Experimental Study On The Effect Of Moxibustion At Shenque Point On The Intestinal Flora Of Mice With Ulcerative Colitis

BACKGROUND:Ulcerative colitis(UC)is a non-specific inflammatory bowel disease that mainly occurs in the mucosa and submucosa of the large intestine.The main clinical manifestations are abdominal pain,diarrhea,mucous pus and bloody stools,and other symptoms such as tenesmus,intestinal perforation,polyps and even colon cancer,of which the incidence rate has increased year by year.Although the pathogenesis of UC is still unclear,people gradually realize the effect of intestinal flora on UC.The intestinal flora plays an important role in t immunity,digestion and metabolism of the body,while the colon is the site of with the most intestinal flora species and density in the human intestine tract.At present,the treatment drugs are concentrated in SASP,glucocorticoids and immunosuppressants,which are relatively limited in use and prone to adverse reactions.Moxibustion,as the traditional green therapy of traditional Chinese medicine,can dredge the meridians with its warmth,regulate the qi and blood,warm the yang to stop diarrhea,nourish the spleen and kidney,and nourish vitality to achieve the purpose of strengthening the body and improving immunity.In previous studies,Guan Yuan(CV4),Zusanli(ST36)and other remote acupuncture points were often selected for the purpose of cultivating the foundation and consolidate the origin.But Shenque(CV8)is more closely related to the gastrointestinal tract in terms of anatomical position,acupoint,and indications by contrast.Shenque(CV8)is close to the intestine,with rich blood vessels and lymphoid tissues.It has the effects of rejuvenating the yang,recuperating the spleen and stomach,clearing the turbidity,and regulating yin and yang.According to records,Shenque(CV8)has a unique effect on diarrhea.A small number of studies have confirmed the effect of mild moxibustion at Shenque(CV8)on some intestinal bacteria through bacterial culture,but the method is still traditional and the exploration is not deep enough,so this experiment uses moxibustion Shenque(CV8)as an intervention method,through 16S rDNA sequencing to explore its effect on the mechanism of intestinal flora in UC model mice.OBJECTIVE:To investisgate the effect of moxibustion at Shenque(CV8)on the structure of intestinal flora in mice with ulcerative colitis.In this experiment,ICR mice were used as the research object,and the mouse model of ulcerative colitis induced by dextran sodium sulfate was used to observe the scores of disease activity index,colon length,colon tissue structure of ulcerative colitis model mice,and the effects of proinflammatory factors IL-6 and TNF-? in serum to evaluate the efficacy of moxibustion at Shenque(CV8)on UC mice.Based on 16S rDNA sequencing technology,observe the microbial diversity information in the mouse flora by annotating and evaluating species,determine the differences in microbial structure between groups by alpha diversity analysis and PCoA analysis,and then by species composition analysis Different classification levels were used to analyze the differences of specific bacteria between different groups to explore the effect of moxibustion at Shenque(CV8)on the intestinal flora of mice with ulcerative colitis.At the same time,it was compared with mesalazine to provide reference for later research and clinical application.Methods:Thirty-two SPF grade male ICR mice(20-23)g were selected as the research object,and 8 mice were randomly selected as the blank group.The rest were modeled by 3.5%DSS self-drinking method for 8 days.After successful modeling,they were randomly divided into model group,moxibustion group and mesalazine group,and each group intervened in the following manner for 8 days:mice in the moxibustion group were grabbed and fixed with a fixture after each day,and mild moxibustion was applied to Shenque(CV8)for 20 min/d;daily grasping and fixing was applied both to the blank group and model group;the mesalazine solution with dose of 0.4 g/kg according to body weight was given to the mesalazine group of mice once a day.After the intervention,the recovery of UC mice was observed by disease activity index score,colon length,HE stained pathological tissue sections and pro-inflammatory factor levels,and the intestinal flora was analyzed by 16S rDNA sequencing.RESULT:1.Compared with the blank group,mice in the model group showed symptoms such as weight loss,bloody stools and loose stools,the DAI score was significantly increased(P<0.05),the length of the colon was shortened(P<0.05),and colon tissue sections showed a lot of inflammation.In the pathological state of cell infiltration,the levels of proinflammatory factors IL-6 and TNF-? in serum were significantly increased(P<0.05).After intervention treatment,compared with the model group,the symptoms of the moxibustion group and the mesalazine group were reduced,and the DAI scores were significantly reduced(P<0.05),colon length increased(P<0.05),colon tissue section returned to normal,serum proinflammatory factors IL-6 and TNF-? were significantly reduced(P<0.05);compared with mesalazine group,the moxibustion group had no significant difference in DAI score,colon length,and IL-6 and TNF-? content.2.Species annotation and evaluation results show that the sample size and data size of this sequencing have reached a sufficiently large and reasonable number,which can reflect the vast majority of microbial diversity information;compared with the blank group,the number of OTUs of model group were decreased,the number of OTUs in the mesalazine group and the moxibustion group were increased,which is similar to the blank group;a diversity analysis showed that,compared with the blank group,the sobs index and shannon index of the model group were significantly decreased(P<0.01);compared with the model group,the moxibustion group and the mesalazine group were significantly improved(P<0.01);There was no significant difference between the shannon index of the mesalazine group and the blank group.PCoA analysis showed that R=0.5831,P<0.01,indicating that there was a significant difference in the composition of the flora between the groupse.The boundary between the model group and the blank group was clear and the distribution was discrete,which indicates that the flora structure of the model group mice has changed.The moxibustion group,mesalazine group and the blank group were closer.Both moxibustion and mesalazine have an effect on the composition of the microflora and change towards the blank group.3.Species composition analysis results show that at the phylum level,compared with the blank group,the proportion of the Bacteroides in the model group increased,the proportion of the Firmicutes decreased,the F/B value decreased,and the Proteobacteria increased.Compared with the model group,the proportion of Bacteroides in the moxibustion group and the mesalazine group decreased,the proportion of Firmicutes increased,the F/B value increased,and the proportion of Proteobacteria decreased.At the genus level,compared with the blank group,the proportion of Lachnospiraceae NK4A136 group,Alloprevotella,unclassified of Lachnospiraceae,Ruminiclostridium 9,Odoribacter in the model group decreased significantly(P<0.05 or P<0.01),the proportion of norank of Muribaculaceae and Lactobacillus decreased but was not statistically significant;the proportion of Bacteroides,Parasutterella,Enterococcus,Klebsiella increased significantly(P<0.05 or P<0.01),the proportion of Blautia increased but was not statistically significant.Compared with the model group,the moxibustion group and the mesalazine group can reduce the proportion of Bacteroides,Parasutterella,Enterococcus,Klebsiella,and Blautia,and increase the proportion of Lachnospiraceae NK4A136 group,Alloprevotella,unclassified of Lachnospiraceae,Ruminiclostridium 9,Odoribacter,norank of Muribaculaceae and Lactobacillus.Compared with the model group,the proportion of Bacteroides,Enterococcus,Klebsiella,Alloprevotella,Lachnospiraceae NK4A136 group,unclassified of Lachnospiraceae in the moxibustion group were significantly different(P<0.05 or P<0.01).Compared with the model group,the proportions of Bacteroides,Klebsiella,Lachnospiraceae NK4A136 group,and unclassified of Lachnospiraceae in the mesalazine group were significantly different(P<0.05 or P<0.01).LEfSe multi-level species discriminant analysis showed that the blank group were characterized by Alloprevotell and norank of Lachnospiraceae;the UC model group were characterized by Bacteroidetes,Bacteroide,Parasutterella,Enterococcus and Klebsiella;the moxibustion were characterized by Firmicutes,Eubacterium fissicatena group and Allobaculum,and the Mesalazine group is characterized by Lachnospiraceae NK4A136 group,Dubosiella and Sporosarcina.CONCLUSION:1.Moxibustion at Shenque(CV8)can reduce the symptoms of UC such as weight loss,bloody stools,diarrhea,colon shortening and other symptoms,lower DAI score,improve colon tissue inflammatory response,reduce the content of proinflammatory factors IL-6 and TNF-?,and promote UC mice recovery;Moxibustion has the same therapeutic effect on UC as mesalazine;2.Moxibustion at Shenque(CV8)can improve the intestinal flora structure of UC mice,restore the diversity and richness of intestinal flora,increase F/B value,inhibit the growth of pathogenic bacteria,increase the proliferation of beneficial bacteria,especially promote the generation of some strains related to short-chain fatty acids.3.The mechanism of moxibustion's therapeutic effect on UC mice is related to the regulation of intestinal flora imbalance and colonic inflammatory response.