To Investigate The Clinical Protective Effect Of LGG/LAC-MMT Combined Application On AFB₁ Mice Based On Intestinal Flora

Aflatoxin B₁（AFB₁）is a secondary fungal metabolite that is widely present in food and feed ingredients and is considered a global public health problem due to its long half-life,strong toxicity and carcinogenic properties.Although lactic acid bacteria have been shown to degrade AFB₁ by modulating the balance of gut microbiota,the poor colonization ability and short survival time of most lactic acid bacteria in the host have been overlooked.Montmorillonite（MMT）is a silicate clay that can not only adsorb AFB₁,but also act as a carrier for lactic acid bacteria to adhere to the tissue surface,which improves the colonization ability of lactic acid bacteria,prolongs the survival time,and reshapes the host microbial ecosystem to achieve the purpose of prevention and treatment.Currently,there are few reports on the combined degradation of AFB₁ by lactic acid bacteria and clay minerals.We found in previous preliminary experiments that when Lactobacillus rhamnosus（LGG）（2×10⁹ cfu/m L）and Lactobacillus acidophilus（LAC）（2×10⁹ cfu/m L）were combined with 0.5 mg/kg MMT When combined treatment,it can effectively alleviate the liver injury caused by AFB₁ in mice.Therefore,this study selected LGG,LAC at a concentration of 2×10⁹ cfu/m L and MMT of 0.5 mg/kg,focusing on the following two experimental purposes:（1）To establish an AFB₁-induced mouse model,using LGG,LAC and MMT intervention was performed to determine whether the combined application of LGG/LAC-MMT could improve tissue damage exposed to AFB_1.（2）Whether the combined application of LGG/LAC-MMT can balance the intestinal microbiota of mice by enhancing the colonization of lactic acid bacteria,thereby alleviating the tissue damage caused by AFB_1.In this experiment,4-week-old healthy male Balb/c mice of 80 were randomly divided into eight groups,MMT（0.5 mg/kg）after 4 weeks of gavage,the experiment ended.The body weight of the mice was counted,and the pathological changes of the liver and intestine were observed by autopsy;the serum liver function and renal function indexes were detected by an automatic biochemical analyzer;the expression of inflammatory factors in the liver was detected by fluorescence quantitative PCR;the liver and intestine were detected by ELISA.16S r RNA amplicon sequencing technology was used to detect the changes of cecal content microflora.The results showed that:AFB₁ induced obvious lesions in the liver and intestinal tissue of mice,with swelling and necrosis of hepatocytes,and obvious shedding of jejunal mucosal epithelial cells;serum liver function indexes AST,ALT,ALP,CHE,TBIL,DBIL and IBIL were significantly increased（P<0.01）;serum renal function indexes,BUN,CR and UA contents were significantly increased（P<0.01,P<0.001,P<0.01）;the levels of serum inflammatory factors IL-1β,TNF-αand IL-10 were significantly increased（P<0.001）;the content of lipid peroxide product MDA in liver tissue increased significantly（P<0.001）,and the activities of antioxidant enzymes SOD,CAT,GSH and GR were significantly decreased（P<0.05）;intestinal tissue lipid peroxidation product MDA content,antioxidant enzymes SOD and GSH activities were significantly increased（P<0.01）;liver tissue inflammatory factors IL-1β,TNF-α,IL-10,IL-2 and the expression of IL-8 m RNA was significantly increased（P<0.01）;the abundance and diversity of bacteria in the cecum contents were significantly decreased（P<0.05）;the content of AFB₁ in the feces of the LGG/LAC-MMT combined treatment group was significantly increased（P<0.01）.However,the addition of LGG,LAC,and MMT all reversed the changes in the above indicators caused by AFB₁ to varying degrees,but the combined application of LGG/LAC-MMT had a more significant effect on inflammatory and oxidative damage.In conclusion,AFB₁ exposure can lead to liver and intestinal tissue damage,increased inflammatory factors,and intestinal flora disturbance.Although LGG,LAC,and MMT can all alleviate AFB₁-induced liver and intestinal damage,reduce the expression of inflammatory factors,and regulate intestinal flora to varying degrees,the combined application of LGG/LAC-MMT has the potential to better degrade the toxicity of AFB₁.Among them,the combined application effect of LAC-MMT is more significant.It shows that the combined application of LGG/LAC-MMT improves the colonization ability of lactic acid bacteria,changes the structure of intestinal flora in mice,and relieves the liver and intestinal damage caused by AFB₁.It also shows that the combined application of lactic acid bacteria and MMT can be used as a compound agent to reduce the toxicity of mycotoxins.