| Purpose:The mouse model of acute radiation enteritis was established,and 16 S r RNA technique was used to study the regulating effect of Gegen Qinlian Decoction(GQD)on intestinal microflora of mouse with radiation enteritis,providing theoretical basis and experimental basis for its clinical application.Material and method:Forty-one male BALB / c mouse of SPF grade were randomly divided into six groups by body weight order,they were blank control group(n=6),model control group(n=7),low dose GQD group(n=7),medium dose GQD group(n=7),high dose GQD group(n=7)and Montmorillonite powder group(n=7).Mouse in each group were fed with SPF grade diet and pure water,and after one week,the mouse model of acute radiation enteritis was established.Mouse were fasted for 24 h before modeling,and were not irradiated except for blank control group.After anesthesia,the other five groups were given a single total abdominal irradiation of 11 Gy by using 6MV high-energy X-ray linear accelerator.All groups were given intragastric administration the next day after irradiation.The blank control group and model control group were given 0.5ml 0.9% normal saline,and the low,medium and high dose GQD groups were given 0.25g/ ml,0.75g/ ml and 1.25g/ ml GQD0.5ml respectively.Montmorillonite powder group was given 0.05g/ ml Montmorillonite powder mixture 0.5 ml,once daily for a total of one week.During the period,the body weight,defecation and free movement of the mouse were observed,and DAI scores were recorded daily.One week later,fresh feces from each mouse were collected and frozen in a-80℃ultra-cryogenic refrigerator for 16 S r RNA high-throughput sequencing,analyzed and compared the composition,therichness and diversity of the intestinal microbiota in each group.The blood of mouse were collected for endotoxin(ET)testing,and the small intestine tissues of each group were divided into two parts.One part was stained for HE staining and the morphological changes of the small intestine were observed under light microscope.The other part was homogenized for nitric oxide(NO)testing.Results:1.General observation of mouse:After irradiation,all groups showed different degrees of decreased dietary and drinking water,reduced body weight,soft stools,unclean perianal area,bowback,messy hair,and less movement,etc,which began to improve on the 6th day after irradiation.2.Results of mouse body weight analysis:On the first day after irradiation,there was no significant difference in mouse body weight of each group(P >0.05).Compared with the blank control group,the body weight of mouse in the other five groups decreased significantly on the 2nd to 5th day after irradiation,and recovered slightly from the 6th day,with statistical significance(P<0.05).3.DAI score results:The DAI score of model control group was significantly higher than that of blank control group,with a significant difference(P<0.0001).The DAI score of each administered group was lower than the model control group,thus being statistically significant(P<0.05).The DAI score of low dose GQD group was significantly higher than that of the high dose GQD group and Montmorillonite powder group,with statistical significance(P<0.05,P<0.0001).The DAI score of the medium dose GQD group was higher than that of the high dose GQD group and the Montmorillonite powder group,and the difference was statistically significant(P<0.05,P<0.0001).4.Histomorphological results of the small intestine:Under light microscope,the small intestinal villi of mouse in blank control group were long and orderly,the crypt was not damaged,the epithelial cells were orderly arranged,the glandular structure was normal and no inflammatory cell infiltration and aggregation were observed.In model control group,the villi of small intestine were shortened,collapsed or absent,crypt and epithelial structure were destroyed,most glands were incomplete or even absent,inflammatory cells were extensively infiltrated,and muscle layer was significantly thinned.The small intestinal villi of mouse in each administration group were slightly shorter,the crypt and epithelial structure were more intact,and a small amount of inflammatory cells were infiltrated.Compared with the model control group,a certain degree of recovery was achieved,which was more obvious in the high dose GQD group.Compared with the blank control group,the height of the small intestinal villi was significantly reduced,with a significant difference(P<0.0001).Compared with the the model control group,the small intestinal villi of medium and high dose GQD group were significantly different(P<0.05,P<0.01).The height of small intestinal villi in the low dose GQD group was less than the high dose GQD group and was statistically significant(P<0.05).The whole thickness of the small bowel wall in model control group was significantly smaller than in blank control group(P<0.001).Compared with the model control group,the whole layer thickness of the small intestine wall of each dosing group was adjusted,the medium dose and high dose group and Montmorillonite powder group were obvious,and the difference was significant(P<0.05,P<0.01).The whole layer thickness of the small intestinal wall in the high dose GQD group and Montmorillonite powder group was significantly greater than that in the low dose GQD group,statistically significant(P <0.05).5.Biochemical index test results:The ET content in model control group was significantly increased compared with blank control group(P<0.0001).Compared with model control group,the content of ET in each administration group was significantly decreased,with statistical significance(P<0.001,P<0.0001).The ET content of low dose GQD group was higher than that of medium dose,high dose GQD group and Montmorillonite powder group,with statistical significance(P<0.05,P <0.0001).The content of ET in the medium dose GQD group was higher than that in the high dose GQD group and Montmorillonite powder group,the results were statistically significant(P<0.05),while the ET content in the high dose GQD group and Montmorillonite powder group had no significant difference(P>0.05).Compared with the blank control group,the NO content in the model control group increased significantly,and the results were significant(P<0.0001).Compared with model control group,the content of NO in each administration group was significantly decreased,with statistical significance(P<0.05),but no significant difference was found in the content of NO in each administration group,with no statistical significance(P>0.05).6.16 S rRNA High-throughput sequencing results:At the level of phylum,the dominant phylum in each group were Firmicutes,Bacteroidetes and Proteobacteria.Compared with the blank control group,Firmicutes(28.42±13.18)and Bacteroidetes(28.12±7.70)were decreased in the model control group(P<0.05).Compared with the model control group,the abundance of Firmicutes increased in each administered group(P<0.05).Although the Montmorillonite powder group also has the upward trend,but there was no obvious difference.The abundance of Bacteroidetes in high-dose GQD group(49.94±13.55)and Montmorillonite powder group(58.31±25.25)increased(P<0.05),ababundance of Actinobacteria in mediumdose GQD group(0.09±0.05)and high-dose GQD(0.09±0.12)reduced(P<0.05).The abundance of Firmicutes in the medium-dose GQD group(53.32±7.20)increased significantly compared with the Montmorillonite group,with statistical difference(P<0.05).In the Alpha diversity analysis,the abundance and diversity of the intestinal flora of the model control group decreased compared with the blank control group,and the high-dose group and Montmorillonite group could improve.The PCo A analysis showed a long distance between the blank control group and the model control group,indicating that the microflora composition was not similar,and the mouse intestinal microbiota was disturbed after mold making.At the genus level,the probiotics such as Lactobacillus(7.65±5.78),Bacteroides(2.14±0.92),Bifidobacterium(0.02±0.02)and Prevotella(0.29±0.22)in model control group were less abundant than those in blank control group(P<0.05),and opportunistic pathogens such as Enterococcus(1.04±0.06)and Escherichia(1.19±1.51)increased(P<0.05).Compared with the model control group,the abundance of Lactobacillus and Bacteroides in eachgroup of GQD was significantly increased significantly(P<0.01,P<0.05),the abundance of Prevotella in the low-dose GQD group(6.45±11.96)and the Montmorillonite group(1.82±1.09)increased significantly(P<0.01,P<0.05),the abundance of Bifidobacterium(0.31±0.01)increased in high-dose group(P<0.05).The abundance of Escherichia and Eschericella was reduced in each administered group(P<0.05),the abundance of Enterococcus in low-dose GQD group(0.05±0.02)and in medium-dose GQD group(0.04±0.01)was significantly lower than that of high-dose GQD group,and the difference was statistically significant(P<0.05),and no significant dose correlation was found in the abundance of other bacteria.Conclusion:1.The mouse model of radiation enteritis was successfully established by local single highdose irradiation of 6MV-X ray.2.GQD can effectively improve the general symptoms and histopathological manifestations of radiation enteritis mouse,reduce the DAI score,ET and NO content,and in different dose experiments,the effect of high-dose GQD group and Montmorillonite group is the most obvious.3.16 S rRNA high-throughput sequencing found that the abundance and diversity of the intestinal flora of the model control group decreased,the abundance of Enterococcus and Escherichia(opportunistic pathogenic bacteria)in the intestinal flora of mice in the model control group increased,Lactobacillus,Bacteroides,Bifidobacterium and the abundance of Prevotella(probiotics)decreased,suggesting that the occurrence of radiation enteritis is closely related to changes in the structure of intestinal flora.It is suggested that the occurrence of radiation enteritis is closely related to the change of intestinal flora structure.The abundance of Enterococcus and Escherichiais positively correlated with the occurrence of radiation enteritis.The abundance of Lactobacillus,Bacteroides,Bifidobacterium and Prevotella was negatively correlated with the occurrence of radiation enteritis。4.GQD by increasing the abundance of Lactobacillus,Bacteroides,Bifidobacterium and Prevotella,reducing Enterococcus and Escherichia abundance,correct the intestinal microecological imbalance,and achieve the purpose of treating radiation enteritis.Among them,the abundance of Bifidobacteria was positively correlated with the drug dose of the GQD,and the high-dose GQD group had certain advantages,Bifidobacteria could be used as a potential marker species of radiation enteritis. |
PDF Full Text Request