Study On The Effect And Mechanism Of Lactobacillus Reuteri FN041 On Radiation-induced Intestinal Injury In Mice

Background and Purpose of the Study:Radiation therapy is an important treatment modality for cancer,but it often leads to radiation-induced intestinal injury in patients with abdominal and pelvic tumors,which severely affects their quality of life.This study aims to establish a mouse model of acute radiation-induced intestinal injury and investigate the potential protective effects of Lactobacillus rhamnosus FN041 intervention on radiation-induced intestinal injury.The study also aims to explore the underlying mechanisms of Lactobacillus rhamnosus FN041 and provide a theoretical basis for nutritional prevention and treatment of acute radiationinduced intestinal injury.Methods:(1)Experiment on the effects of radiation on mouse intestinal mucosal injury and plasma metabolites: Twenty 8-week-old male C57BL/6J mice were randomly divided into healthy control group(CN)and radiation model group(TAI)according to body weight.After one week of feeding,the mice were subjected to 12 Gy abdominal irradiation using a 6-MV linear accelerator,while the CN group was subjected to sham irradiation in the same environment.Pathological changes in the jejunum,ileum,and colon tissues were observed using the HE staining method.The villus length and crypt depth were measured,and the intestinal tissue damage was evaluated according to the Chiu’s histopathological injury scoring system.The distribution and changes of tight junction-related proteins including zonula occludens-1(ZO-1),Occludin,and mucin 2(MUC2)in different parts of the intestine were observed using immunofluorescence staining.The impact of radiation on plasma metabolites was analyzed using untargeted metabolomics with UHPLC-QTOFMS.(2)The therapeutic effect and mechanism of Lactobacillus rhamnosus FN041 on acute radiation-induced intestinal injury in mice: A total of 40 eight-week-old male C57BL/6J mice were divided into four groups: healthy control group(CN),radiation model group(TAI),intervention group with Lactobacillus rhamnosus GG(LGG),and intervention group with Lactobacillus rhamnosus FN041(FN041),with 10 mice in each group.Oral gavage was administered for one week prior to radiation exposure and continued until mice were sacrificed at 3.5 days after radiation.The CN and TAI groups were given phosphate-buffered saline(PBS,p H=7.3,0.2ml/day),while the LGG and FN041 groups were given Lactobacillus rhamnosus GG and Lactobacillus rhamnosus FN041(1×10^9 CFU/day,0.2ml/day),respectively.The TAI,LGG,and FN041 groups were exposed to 12 Gy abdominal radiation using a 6-MV linear accelerator,while the CN group received sham radiation in the same environment.The pathology of colon tissue was observed by hematoxylin-eosin staining.Immunofluorescence staining was used to observe the distribution and changes of tight junction-related proteins ZO-1,Occludin,and MUC2 in different intestinal segments.Alcian blue-PAS staining was used to observe goblet cells and mucus layer.The composition and changes of intestinal flora in the cecal contents of mice at 3.5 days after radiation were detected by 16 S r DNA.The composition of shortchain fatty acids in the cecal contents was detected by GC-MS(Agilent,7890B-5977 B GC/MSD,USA).ELISA assay was used to detect the intestinal barrier indices in mice,and changes in plasma endotoxin(LPS)and diamine oxidase(DAO)were measured.The expression levels of the inflammatory factors interleukin-6(IL-6)and tumor necrosis factor in mouse intestinal tissue were measured.Results:(1)Effects of radiation on small intestine mucosal injury and plasma metabolites in mice: At 3.5 days after radiation induction,compared with the CN group,the TAI group showed a significant decrease in body weight(P < 0.01).The histopathological injury scores of the CN group’s jejunum,ileum,and colon were 0.35 ± 0.06,0.37 ± 0.06,and 0.36 ± 0.11,respectively.In contrast,the TAI group’s jejunum,ileum,and colon had significantly higher histopathological injury scores of 3.32 ± 0.36,2.91 ± 0.16,and 3.34 ± 0.3,respectively,with a significant increase in intestinal tissue pathological injury(P < 0.01).The mice in the TAI group showed broken and shortened villi,deformed crypts,and increased inflammatory infiltration in colon tissue,indicating significant damage to the intestinal mucosa.Villus length and crypt depth were both significantly lower than in the control group(P < 0.01).The expression of tight junction proteins ZO-1 and Occludin was reduced and distributed abnormally in the jejunum,ileum,and colon of the TAI group.MUC2 protein expression was also significantly decreased(P < 0.01).Compared with the CN group,the TAI group showed significant downregulation of FADH and riboflavin metabolites in plasma(P < 0.05),as well as downregulation of metabolites such as L-Rhamnose,Sphingosine-1-phosphate,Dimethyl sulfone,CDP-ethanolamine,Sarcosine,and L-Tyrosine(P < 0.05).(2)The beneficial effects and mechanisms of Lactobacillus rhamnosus FN041 on acute radiation-induced intestinal injury in mice: After intervention with Lactobacillus rhamnosus FN041,compared with the TAI group,the FN041 intervention significantly reduced body weight loss(P < 0.05)and alleviated intestinal mucosal damage,significantly reduced intestinal tissue pathological injury scores(P < 0.01),increased the expression of tight junction proteins ZO-1 and Occludin,and significantly increased the staining intensity of the intestinal mucous layer by 10.76%(P < 0.001).It also significantly enhanced the expression of colonic MUC2(P < 0.01)compared to the intestinal flora in the CN group,pathogenic bacteria such as Desulfovibrio and Paeniclostridium had higher relative abundances in the TAI group.Compared with the TAI group,the FN041 intervention group had higher relative abundances of Ruminococcaceae-UCG-010,Acinetobacter,and other genera.The total amount of shortchain fatty acids(SCFAs)in the TAI group was significantly lower than that in the CN group after radiation(P < 0.01),and except for butyric acid,the contents of acetic acid,propionic acid,isobutyric acid,valeric acid,isovaleric acid,and hexanoic acid were significantly lower than those in the control group(P < 0.01).Compared with the TAI group,the FN041 intervention group significantly increased the contents of acetic acid,propionic acid,isobutyric acid,valeric acid,isovaleric acid,and hexanoic acid(P < 0.05).Although the SCFA content in the LGG intervention group showed an increasing trend,there was no significant difference compared with the TAI group(P > 0.05).The FN041 group showed a significant reduction in LPS level compared to the TAI group(P < 0.001).The IL-6 level in intestinal tissue was significantly reduced(P < 0.05),and there was no significant difference in the expression levels of DAO and TNF-α in the plasma and intestinal tissue compared to the TAI group.After intervention with the probiotic LGG,it significantly reduced the levels of LPS and IL-6 compared with the TAI group(P < 0.05),and there was no statistically significant difference in the expression levels of DAO in the plasma and TNF-α in intestinal tissue between the LGG group and the TAI group.Correlation analysis of intestinal mucosal barrier indicators and SCFAs showed that MUC2 expression in the colon was positively correlated with propionic acid,isobutyric acid,valeric acid,and isovaleric acid(r = 0.529,0.463,0.529,0.541,P < 0.05),and significantly positively correlated with acetic acid(r = 0.675,0.664,P < 0.01).Conclusion:(1)Radiation induces varying degrees of damage to the small intestine,jejunum,and colon in mice,leading to intestinal mucosal barrier dysfunction and changes in plasma metabolic products.(2)Lactobacillus reuteri FN041 can improve radiation-induced intestinal mucosal barrier damage in mice,which may promote the production of short-chain fatty acids by affecting the change of intestinal flora by radiation,increase the expression levels of tight junction protein ZO-1 with Occludin and mucin MUC2,and supplementation with Lactobacillus reuteri FN041 can downregulate plasma LPS levels to reduce radiation-induced intestinal mucosal damage and inflammation,thereby enhancing the integrity of the intestinal epithelial barrier and reducing radioactive intestinal injury.In summary,Lactobacillus reuteri FN041 can improve radiation intestinal injury and has the potential of nutritional intervention to improve radiotherapeutic intestinal injury.