Improvement And Mechanism Of Lactobacillus Rhamnosus GG On Zebrafish Against Oxytetracycline-induced Toxicities

Oxytetracycline(OTC)as a tetracycline antibiotic,was widely used in poultry industry and aquaculture,which had the potential to increase agricultural productivity.OTC could migrate and accumulate through surface water and the digestion and absorption of animals,then entered human body through the food chain.OTC could accumulate in human body,induced oxidative damage,led to gut microbiota imbalance and immune system disorders,and seriously threatened the health of human.It is meaningful to seek safe and effective ways of dietary intervention to alleviate OTC-induced damages in human or animals.As living and supplementary microorganisms,probiotic Lactobacillus rhamnosus GG(LGG)could improve human health status via balancing the gut microbiota,modulating the intestinal motility,and facilitating digestion and absorption of nutrients.This study started from the early developmental toxicities caused by OTC exposures at environment-related concentrations in the model animal zebrafish,through the intervention of probiotic LGG,the research was based on social behavior,oxidative stress and the function of gut motility to explore the amelioration and mechanism of LGG intervention on abnormal behaviors,damages or dysfunction in zebrafish after OTC exposures.Additionally,in view of the application limitations of probiotics,LGG was encapsulated with a layer-by-layer method to explore the amelioration and mechanism of encapsulated LGG intervention on the adverse intestinal environment after OTC exposures.This study provided a basis for probiotic products to prevent toxic damages in animals induced by adverse intestinal conditions and the development of functional food.The main innovation work and results were as follows:1.The effects of antibiotic OTC exposures at environmentally relevant concentrations on early development of zebrafish were investigated.The results showed that OTC exposures inhibited the hatching of larvae,caused the decrease in heart rate,affected morphological development including the decrease of body length,head area and eye area in larvae,significantly increased the degree of apoptosis,increased the area of intestinal lumen and caused the abnormal ability of movement in zebrafish.However,exposure to the low concentration of OTC(10 μg/L)had no significant effects on hatching rate,morphological indicators and the area of intestinal lumen in larvae.Therefore,OTC exposures produced toxic effects on early development in zebrafish.2.The improvement effects of LGG on OTC-induced abnormal behaviors in zebrafish were studied.The results showed that antibiotic OTC exposure altered the gut microbiota,decreased the dopamine levels in the brain and decreased the sociability and weakened shoals’ preference for all color combinations.When given a supplementation of LGG after OTC exposure,zebrafish normalized the gut microbiota,dopamine levels of brain and social interaction and color proference.Meanwhile,dopamine receptor antagonist,SCH23390,could inhibit dopamine production,and disrupt the social interaction which led to the deficits of color preference in shoals.Therefore,shoaling behavior of zebrafish affected the color preference of shoals,indicating that shoals tended to choose a favorable color environment that benefited social contact.Additionally,the dopamine levels in brain of zebrafish could mediate both social interaction and color preference.This study suggested that dopamine affected social behavior of zebrafish possibly through gut microbiota.3.The improvement effects of LGG on OTC-induced oxidative damage and gut motility dysfunction were investigated.The results showed that 4-day OTC exposure on the zebrafish embryos at 1 day post fertilization led ROS accumulation through decreasing antioxidant capacity,and decreased the intestinal peristalsis through decreasing the serotonin(5-HT)content.Under the LGG administration,OTC-evoked oxidative damage could be alleviated via increasing antioxidant capacity and gut dysmotility could be normalized via increasing 5-HT secretion.In order to further prove the important role of 5-HT in improving gut dysmotility,exogenous5-hydroxytryptophan was a chemical precursor of 5-HT,which could also rescue gut dysmotility in OTC-treated zebrafish.This study demonstrated that LGG improved oxidative damage and prevented intestinal dysmotility associated with low levels of5-HT.4.The improvement effects of encapsulated LGG on OTC-induced intestinal damage and gut dysmotility were studied.As probiotics were vulnerable to acid and bile salt damage and were limited in intestinal colonization,choosing chitosan/alginate to coat on the surface of probiotics could effectively protect them from the undesirable enteric environments induced by OTC.The results demonstrated that the encapsulated LGG could significantly improve its survival rate in simulated gastric fluid and bile salt solution.The coated LGG for only 2 h administration could significantly improve the colonization rate of Lactobacillus in the intestine of OTC-treated larvae.After OTC exposure,the coated LGG for 2 h administration diminished ROS generation via enhancing antioxidant capacity and recovered gut motility via increasing 5-HT synthesis.Therefore,the application of this encapsulated probiotic method may be a promising prophylactic approach to alleviate antibiotic-induced damage and dysfunction.