| The theory of "correspondence between heaven and man" is the most important embodiment of the overall view of traditional Chinese medicine,and its core content is the synergistic seasonal changes between the human body and nature.In "Cihai",late summer season refers to the sixth month.The climate of this season is mainly characterized by high temperature and high humidity,which often leads to a series of diseases due to the invasion of dampness and heat.Ulcerative colitis,as a kind of inflammatory bowel disease,often occurs in the late summer season.Traditional Chinese medicine believes that this seasonal pattern is closely related to the abnormal spleen main transport function caused by high temperature and high humidity environment in late summer season,but its specific pathogenesis has not been clarified.Based on this,this topic intends to explore the internal mechanism of high incidence of ulcerative colitis in late summer season by combining the modern research progress of intestinal microecology and immune environment,in order to provide theoretical and experimental basis for the prevention and treatment of ulcerative colitis.1 Research purposeThrough the analysis and collation of the literature,combined with the understanding of the onset of ulcerative colitis,the core pathogenesis of the high incidence of ulcerative colitis in late summer season is explored.In the experimental study,the UC mice model was established in the environment of room temperature and late summer,and the intervention was used to study the molecular mechanism of intestinal macrophage polarization imbalance caused by intestinal flora imbalance,and provide a new diagnosis and treatment target and experimental basis for the prevention and treatment of clinical intestinal inflammatory diseases such as ulcerative colitis.2 Research methods2.1 Theoretical researchUsing literature combing and theoretical discussion method,to collect,organize,summarize and summarize ulcerative colitis,intestinal flora and its metabolites,reactive oxygen species change of intestinal cells and macrophage polarization,to explore the internal mechanism of high incidence of ulcerative colitis in late summer.2.2 Experimental study2.2.1 Temperature and humidity settingArtificial climate simulation box is used to simulate the late summer environment.According to the temperature and humidity data of late summer season in Beijing provided by China Meteorological Network,the parameter of late summer season temperature is 30℃,relative humidity is 81%.The parameter of room temperature is 20℃,relative humidity is 50%.2.2.2 Experimental animal and model replicatesSixty-two male SPF grade C57BL/6J mice with a body mass of 20±2 g were randomly divided into four groups after 7 days of adaptive feeding,namely blank group of room temperature,model group of room temperature,blank group of late summer,late summer model group,including 9 mice in blank group of room temperature,10 mice in model group of room temperature;10 mice of blank group and 34 mice of model group were kept in artificial climate simulation box with late summer season.Mice in the room temperature blank and loate summer blank groups freely drank autoclaved water for 7 days,and the other groups freely drank 3%dextran sodium sulfate(DSS)solution for 7 days,the body weight,stool shape and disease activity index(DAI score)of various mice were recorded.After 7 days,three mice from each group were randomly selected for model evaluation.After successfully copying of UC model,the model group of late summer season was randomly divided into 3 groups,namely model group(6),5-ASA group(9)and LLT group(11),5-ASA group was gavaged by mesalazine,LLT group was gavaged by Lianlitang,and model group was gavaged by saline.2.2.3 Index detection method(1)The general condition was observed and recorded of mice,including body weight,anal temperature,drinking water volume,stool shape and fecal occult blood,etc.,and the disease activity index of each group of mice was compared and analyzed.(2)The colon tissue of each group of mice was observed under an optical microscope and scored according to the degree of histological lesions of the colon samples.(3)16S rDNA gene sequencing was used to detect the composition of fecal flora of mice,and LC-MS was used to detect the colonic metabolites of each group.(4)The ROS levels of colonic tissues in each group of mice were detected by fluorescent probe labeling.(5)Immunofluorescence polarization typing of macrophages in colon tissues of each group of mice was detected by staining(CD206 was used for surface labeling of M2 macrophages and CD68 was used for surface labeling of M1 macrophages).2.2.4 Statistical methodData analysis was performed by using SPSS20.0.When the data followed a normal distribution,the independent sample T-test was used to calculate the mean and standard deviation of each group,Results are presented as the mean ± standard deviation(x ± s),When comparing between multiple groups,first judge whether each group meets the normal distribution.If any belong,Test for homogeneity of variance was performed,compared between groups using one-way ANOVA for alignment of variance,pairwise comparisons with LSD;if the data is normal but the variance is uneven,compared between groups using Welch,pairwise comparisons with Dunnett T3;if the data does not fit the normal state,for comparison between groups using Kruskal Wallis test,also do Mann Whitney test for pairwise comparisons.P<0.05 is statistically significant.3 Research resultsExperiment 1:Effect of late summer and room temperature environment on the UC mice.Both the mice in the room temperature model group and the late summer season model group showed decreased body mass,loose stool and hematochezia,indicating the success of mold making.Compared with the room temperature blank group,the anal temperature decreased(P<0.01),body mass decreased(P<0.05),and DAI score increased(P<0.01).The optical microscope showed the room temperature model group,crypt swelling and inflammatory cell infiltration,and the histological lesion score increased(P<0.05).Compared with the blank group,the anal temperature decreased,the body mass decreased significantly(P<0.001),the DAI score increased significantly(P<0.001),the model group had extensive enlargement and widespread loss,significant mucosal edema with extensive inflammatory cell infiltration,and the histological lesion score increased(P<0.05).Compared with the room temperature model group,the anal temperature of the late summer season model group increased(P<0.05),and the DAI score and histological score showed a higher trend,but there was no significant statistical significance..Experiment 2:Treatment effect of Lian Li Tang in mice with ulcerative colitis of late summerDuring the medication period,the body mass of mice in model group were decreased,thin stool and blood in stool were decreased;compared with the model group,mice in the 5-ASA group were increased in body mass(P<0.05)and a significant decrease in DAI score(P<0.05).Compared with the model group,the mice in the LLT group increased significantly(P<0.01),and the DAI score decreased significantly(P<0.01).Under the optical microscope,mesalazine and the mucosal structure was basically recovered,the intestinal inflammation was significantly improved,and the histological score of pathological sections decreased(P<0.05).Experiment 3:Effect of Lian Li Tang on intestinal flora and its metabolites in mice of ulcerative colitisThe detection of the intestinal flora of each group found that the composition of the intestinal flora of mice in the model group was changed,increasing the abundance of Bacteroidetes and decreasing the relative abundance of Firmicutes at the phylum level,and increasing the abundance of[Eubacterium]_fissicatena_group and Erysipelatoclostridium at the genus level.After drug intervention,the intestinal flora of mice in LLT group increased the abundance of Proteobacteria at the phylum level and decreased the abundance of[Eubacterium]_fissicatena_group、Erysipelatoclostridium、Parabacteroides and Roseburia at the genus level.Enrichment analysis of differential metabolites in each group of mice showed that the expression of Trehalose and Succinic acid increased in the model group,and the differential metabolic pathways between blank group and model group were mainly about D-amino acid metabolism and ABC transporter(P<0.01);the intestinal Taurocholic acid 3-sulfate and Palmatine were increased in the LLT group,and the differential metabolic pathways involved between LLT group and the model group were mainly about the metabolism of ABC transporter and Taurine and hypotaurine metabolism(P<0.01).Experiment 4:Effect of Lian Li Tang on intestinal mitochondrial ROS and M1/M2 polarization in ulcerative colitis mice of late summerThe intestinal fluorescence staining images of mice in each group showed increased colon ROS in the model group(P<0.001),and the colon ROS in the model group was decreased compared with the model group(P<0.05).Comprehensive analysis of the images of each group showed that the model group had the lowest ratio of CD206/CD68,the mean CD68 in the blank group and the soup group were significantly reduced(P<0.001),and the CD206/CD68 was significantly higher(P<0.001).4 Research conclusion4.1 Theoretical researchBy combing and summarizing the relevant literature,the research hypothesis:the seasonal incidence rule of UC involves the temporal rhythm study of intestinal flora and intestinal immunity.The Institute of Traditional Chinese Medicine emphasizes that the normal function of the "spleen" is the key to maintain the intestinal microecological environment and the immune balance.The hot and humid environment in the late summer has aggravated the spleen deficiency,leading to the intestinal microflora dysbiosis,an increase in proinflammatory metabolites,and the balance of M1/M2 polarization in intestinal macrophages mediated by the ROS pathway is an intrinsic mechanism for the formation of ulcerative colitis.And the Lian Li Tang can improve the "spleen" function,regulating the ROS and intestinal macrophage polarization balance by changing the content of intestinal microbiota and their metabolites,this may be the key to its treatment of UC.4.2 Experimental study(1)The lesion severity,DAI score and histopathological score of UC mice in late summer were increased compared with UC mice at room temperature.(2)Lian Li Tang can reduce DAI score and histopathological score of UC mice,and improve UC symptoms.(3)Lian Li Tang can reduce intestinal ROS expression,inhibit M1 type macrophage polarization,and promote the enhancement of M2 type macrophage fine polarization.(4)The imbalance of polarization of intestinal macrophages in late summer UC mice may be related to the abnormal D-amino acid metabolism caused by the increased intestinal production of SCFAs bacteria in mice.(5)The mechanism of immunotherapy in Lian Li Tang may be related to the changed regulation of Taurine and hypotaurine metabolism of macrophage polarization balance. |
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