Molybdenum And Cadmium Co-induce Spleen Inflammation Via TLR4/MyD88/NF-κB Signaling Pathway In Sheep Spleen

To explore the effects of molybdenum and cadmium co-exposure on spleen injury and TLR4/MyD88/NF-κB signaling pathway in sheep.In this study,the Mo,Cd and their combination stress model of spleen and primary splenic lymphocytes in sheep were established.In vivo experiments,48 two-month-old sheep were divided into four groups,as follows:control group;Mo(45 mg Mo·kg^-1·BW)group;Cd(1 mg Cd·kg^-1·BW)group;Mo+Cd(45 mg Mo·kg^-1·BW+1 mg Cd·kg^-1·BW)group.The sheep were gavage for 50 days.In vitro experiments,sheep splenic lymphocytes were treated with Mo（100μM）and Cd（3μM）for 24 h.The structure and the morphological changes of spleen and splenic lymphocytes were observed through tissue sections and transmission electron microscopy.Immunofluorescence was used to observe the co-localization expression of TLR4 and MyD88.Change in antioxidant capacity levels and immunoglobulin levels were detected by using antioxidant kits and ELISA kit.The expression levels of TLR4/MyD88/NF-κB signaling pathway and inflammatory factors were detected by RNA-seq and western blot.In addition,the fluorescence microscope was used to observe the changes of intracellular ROS levels.The in vitro inhibition experiment of TLR4 inhibitor was conducted.The results showed that Mo,Cd and their combination damaged the morphological structure of the spleen and significantly reduced the antioxidant capacity and immunoglobulin levels（P<0.05）.TLR4 and MyD88 co-localization and intracellular ROS levels were significantly enhance.Mo and Cd activated the TLR4/MyD88/NF-κB signaling pathway（P<0.05）,significantly up-regulates the levels of inflammatory factors（P<0.05）,and ultimately induced inflammation.In addition,the addition of TAK-242 in Mo and Cd combined group significantly inhibited the TLR4 levels which was up-regulated by Mo and Cd,and thereby significantly reduced the induction of the above factors by Mo and Cd.The above results indicated that co-exposure of Mo and Cd induced spleen injury and up-regulated the expression of inflammatory factors by activating TLR4/MyD88/NF-κB signaling that eventually led to spleen inflammation in sheep.