Nonylphenol-induced Intestinal Injuries And Dysbacteriosis And The Alleviative Effect Of Lactic Acid Bacteria On Its Toxicity

As an endocrine disruptor,nonylphenol(NP)is widely present in daily life and is absorbed in the body through food intake.NP can accumulate in bodies and cause damage to the endocrine system,reproductive system,immune system and central nervous system.As the main absorption site of NP,the intestine is also one of its target organs,and when NP damages the intestine,it may aggravate the central nervous system damage through the gut-brain axis.However,the previous research on NP-induced intestinal injury is still relatively limited,and the clearing methods for NP that have been reported cannot be applied in the food field.Lactic acid bacteria,as a kind of beneficial bacteria,can protect intestinal health and have been extensively studied in the removal of heavy metals,polycyclic aromatic hydrocarbons,bisphenol A and other environmental pollutants.Based on the above,this study will explore the following three aspects to find lactic acid bacteria that can efficiently remove NP and alleviate the toxicity of NP.First,a subchronic exposure experiment was carried out by using NP range from 30?g/kg to 50 mg/kg.The effects of NP on the intestinal barrier,intestinal immunity and intestinal microecology of rats and the potential dose-effect relationship were analyzed.The results of colonic pathology showed that NP could damage the intestinal mucosa and induce inflammation of the colon.Based on these results,we further analyzed the changes in the transcription level of colonic tight junction protein.The results proved that 15 mg/kg and 50mg/kg NP significantly down-regulated the m RNA of ZO-1 and Claudin-1.The effects of different doses of NP on tight junction proteins and specific inflammatory factors were measured by Enzyme-Linked Immuno Sorbent Assay(ELISA).It was found that the down-regulation of ZO-1,Claudin-1 protein expression and the increase of TNF-?,IL-1? all showed a dose-dependence on NP.The changes of the intestinal flora after NP exposure were analyzed by 16 s r RNA,and the results showed that the abundance of the flora related to the production of short-chain fatty acids(SCFAs)decreased,such as Allobaculum and Coprococcus,and the SCFAs in feces were further analyzed.The content of butyric acid decreased significantly corresponding to the results of the flora.Then,lactic acid bacteria with the ability to efficiently eliminate NP were screened and cell experiments was carried out to verify its mitigation effect on NP toxicity in vitro.In this study,a strain of Bifidobacterium longum CCFM1077 with a NP reduction rate of 60.56%within 6 hours was selected.The reduction rate of NP by CCFM1077 is 6 times that of the lactic acid bacteria with poor reduction ability.By comparing the reduction rate of living and dead bacteria,it is preliminarily determined that the reduction mode of CCFM1077 was adsorption.The study further explored the adsorption kinetics and adsorption thermodynamics of NP by CCFM1077.Results showed that there were both physical and chemical adsorption.Based on the Caco-2 cell model,the effect of lactic acid bacteria on the alleviation of NP toxicity was preliminarily verified.The CCK-8 method confirmed that the survival rate of Caco-2 cells that exposure to NP for 6 h increased about 30%by CCFM1077.Based on the above results,animal experiments were carried out to verify the alleviating effect of CCFM1077 on NP toxicity in vivo.Through open field experiments,it was found that CCFM1077 could markedly improve the behavioral obstacles caused by NP.At the same time,quantitative real-time polymerase chain reaction(QPCR)analysis found that the bacteria significantly reduced TNF-? in the brain.CCFM1077 significantly increased not only the excretion of NP in feces,but also the content of tight junction protein ZO-1,which protects the integrity of the colonic mucosa.Also it down-regulated the content of IL-1? and TNF-?,reducing inflammation damage to the colon.In addition,CCFM1077 dramatically increased the abundance of SCFAs producing bacteria such as Turicibacter in intestine and the production of butyric acid,effectively maintaining intestinal homeostasis.To sum up,this subject has studied the damage of different doses of NP to rats.Through correlation analysis,it is found that the decrease of butyric acid caused by NP and the decreased expression of colonic tight junction protein as well as the increase of colonic IL-1?had significant correlation.Then,the mitigation effect of the Bifidobacterium longum CCFM1077 was verified from both in vitro and in vivo perspectives.This provides a new method to treat NP injuries.