Toxic Effect Of AFB₁ On Primary Mouse Peritoneal Macrophages And Mechanism Of Iron Homeostasis

Aflatoxin B₁?AFB₁?is a mycotoxin that widely pollutes food and feed.It is a serious threat to human food safety and the healthy development of aquaculture,and can be transmitted through the food chain to endanger human health.AFB₁ can lead to immune system dysfunction after long-term exposure.Macrophages,the"first line of defense"of the natural immune system,which are also important effector cells of the specific immune system.They serve a variety of immune functions such as phagocytosis,chemotaxis,adhesion,antigen presentation,secretion of bioactive substances and tumor inhibition.AFB₁ is toxic to macrophages,which can inhibit the phagocytosis of macrophages and affect the secretion of inflammatory factors,thus leading to abnormal immune dysfunction.Iron homeostasis is the main toxic mechanism of oxidative stress and macrophage damage,but it is unclear whether AFB₁ produces toxic effects by causing iron imbalance in macrophages.This study is theoretical foundation with AFB₁ can disturb the iron homeostasis of macrophages,and primary mouse peritoneal macrophages were selected as subjects.Firstly,the expression of F4/80 was determined by flow cytometry to determine the purity of primary mouse peritoneal macrophages.Secondly,primary mouse peritoneal macrophages were treated with graded concentration of AFB₁ for 24 h,then the median inhibitory concentration(IC₅₀)was calculated to determine the appropriate dose of AFB₁ in subsequent experiments.At last,primary mouse peritoneal macrophages were treated with AFB1 of 0?control group,CG?,6.25?M?low dose group,LG?,12.5?M?medium dose group,MG?,and 25?M?high dose group,HG?for 24 h.The structure,function,oxidative stress level,iron content and iron-related protein expression of primary mouse peritoneal macrophages were detected for evaluating the relationship between the toxic effect of AFB₁ on primary mouse peritoneal macrophages and its relationship with iron homeostasis imbalance,aiming to provide theoretical basis for the elimination of AFB₁ macrophage toxicity.The test results are as follows:?1?The average purity of primary mouse peritoneal macrophages extracted was 98.7%.?2?The IC₅₀ of primary mouse peritoneal macrophages treated with AFB1 for 24 h was 74.5?M.?3?Compared with the CG,the activity of primary mouse peritoneal macrophages in the MG and HG decreased significantly?p<0.05;p<0.01?,indicating that AFB₁ can inhibit the growth of primary mouse peritoneal macrophages.?4?In SEM,we can see,there were many micro folds and a large number of extended pseudopods on the surface of mouse peritoneal macrophages of the CG,which were in good growth state and present typical macrophage morphology.The folds of mouse peritoneal macrophages of the LG were maintained well,but pseudopodia was shortened.The surfaces of mouse peritoneal macrophages of the MG and the HG were smooth and pseudopodia were broken or even disappear,indicating that AFB₁ damages the morphology of mouse peritoneal macrophages.Detection of LDH release showed that compared with the CG,LDH release from mouse peritoneal macrophages in AFB₁ exposure group was significantly increased?p<0.01?,indicating that AFB₁ resulted in cell membrane structure destruction of mouse peritoneal macrophages.The above results directly and indirectly show that AFB₁ has damaging effects on the structure of mouse peritoneal macrophages.?5?Compared with the CG,the phagocytic function of mouse peritoneal macrophages in the MG and HG decreased significantly?p<0.01?,and the contents of inflammatory factors?IL-1,IL-6and TNF?in culture medium increased significantly?p<0.05;p<0.01?,indicating that AFB₁exposure can disturb the phagocytic function and inflammatory factor secretion function of mouse peritoneal macrophages.?6?Compared with the CG,the levels of ROS and MDA of mouse peritoneal macrophages in the MG and HG increased significantly?p<0.05;p<0.01?;the activities of SOD and GSH-Px in each AFB₁ exposed groups decreased significantly?p<0.05;p<0.01?,indicating that AFB₁ exposure can induce oxidative stress of mouse peritoneal macrophages.?7?Compared with the CG,the iron content,the expression of iron uptake protein?Tf R1 and DMT1?,iron export protein?FPN1?and iron storage protein?Fth and Ftl?were increased significantly in mouse peritoneal macrophages treated with AFB₁?p<0.05;p<0.01?,indicating that AFB₁ exposure can disturb iron homeostasis of mouse peritoneal macrophages.The above results show that AFB₁ exposure can induce structural and functional damage and oxidative stress of primary mouse peritoneal macrophages,and is related to iron homeostasis imbalance.