Investigating The Role Of Bacillus Amyloliquefaciens TL In Inducing Macrophage Polarization And Regulating Intestinal Flora

Bacillus amyloliquefaciens has been identified as a safe and probiotic strain.Previous research in this laboratory found that B.amyloliquefaciens TL can promote protein synthesis,enhance lipid metabolism,and promote the development of immune organs,thereby achieving a growth-promoting effect similar to chlortetracycline.Macrophages are important immune cells in the body.When stimulated by different signals,macrophages will polarize into two types:classic macrophages(M1)or alternative macrophages(M2).In addition,the interaction between B.amyloliquefaciens and macrophages remains to be determined.This study aimed to investigate the effects of the probiotic B.amyloliquefaciens TL on the polarization of mouse macrophages,and to explore the effects of B.amyloliquefaciens TL on the host intestinal microbial structure.The main research contents and results are as follows:(1)Bacillus amyloliquefaciens TL stimulated RAW264.7 cells in vitro to detect cytotoxicity.The results showed that B.amyloliquefaciens TL at a concentration of 10~7CFU/m L could promote the proliferation of RAW264.7 cells within a certain period of time,and the effect was best at 4 hours.Lipopolysaccharide(LPS)and TL stimulated RAW264.7 cells for 4 hours,respectively,and detected the macrophage activation-related genes:COX-2,MCP-1,M1 marker genes:i NOS,IL-6,IL-1?,TNF-?and M2 marker genes:IL-10,Arg-1 expression levels using reverse transcription quantitative polymerase chain reaction(RT-q PCR).The results showed that compared with the control group,the expression levels of COX-2,MCP-1,i NOS,IL-6,IL-1?and TNF-?genes in the TL group and the LPS group were significantly increased.There was no significant change in the expression levels of IL-10 and Arg-1 genes.It showed that both TL and LPS could activate RAW264.7 cells and induce RAW264.7 cells to polarize to M1.(2)Forty C57BL/6 mice(female)were randomly divided into four groups,namely the control group,the B.amyloliquefaciens TL group,the prevent group and the ETEC group.Mice in the control group were given 200?L of sterile saline by intragastric administration on days 1-14;mice in the B.amyloliquefaciens TL group were administrated with a concentration of 5×10~7 CFU/m L of TL on days 1-14 200?L;mice in the prevent group were administered with 200?L of TL at a concentration of 5×10~7 CFU/m L on days1-14,and administered with 200?L of enterotoxigenic Escherichia coli(ETEC)K88ac at a concentration of 1×10~8 CFU/m L on days 15-17;mice in the ETEC group were administered 200?L of sterile saline on days 1-14,and administered with 200?L of ETEC K88ac at a concentration of 1×10~8CFU/m L on days 15-17,and intragastric administration once a day.None of the mice died in each group during the experiment.Compared with the prevent group,the spleen lesions in the ETEC group were obvious.Flow cytometry was used to detect the proportion of T lymphocytes and macrophages in the spleen mononuclear cells of mice.It was found that B.amyloliquefaciens TL had no significant effect on the ratio of CD4~+T cells to CD8~+T cells,but TL increased the proportion of macrophages.The number of M1 macrophages and M2 macrophages in colon tissue was detected by immunohistochemistry.The results showed that B.amyloliquefaciens TL can promote the polarization of macrophages to M1,and has no significant effect on M2 polarization.The serum cytokine test results showed that B.amyloliquefaciens TL had no significant effect on some cytokines,but increased IL-12 in the blood.The results of RT-q PCR of colon genes showed that TL down-regulated the expression of M2 marker gene Arg-1 in the mouse colon,and the M1 marker gene i NOS had an up-regulated expression trend.It is speculated that B.amyloliquefaciens TL may play an immunoregulatory role by activating macrophages and inducing macrophages to polarize to M1 to enhance the body's ability to resist ETEC K88ac infection.(3)16S r RNA amplicon sequencing technology was used to analyze the structure of cecum flora in mice.The results showed that compared with the control group,B.amyloliquefaciens TL had a certain effect on the structure of the cecum flora in the TL group.And there was no significant change in the intestinal flora between the prevent group and the ETEC group.Bacteroidetes,Firmicutes,Proteobacteria and Actinobacteria were the dominant bacteria in the cecum flora of mice.Compared with the control group,B.amyloliquefaciens TL can significantly reduce the proportion of Proteobacteria and Actinobacteria in the cecum flora.At the genus level,the proportion of Helicobacter,Dubosiella and Intestinimonas in the TL group was lower than that in the control group,and the proportion of Bacteroides and Lactobacillus was higher than that of the control group.The results show that B.amyloliquefaciens TL helps the body to establish a normal intestinal microbial structure by inhibiting the colonization of pathogenic bacteria and promoting the growth of beneficial bacteria.