| Objective:Under the guidance of the theory of"dysfunction of spleen in transportation,mutual accumulation of phlegm and blood stasis",combined network pharmacology and 16s r DNA sequencing analysed that the TLR-4/My D88/NF-κB pathway may be closely related to the potential mechanism of Huayu Qutan Formula in interfering with AS intestinal mucosal injury.We used animal experiments to construct an AS model by high-fat feeding Apo E-/-mice,and cellular experiments with LPS stimulation of porcine small intestinal epithelial IPEC-J2 cells to construct an inflammatory injury model,to explore the effects and mechanisms of this formula on the intestinal flora and intestinal mucosal barrier of AS.Materials and Methods:Experiment 1:Based on the theoretical foundation of"dysfunction of spleen in transportation,mutual accumulation of phlegm and blood stasis.",the theoretical research on the method of strengthening the spleen,expelling phlegm and removing stasis to regulate intestinal flora to prevent and control atherosclerosis was explored.Experiment 2:Taking the intersection of the action targets of Huayu Qutan Formula with those of AS and intestinal mucosal injury,we obtained the potential therapeutic targets of this formula for the treatment of intestinal mucosal injury of AS,and drew the network map of disease,drug,and target gene to construct the PPI network and further screened the key targets to get access to the biological processes,cellular components,molecular functions,and cellular signalling pathways.Experiment 3:After 1 week of acclimatisation of 40 Apo E-/-mice and 10 C57BL/6 J mice,Apo E-/-mice were grouped according to the random number table method and given high-fat chow for model replication.The Chinese medicine treatment group was converted to administer the equivalent dose according to the ratio of human to mouse body surface area,and the drug was administered by gavage once a day for 30 consecutive days starting from the first day of successful modelling.It was divided into normal control group,model group,and resolving stasis and expectorant formula group.16s r DNA sequencing analysis of mouse intestinal flora changes included:valid data statistics and distribution of quality sequences;OTU clustering results;construction of dilution curves;Shannon-Wiener curves;Specaccum species cumulative curves;analysis of the composition of the mouse intestinal flora in each group and Alpha diversity analysis;Principal Component Analysis（PCA analysis）;NMDS analysis;Heatmap based on Uni Frac;and analysis of structural variability in the composition of the species of the intestinal flora of the mouse intestinal flora in each group.Experiment 4:Ten C57BL/6 J mice were classified as the normal group,and 40 Apo E-/-mice were divided into the model group,and the low,medium,and high dose groups of traditional Chinese medicine;the model preparation was the same as that of experiment 3.We will first use HE staining to assess the degree of pathological damage to the intestinal mucosal barrier,ELISA to detect the level of LPS in the serum and cecum contents of mice in each group,and immunohistochemistry to detect the expression of ZO-1,Claudins and Occludins in the intestinal tissues of mice in each group.Secondly,Western Blot was applied to detect the protein expression levels of ZO-1,Claudins,Occludins,and TLR-4,My D88,and NF-κB in the intestinal tissues of mice in each group,and WES was used for fully automated protein quantification.Finally,the expression levels of ZO-1,Claudins,Occludins,TLR-4,My D88and NF-κB m RNA in the intestinal tissues of mice in each group were detected by Realtime RT-PCR.Experiment 5:Cells were divided into normal control groups:IPEC-J2 cells;Model group:50ug/ml LPS+IPEC-J2 cells;Chinese medicine containing serum group:50ug/ml LPS+IPEC-J2 cells+rat herbal drug-containing serum;Inhibitor group:50ug/ml LPS+IPEC-J2cells+rat Chinese drug-containing serum+TLR-4 inhibitor（TAK-242）.Effect of MTS on cell viability;Effect of different concentrations of LPS on CLND1 and TJP1;Immunofluorescence detection of ZO-1 protein expression in each group of cells;The levels of TLR-4,My D88,NF-κB,ZO-1,Claudin-1,and Occludin m RNA were detected by q RT-PCR;and the levels of TLR-4,My D88,NF-κB,ZO-1,Claudin-1,and Occludin proteins were detected by Western Blot.Result:Experiment 1:Guided by the Chinese medical mechanism of AS,"dysfunction of spleen in transportation,mutual accumulation of phlegm and blood stasis.",in-depth exploration of the mechanism of intestinal Jianpi Huayu Qutan method regulate intestinal flora against atherosclerosis,to contribute to the further elucidation of the scientific meaning of"phlegm and blood atasis"in atherosclerosis.Experiment 2:113 active ingredients and 412 corresponding targets of Huayu Qutan Formula were collected in TCMSP and BATMAN-TCM.After the intersection of potential targets of Huayu Qutan Formula with AS-related targets and targets related to intestinal mucosal injury,192intersecting genes were obtained,and the network of"Huayu Qutan Formula-Active Ingredient-AS-Injury"was constructed.Protein interaction network relationships,GO gene function ontology enrichment analysis,KEGG pathway enrichment analysis yielded 2827entries,as well as enrichment to 174 pathways.Experiment 3:The results of the dilution curves of the 16s flora in each group of mice are as follows:The curves of each group tended to be flat,indicating that the amount of sequencing data was reasonable and reliable,and the results of Shannon-Wiener curves tended to be flat at the level of 20,000 Reads,and all the samples entered the plateau period,which indicated that the amount of sequencing data in this study was sufficiently large to adequately reflect the microbiological information of the tested samples.Specaccum species accumulation curve results are shown below:indicating that the sample size of this study was sufficient to analyse Alpha diversity;Analysis of the composition and Alpha diversity of the intestinal flora of mice in each group showed that OTUs and Chao1indices were significantly lower in the model group compared with the normal control group,and the differences were statistically significant（P<0.05 or P<0.01）Compared with the model group,the OTUs and Chao1 indices were significantly lower and the Shannon and Simpson indices were higher in the group of the formula,and the difference was statistically significant（P<0.01）;Secondly,principal component analysis（PCA analysis）illustrated that there was variability between groups.NMDS analyses illustrated small microbial differences within groups and large differences between groups,Uni Frac-based heatmap showed that Beta diversity comparisons between groups showed differences,with the control group and Huayu Qutan Formula group having closer matrices,higher similarity,and showing a trend of decreasing differences in Beta diversity,while the model group showed significant differences in Beta diversity compared to the other two groups,with matrices that were farther apart.3.Analysis of the structural variability of the species composition of the intestinal flora of mice in each group showed:Firmicutes and Bacteroidetesother remained the major components of the intestinal flora in all groups of mice.Comparing the proportions of bacteria at these phylum levels in the cecum contents of mice in each group,the proportions of Firmicutes and Actinobacteria were increased in the model group,while the proportions of Bacteroidetesother and Aspergillus were decreased;the proportions of Firmicutes and Actinobacteria were decreased,while the proportions of Bacteroidetesother and Aspergillus were increased in the intervention of Huayu Qutan Formula,as compared with the model group.The results of the LDA Effect Size analysis showed that the species that differed in abundance in the normal control group were:f-Bacteroidale-S24-7-group,f-Prevotellaceae,o-Bacteroidales,c-Bacteroidia,f-Lactobacillaceae,and f-Ruminococcaceae;Species that differed in abundance in the Huayu Qutan Formula group were:f-Bacteroidaceae,f-Staphylococcaceae,o-Bacillales,f-Enterococcaceae,o-Lactobacillales,c-Bacilli,f-Enterobacteriaceae,o-Enterobacteriales,c-Gammaproteobacteria.Experiment 4:1.Pathomorphological results:The intestinal epithelial tissue of normal control mice was structurally intact,with no obvious damage to the mucosal layer and tightly arranged epithelial cells.The intestinal epithelium of mice in the model group could be seen as a large area of villi detached,and the lamina propria was exposed;Huayu Qutan Formula low-dose group could be seen as a widening of the subepithelial gap in the staining,and the epithelial layer was separated from the lamina propria,accompanied by a small amount of villi detached;the upper and lower epithelial gaps at the tip of the villi could be seen as a slight widening in the middle-dose group of Huayu Qutan Formula;the upper epithelial gap at the tip of the villi was seen as a slight widening in the high-dose group of Huayu Qutan Formula.In Huayu Qutan Formula medium-dose group,the upper and lower cortical gaps at the tip of villi were slightly widened;in Huayu Qutan Formula high-dose group,the subepithelial gap was slightly widened and the epithelial layer was slightly separated from the lamina propria.2.results of cecum contents and serum LPS levels of mice in each group:Compared with the normal control group,the levels of LPS in serum and cecum contents of mice in the model group were significantly decreased（P<0.01）;compared with the model group,the levels of LPS in serum and cecum contents of mice in the medium-dose and high-dose groups of Huayu Qutan Formula were significantly decreased（P<0.05 or P<0.01）.3.Levels of inflammatory factors IL-6,IL-1βand TNF-αin intestinal tissues of mice in each groupThe results showed that the expression of TLR-4,My D88 and NF-κB proteins in the intestinal tissues of the model group was significantly higher compared with that of the normal control group（P<0.01）;the expression of TLR-4,My D88 and NF-κB proteins in the Huayu Qutan Formula low,medium and high dose groups was significantly lower compared with that of the model group（P<0.01）.4.Immunohistochemical detection of ZO-1,Occludin and Claudin1 protein expression in the intestinal tissues of mice in each group.The expression of ZO-1,Occludin and Claudin1 proteins in the intestinal tissues of the model group was significantly lower compared with the normal control group;the expression of ZO-1,Occludin and Claudin1 proteins in the low,medium and high dose groups of Huayu Qutan Formula was significantly higher compared with the model group.5.Expression of TLR-4/My D88/NF-κB pathway-related proteins and m RNAs in mice of all groupsComparison of TLR-4,My D88,and NF-κB protein expression in the intestinal tissues of mice in each group was shown:Compared with the normal control group,the expression of TLR-4,My D88 and NF-κB proteins in intestinal tissues of the model group was significantly higher（P<0.01）;compared with the model group,the expression of TLR-4,My D88 and NF-κB proteins in the Huayu Qutan Formula low,medium and high dose groups was significantly lower（P<0.01）;Comparison of intestinal TLR-4,My D88,and NF-κB m RNA levels in each group of mice showed that the expression of TLR-4,My D88,and NF-κB m RNA in the intestinal tissues of the model group was significantly higher compared with that of the normal control group（P<0.01）;Compared with the model group,the expression of TLR-4,My D88,and NF-κB proteins was significantly reduced in the low,medium,and high dose groups of Huayu Qutan Formula（P<0.01）.6.Expression of proteins and m RNAs related to tight junction proteins between intestinal epithelial cells of mice in various groupsComparison of mouse intestinal ZO-1,Occludin,and Claudin1 protein expression in each group was shown:The expression of ZO-1,Occludin and Claudin1 proteins in the intestinal tissues of the model group was significantly decreased compared with that of the normal control group（P<0.01）.Compared with the model group,the expression of ZO-1 in the low and medium dose groups of Huayu Qutan Formula and the expression of Occludin and Claudin1 proteins in the medium dose group of Huayu Qutan Formula were significantly higher（P<0.01）.Comparison of intestinal ZO-1,Occludin,and Claudin1 m RNA levels of mice in each group showed that the expression of ZO-1,Occludin,and Claudins m RNA in the intestinal tissues of the model group was significantly decreased compared with that of the normal control group（P<0.01）.Compared with the model group,the expression of ZO-1 in the low and middle dose groups of Huayu Qutan Formula and the expression of Occludin and Cludins m RNA in the middle dose group of Huayu Qutan Formula were significantly higher（P<0.01）.Experiment 5:1.CCK8 screening of LPS（10ug/ml、50ug/ml）intervention in IPEC-J2 cells for 24 h.MTS screening showed that 50μg/m L LPS was the optimal dose to stimulate CLDN1 and TJP1;2.Levels of inflammatory factors IL-6,IL-1β,and TNF-αin cell supernatants and cells of each groupThe results showed that compared with the normal control group,the levels of IL-6,IL-1β,and TNF-αwere significantly higher in the model group（P<0.05）;compared with the model group,there was no significant difference in the levels of IL-6,IL-1β,and TNF-αin the blank drug-containing serum group（P>0.05）,and the levels of IL-6,IL-1β,and TNF-αin the traditional Chinese medicine drug-containing serum group were significantly lower（P<0.05）;3.Immunofluorescence detection of the distribution of tight junction protein ZO-1 in various groups of cells.The results showed that the expression of tight junction protein ZO-1 was significantly lower in the model group compared with the normal control group,and the expression of tight junction protein ZO-1 was significantly higher in the traditional Chinese medicine-containing serum group and the inhibitor group compared with the model group,and the expression of tight junction protein ZO-1 was significantly lower in the inhibitor group compared with the traditional Chinese medicine-containing serum group;4.Expression of TLR-4/My D88/NF-κB pathway-related proteins intestinal epithelial intercellular tight junction proteins and m RNAs in various groups of cellsCompared with the normal control group,the expression of TLR-4,My D88 and NF-κB was significantly increased and the expression of ZO-1,Occludin and Claudins m RNA was significantly decreased in the cells of the model group（P<0.01）.Compared with the model group,the expression of TLR-4,My D88 and NF-κB was significantly down-regulated,and the expression of Occludin and Cludins m RNA was significantly elevated in the traditional Chinese medicine-containing serum group and the inhibitor group（P<0.05 or 0.01）.Compared with the normal control group,the expression of TLR-4,My D88,and NF-κB was significantly increased,and the expression of ZO-1,Occludin,and Claudins m RNA was significantly decreased in the cells of the model group（P<0.01）.Compared with the model group,the expression of TLR-4,My D88,and NF-κB was significantly down-regulated,and the expression of Occludin and Cludins m RNA was significantly elevated in the Chinese medicine-containing serum group and the inhibitor group（P<0.05 or 0.01）.Conclusion:1.Huayu Qutan Formula has a corrective effect on intestinal flora disorders in Apo E-/-AS mice.2.Huayu Qutan Formula has a protective effect against intestinal mucosal barrier damage in Apo E-/-AS mice.3.Huayu Qutan Formula improves intestinal mucosal barrier damage in AS state by inhibiting inflammatory response through modulating TLR-4/My D88/NF-κB pathway. |
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