Study On The Combined Effects Of Liver Injury And The Immune Interaction Mechanism Induced By Aflatoxin B₁ And Microcystin LR At Low Dose & Sub-chronic

About 600,000 people died for liver cancer?HCC?annually in the world and HCC?14.56%?is second leading cause of death in China.The precursor factors of inducing HCC are very complex.According to epidemiological investigation and other studies,it has been preliminarily confirmed that dietary exposure to aflatoxin B₁?AFB₁?and microcystin LR?MC-LR?in contaminated food may induce combined hepatotoxicity and promote subsequent HCC outcomes.The main target organ of AFB₁ and MC-LR is liver.However,there is still a lack of research on the combined hepatotoxicity of AFB₁+MC-LR.Firstly,the intensity,characteristics?synergism,antagonism,etc.?and distribution of hepatotoxicity/HCC induced by co-exposure AFB₁+MC-LR are still unknown.Secondly,combined toxicity caused by AFB₁+MC-LR under low dose&sub-chronic exposure often present the different characteristics compared with that under acute exposure,including reversibility of liver injury and inflammation,randomness between gene mutation and liver cancer induction.Studies have shown that immunity can affect the expression of hepatotoxicity.Therefore,furthering the related researches maybe very important to elucidate the hepatotoxicity interaction of AFB₁+MC-LR.The following conclusions show as follow from this paper:1.Based on CI model,the combined effects of mice induced by co-exposure to AFB₁+MC-LR under low-dose&sub-chronic was predicted.The results showed that the occurrence probability of combined dose and CI value mainly concentrated in the range of0?20 mg/kg bw and 0?1.5 respectively,the combined effects showed slight antagonistic,slight synergistic or additive action modes,and the possibility of strong antagonism or strong synergistic effects was very low,and the probability of additive effect was relatively higher.Meanwhile,the combined effects of AFB₁+MC-LR in dietary pathway was predicted based on IORP model,and the risk of co-exposure was higher than that of single exposure.The order from highest to lowest was:Elderly?54.638%?>children?54.172%?>adults?51.865%?.The combined toxicity effect coefficient(c₁₂=54.827)showed that the combined effect of liver cancer induced by co-exposure to AFB₁+MC-LR was synergetic and the risk might be amplified.2.The expression of AKP,ALT,AFP,TBIL and AST in male wild-type C57BL/6 mice co-exposed to AFB₁+MC-LR with low-dose&sub-chronic conditions showed that:?1?With the dose ratio of AFB₁/MC-LR when AFB₁ and MC-LR are achieved similar response respectively,the overall expression level of co-exposure group in serum and liver was slightly higher than that of single exposure group,the expression level of AFB₁ in serum was higher than that of MC-LR?p<0.05?,while MC-LR in liver was higher than AFB₁?p<0.05?;based on Principal Component Analysis,it was found that the co-exposure group showed slight synergistic or slight antagonistic effect,but the occurrence probability of synergism was higher than that of antagonism;?2?With the dose ratio of AFB₁/MC-LR based on the ED₅₀inducing liver cancer of AFB₁ and MC-LR,the dose changes of any mycotoxin and MC-LR in the serum for co-exposure group did not significantly affect the expression of TBIL and AFP in the co-exposure group?p>0.05?,while the other biomarkers were significantly affected by the single mycotoxin?p<0.05?;meanwhile,dose change of any single mycotoxin in liver had a significant impact on 5 biomarkers in the co-exposure group?p<0.001?.Those results suggest that the liver may be the key target organ of AFB₁+MC-LR for its different synergistic effect of combined hepatotoxicity.3.The immune response and influence the liver immune microenvironment in male wild-type C57BL/6 mice co-exposed to AFB₁+MC-LR with extremely low dose and sub-chronic conditions showed that:?1?Based on the"time-effect"analysis of cytokine,the pathways of liver injury and immune response may be:a)In the early stage?0?5wks?:IFN-?receptor signal is to promote and enhance expression of IFN-?and IL-2,meanwhile,activating factor for macrophage was induced by IFN-?.IL-18,as an upstream event,promotes the directional differentiation of Th1 and induces the production of IFN-?induced by Th1.The combined toxicity show slight antagonism or antagonism effect;b)In the early and middle stage?5?7wks?:with the increase of exposure time,dose and frequency,AFB₁+MC-LR antigen presented,immune response entered the platform competition stage,slight antagonism or antagonism gradually became additive effect;c)In the late metaphase?7?11wks?:AFB₁+MC-LR"time-dose"accumulation,which can enhance IL-4 and IFN-?significantly inhibited;d)In the late stage?11?13wks?:the immune response continue to increase,and the balance of Th1/Th2 may be the key mechanism.At the same time,there are many uncertainties in the"time-dose-effect"process,which makes the liver cancer outcome become a probability event.This conclusion confirms the results of model simulation in Chapter.?2?Based on the"time-effect"analysis of liver immune,At the 9th and 13th week,the abundance of T cells in the liver was the highest.In the blank group,AFB₁ group,MC-LR group and AFB₁+MC-LR group,the percentage of T cells in total immune cells was 26.26%,36.70%,34.65%and 30.58%;33.53%,20.37%,39.08%and 27.11%,respectively.It shown that there is no significant difference between different experimental groups and blank groups,and the abundance of T cells in AFB₁+MC-LR group gradually decreased.In addition,there were significant differences in neutrophil abundance between single and co exposed groups at9 weeks?p<0.01?,macrophages?p<0.05?,and dendritic cells?DC?;at 13 wks,there were significant differences between B cells?p<0.001?,and macrophages?p<0.01?.At the same time,the ratio of CD4+/CD8+related with AFB₁ and MC-LR?0.947 and 0.466,respectively?decreased with the increasing of exposure time,but increased in AFB₁+MC-LR group?1.081?.The decrease of the ratio indicated that the immune system disorder trend of AFB₁+MC-LR group was more significant,and the combined effect was gradually changed from additive and slight synergism to slight antagonism,which was basically consistent with the trend of cytokine deduction.It is also confirmed that CD4 cells may undergo T-Tfh,Th1-Th2 and T-Treg pathways leading to the characterization of combined effect during 11-13 weeks.4.The molecular mechanism of hepatotoxicity and immune response in male wild-type C57BL/6 mice co-exposed to AFB₁+MC-LR with extremely low dose and sub-chronic conditions showed that:?1?Compared with the single mycotoxin,the number and level of significant m RNA expression in the AFB₁+MC-LR group were significantly up-regulated,and mainly concentrated in Cyp4a14,Cyp4a10,Cyp4a10,EGR1,NOX4,gadd45g,HSPA 1b,zfp809,GDF15 and CTGF.At the same time,the synergistic effect was observed before the13th week in the AFB₁+MC-LR group,while the iigp1 and other differential genes were up-regulated in MC-LR group compared with AFB₁ group at the 9th week,which may directly and/or indirectly participate in the subsequent expression of AFB₁+MC-LR group,which is a synergistic effect;?2?The differential genes of single group and AFB₁+MC-LR group are rich in tumor necrosis factor TNF,RNA transport and Nod like receptor,phosphatidylinositol signaling pathway.However,the cholesterol metabolism pathway,arachidonic acid signaling pathway,phosphatidylinositol signaling pathway and vascular smooth muscle contraction signal pathway were enriched between the blank group,the single and AFB₁+MC-LR groups.It is suggested that arachidonic acid pathway may be one of the most important molecular mechanisms affecting the hepatotoxicity of AFB₁+MC-LR.