| Objective: Astragalus and Scutellaria are classical Chinese herbs commonly used in clinical practice and can be used in combination to treat a variety of diseases.Professor Qiao Baozhang and Associate Professor Zheng Jin,experts in Chinese internal medicine,have made good use of the combination of Astragalus and Scutellaria(3:1)in the treatment of acute radiation intestinal injury(ARII)with good clinical results.The results of the preliminary experiments suggest that the combination of Astragalus and Scutellaria 3:1 can significantly alleviate the inflammatory response in ARII mice.Therefore,this experiment is intended to further explore the effect and mechanism of combining Astragalus and Scutellaria to improve ARII in mice,and reveal that this drug may alleviate ARII by regulating the inflammatory response and intestinal flora.Methods: C57BL/6 mice were randomly divided into 6 groups,namely,10 mice in the blank group(Sham group),10 mice in the model group(ARII group),10 mice in the hormone group(DXT group),10 mice in the TCM low concentration group(TCM-L group),10 mice in the TCM medium concentration group(TCM-M group)and 10 mice in the TCM high concentration group(TCM-H group).The ARII model was constructed by irradiating the mice with a single abdominal dose of 14 Gy.After the successful construction of the model was confirmed by the results of inflammatory factors(TNF-α,IL-1β)and HE morphological staining of colonic tissues,the mice were given drug intervention for 14 consecutive days(10% dexamethasone sodium phosphate injection intraperitoneally 0.2ml/time in the DXT group;TCM-L,TCM-M and TCM-H groups were given different concentrations of herbal medicine solution by gavage 0.39 ml/time;Sham group and ARII group were given equal amount of saline by gavage).After the detection of TNF-α,IL-1β and IL-10 by ELISA;HE morphological staining of mouse colon tissue,the analysis of mouse intestinal flora by 16 S rRNA gene amplification technique,and the main compounds of the Chinese medicine solution were analyzed and identified by liquid-quality coupling technique,the improvement of the intestinal inflammatory response in mice with acute radiation intestinal injury was observed.Results: 1.Mouse colonic inflammatory factors and anti-inflammatory factors assay:(1)TNF-α results: compared with the ARII group,TNF-α in mice was significantly reduced in DXT group,TCM-M group and TCM-H group(P < 0.0001,P < 0.0001,P < 0.0001),and the treatment effect of the TCM-H group was significantly better than that of TCM-L group and TCM-M group(P < 0.0001,P < 0.05),and no significant differences were seen in the DXT group compared with the TCM-H group;(2)IL-1β results: compared with the ARII group,the expression of IL-1β was significantly reduced in the DXT group(P < 0.0001),and IL-1β was inhibited to different degrees in all groups of traditional Chinese medicine,with a significant reduction in the TCM-H group compared with the ARII group(P < 0.0001),and The improvement of IL-1β in the TCM-H group was significantly better than that in the TCM-L and TCM-M groups(P < 0.0001,P < 0.05),and no significant difference was seen in the DXT group compared with the TCM-H group;(3)IL-10 results: compared with the ARII group,the expression of IL-10 in the TCM-M,TCM-H,and DXT groups were significantly increased(P < 0.0001,P < 0.0001,P < 0.0001),and the expression of intestinal IL-10 in the TCM-H group was significantly higher than that in the TCM-M group(P < 0.05),while no significant differences were seen between the DXT and TCM-H groups.2.HE morphological staining of mouse colon: compared with Sham group,the intestinal epithelial barrier structure of ascending,transverse and descending colon of ARII group mice was incomplete,epithelial cells were shed,intestinal mucosal glands were shortened,crypt structure was changed,and the number of crypt and cup cells tended to decrease;the intestinal epithelial mucosa of ascending,transverse and descending colon of DXT group mice was more complete than ARII group,and the structure of glands and crypt was also restored to some extent.The intestinal epithelial mucosa of the ascending,transverse and descending colon in the DXT group was more intact than that in the ARII group,and the glandular and crypt structures were also restored to a certain extent,indicating that the dexamethasone could effectively restore the intestinal epithelial barrier structure in mice;while the intestinal epithelial mucosa of the ascending,transverse and descending colon in the TCM-L group was still disrupted,and the glandular and crypt structures were not effectively improved compared with those in the ARII group;the intestinal epithelial mucosa barrier of the ascending,transverse and descending colon in the TCM-M group and the ascending,transverse and descending colon in the TCM-H group were restored to a certain extent.3.Analysis of mouse intestinal flora: There was no significant change in the α-diversity of mice between groups;the β-diversity of intestinal flora of Sham group was significantly different from that of the rest of the group,and radiation significantly changed the β-diversity of intestinal flora of mice;the species composition of intestinal flora at the level of phylum,order,family and genus also differed among groups of mice;a total of nine microorganisms with statistically significant differences were analyzed by the test for significant differences between groups.The microflora that were statistically different between groups(LDA > 4)were identified by LEfse analysis,and those significantly enriched in the Sham group were: c＿Actinobacteria,g＿Bifidobacterium,o＿Bifidobacteriales,f＿Bifidobacteriaceae;those significantly enriched in the ARII group were: f＿Muribaculaceae,g＿norank＿f＿＿Muribaculaceae;The significantly enriched bacteria in the DXT group were: f＿Bacteroidaceae,g＿Bacteroides;the significantly enriched bacteria in the TCM-H group were: p＿Proteobacteria,c＿Gammaproteobacteria,f＿Enterobacteriaceae,o＿Enterobacterales,g＿Escherichia Shigella,f＿Rikenellaceae,o＿Oscillospirale,g＿Rikenellaceae＿RC9＿gut＿group.4.Analysis of the results of liquid mass spectrometry(LCMS)identification of 3:1 pairs of highly concentrated Astragalus and Scutellaria: We matched a total of 598 compounds in the mzCloud database.149 compounds in the mzCloud best match had mzCloud scores greater than 90,and we screened 55 compounds with scores greater than 99 against 55 compounds with the top peak area ranking.We overlapped the 55 compounds with scores above 99 with the 55 compounds with the top peak areas and obtained 29 compounds,including adenosine,L-Phenylalanine,nicotinic acid,salicylic acid,ononin,formononetin,baicalin,wogonin,baicalein,etc.Conclusions: 1.The 3:1 combination of medium and high concentrations of Astragalus and Scutellaria improved the inflammatory response to acute radioactive intestinal injury in mice,and the therapeutic effect of the herbal medicine was more significant in the high concentration group.2.High concentrations of Astragalus and Scutellaria in a 3:1 dosing ratio for treatment of ARII in mice can increase the abundance of f＿Rikenellaceae,o＿Oscillospirale,g＿Rikenellaceae＿RC9＿gut＿group and Bacteroides,Alistipes,Akkermansia,and Parabacteroides and others to adjust intestinal microecology and inhibit the progression of inflammation in mice.3.After the liquid mass spectrometry(LCMS)identification of high concentration of Astragalus and Scutellaria in 3:1 preparation,a total of 29 compounds with high peak area ranking and scores greater than 99 were screened,including adenosine,LPhenylalanine,nicotinic acid,salicylic acid,ononin,formononetin,baicalin,wogonin,baicalein,etc.4.The results of this study suggest that the mechanism of high concentrations of Astragalus and Scutellaria 3:1 in the treatment of acute radiation intestinal injury in mice may work by inhibiting the secretion of inflammatory factors,promoting the expression of anti-inflammatory factors and increasing the abundance of beneficial intestinal bacteria. |
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