology.The acupuncture treatment of obesity in the first7 days gained the most obvious effect,in 21 days to reach the most stable state,Acupuncture can effectively lose weight based on the regulation of intestinal TLRs and the key elements of downstream pathways,such as MyD88 and TRAF6,and improvement of the structure and function of intestinal flora,and promote the establishment of a healthy micro-ecology.