Regulatory Effect Of Bacillus Licheniformis BL63516 On Intestinal Mucosal Barrier Function

ObjectiveThis study explored the regulatory effect of Bacillus licheniformis BL63516 on the intestinal mucosal barrier dysfunction induced by different ways and provided an understanding of the function of the gut microbiota in the development and maintenance of the intestinal barrier,which may facilitate the treatment of clinical diseases.The 1st part:the regulatory effect of Bacillus licheniformis BL63516 on antibiotic-induced intestinal barrier damaged in mice1.Materials and methods30 male BALB/c mice（body weight:20±2 g）were randomly divided into 3 groups:the control group（CON group）,model group（CS group）,and BL administrated group（BL group）.The mice in the CON group were gavaged with 0.2 ml normal saline in the first 8 days,twice a day.To destroy the intestinal barrier,CS and BL group were gavaged with ceftriaxone sodium 300 mg/ml,0.2 ml for 8 days,twice a day.BL group was administrated with 3×10⁷ CFU/ml Bacillus licheniformis 0.2 ml from the 8 day while CON group and CS group were given 0.2 ml of normal saline.The body weight of mice was recorded everyday,and the mental activity,hair color and fecal properties were evaluated.The liver,serum and colon tissues were collected.The levels of cytokines and LPS in serum and liver were detected by ELISA.The structure of intestinal microbiota in mice was determined by PCR-DGGE and 16S rRNA high throughput sequencing.2.ResultsComparing with CON group,CS group presented significantly higher serum LPS level,significantly lower bacterial abundance and diversity in their gut.The abunance of bacterial phyla,including Bacteroides,Firmicute,Proteobacteria and Verrucomicrobia,in gut of BL group of mice decreased significantly,and the abundance of Firmicute became the dominant bacteria.Bacillus licheniformis administration significantly reduced the permeability of intestinal barrier,protected the function of intestinal barrier and improved the disorder of intestinal flora.Compared with the CS group,the inflammatory infiltration of intestinal mucosa in BL group was alleviated,the level of serum LPS was significantly reduced,the abundance and diversity of intestinal flora were significantly increased,the abundance of Verrucomicrobia was significantly decreased,and the abundance of Lactobacillus was found significantly increased.3.ConclusionThe results of this study suggest that Bacillus licheniformis BL63516 can effectively improve the intestinal dysbiosis caused by ceftriaxone sodium and regulate the intestinal barrier function.The 2nd part:the regulatory effect of Bacillus licheniformis BL63516 on intestinal barrier function in colitis mice1.Materials and methods30 BALB/C female mice（body weight:20±2 g）were divided into three groups:the control group（CON group）,model group（DSS group）and Bacillus licheniformis treated group（BL group）.The mice of DSS and BL groups were given 3%（m/v）DSS,free drinking for 7 days.At same time,the BL group were administrated with 3×10⁷CFU/ml Bacillus licheniformis while the control group was gavaged with 0.2 ml normal saline.The levels of cytokines in serum and colon tissue were detected by ELISA.PCR-DGGE and 16S rRNA high throughput sequencing were used to detect the intestinal microbial structure in mice.2.ResultsThe DSS group of mice presented severe deficiency of intestinal mucosal barrier function and significantly alteration of the richness and composition of intestinal microbiota,specifically,the higher DAI index,pathological score of colon,serum MPO level,and lower expression level of intestinal tight junction protein,and intestinal microbiota richness.Compared with the mice in DSS group,the disease activity index and colonic pathological score of BL group were significantly lower revealing that the symptoms of colitis were relieved;the inflammatory infiltration of intestinal mucosa was reduced,the level of serum IL-10 was significantly increased,the level of serum LPS was significantly reduced,and the expression level of occludin was significantly increased,indicating that the administrated of Bacillus licheniformis protected the intestinal immune system and intestinal barrier function in BL group;also the abundance of intestinal microbiota increased significantly,the abundance of verruca microflora increased,the abundance of Bacteroides decreased.3.ConclusionBacillus licheniformis BL63516 can effectively improve the symptoms of DSS induced colitis,and its mechanism is closely related to the regulation of intestinal dysbiosis.The 3rd part:the regulatory effect of Bacillus licheniformis BL63516 on intestinal barrier function in hyperlipidemia rats1.Materials and methods18 male SD rats were randomly divided into control group（CON group）,high fat diet group（HF group）and Bacillus licheniformis BL63516 intervention group（BL group）.The CON group was fed with normal diet,the HF group and BL group were fed with high-fat diet.At the same time,BL group were gavaged 7.5×10⁸ CFU/ml Bacillus licheniformis 0.4 ml,HF and BL groups were gavaged with 0.4 ml normal saline from the first day until the 21^st day.The body weight,the amount of food and water,the character of feces were recorded.The serum,colonic tissue and intestinal contents of rats were collected,visceral fat was weighed.The serum lipid level was measured by enzyme method,the LPS level in serum and liver tissue solution was measured by ELISA.The expression level of tight junction protein was detected by Western blot and the intestinal microbiota was analyzed by PCR-DGGE and 16S rRNA high-throughput sequencing.2.ResultsHyperlipidemia and gut microbiota disorder were found in rats fed with high-fat diet.Compared with the CON group,the HF group’s body weight increased rapidly,their Lee’s index and visceral fat ratio also increased significantly,serum T-CHO,TC,LDL-C levels increased significantly and the abundance of intestinal microbiota decreased significantly.The treatment of Bacillus licheniformis BL63516 significantly reduced the level of obesity and hyperlipidemia in rats,enhanced the intestinal barrier function.In BL group,the Lee’s index,visceral fat proportion,serum TC,LDL-C level,serum LPS and liver LPS levels were significantly reduced comparing to HF group and the expression level of occludin was significantly improved.The regulatory function of Bacillus licheniformis BL63516 on intestinal microbiota composition structure was indentified,especially,the abundance of Parabacteroides were significantly increased and the abundance of Desulfovibrionaceae were significantly decreased.3.ConclusionBacillus licheniformis BL63516 can regulate the high fat diet-induced dyslipidemia in rats and improve the function of intestinal barrier.