Melatonin Alleviates Lead-induced Fatty Liver In Common Carps Via Gut-Liver Axis

Lead(Pb)is one of the most toxic environmental heavy metal contaiminant,that has been leabeld as hazardous substances by WHO,and identidied as one of the top 10 most toxic chemicals to public health.Environmental Pb mainly comes from human activites.Dut to ore and metal manufacturing,leaded gasoline and paint consumption,industrical and agricultural wastes,aquatic Pb contaiminat has been serious threat to fish.As gut is the main target organ to intake Pb,the gut mucosal baerrier plays the role as the fist defense line aginst pathogens.While,the damage of gut mucosal baerrier permits the microbiota and metabolites translocated to the liver via portal vein,and causing hepatic inflammatory injury.Metatonin is natural hormone secreted by the pineal gand,and performed antioxidation effects as endogenous free radical scavenger.MT protect gut barrier through effects on gut microbiota,and palys therapeutic role to hepatic oxidative stress.But the study about the effects of Pb on the gut and liver tissue of fish was very rare,and the function of MT on Pb also need to be further explored.Therefore,the common carps were used as experimental animals,and Pb nitrate and MT were used to established Pb-poisoinng and Pb.MT model.H&E and oil red O staining,scan electron microscopy and transmission electron microscopy,metagenome.Transcriptome,real-time fluorescent quantitative PCR,western blot,immunofluorescence staining,flow cytometry,enzyme linked immunosorbent assay and fatty acid target detection were performed to observe pathophysiological changes of intestinal and liver tissues,to detect gut microbiota alternation and gene experession difference,to anlysis expression of related genes and fatty acid compounent in liver,and the effects of MT on Pb were verified in EPC and L8824 cells.The aim of this study was to detect the toxicity mechanism of Pb on the gut and liver in the common carps,and to illuminate the effects of MT on Pb-induced toxicty.The main results are as follows:(1)The intestinal histopathology results shown that Pb exposure induced gut inflammatory injury.Microscopic observations showed that Pb exposure resulted in the abrosion of the villi brush margin,diffuse infiltration of lymphocytes and insufficient mucous particles in the lumen.Ultrastructural observation showed that Pb exposure resulted in intestinal villus loss,mitochondrial aggregation and shrink,and pyrosomes formation.(2)The intestinal metagenomic analysis results shown that Fusobacteria was the dominant phyla of the common carps,and Pb exposure performed an augmentation in Proteobacteria and Firmicutes phyla,and an elimination of Fusobacteria and Bacteriodets phyla results in the decline of ratio of Firmicutes to Bacteriodets.The elevation of Actinobacteria phyla were assessed with MT supplement.The functional analysis of microbiota shown the microbiota gene functions related to LPS biosynthesis(ko00540)were remarkable higher after Pb exposure.While MT supplement alleviated Pb induced high LPS content in serum,gut and liver tissue,indicating MT alleviated Pbinduced LPS synthesis.(3)Gut transcriptome analysis results shown that Pb exposure significantly upregulated 451 genes and downregulated 311 genes,and enrichment analysis showed that differential genes were mainly involved in necroptosis and pyroptosis pathways.In vitro and in vivo experiments shown that MT alleviated Pb-inbuced necroptosis and pyroptosis pathways,upregulated tight junction protein expression,improved intestinal permeability and reduced intestinal inflammatory injury.(4)The liver histopathological observation and oil red staining result shown that Pb exposure caused the lipid accumulation.Microscopic observations showed that Pb exposure resulted in hepatic cell disorder,steatosis and cell death.Ultrastructural observation showed that Pb exposure resulted in mitochondria shrunk,and ferroptosis.(5)The fatty acid target detection resulte showed that Pb exposure significantly upregulated polyunsaturated fatty acids synthesis of linoleic acid,α-linolenic acid,γ-linolenic acid,eicosapentaenoic acid,arachidonic acid and eicosapentaenoic acid,while the contents of methyl docosahexaenoic acid decreased.MT alleviated Pb-induced l the imbalance of polyunsaturated fatty acid homeostasis in liver tissue.(6)In vitro and in vivo experiments shown that Pb induced ROS generation,promoted mitophagy and auto-lysosome formation,destroyed lysosome and iron homeostasis,resulting in autophagy and ferroptosis.(7)The glucolipid metabolic pathways related gene expression testing found that Pb exposure inhibited AMPK phosphorylation promoted fat synthesis and glycolysis,reduced adipose decompose and glycogen synthesis,leading to hepatic lipid accumulation.MT actived AMPK phosphorylation,relieved lipid accumulation,alleviated Pb-induced fatty liver.In conclusion,Pb exposure altered intestinal mcrobiota homeostasis caused intestinal inflammatory damage and barrier permeability elimination,promoted LPS synthesis,resulted in fatty liver via gut-liver axis.MT inhibited Pb-induced mitophagy and ferroptosis via activiting AMPK signaling pathway,improved Pb-induced lipid accumulation and fatty liver.These results will enrich the toxic mechanism of Pb exposure to the common carps,revealed the new mechanism of MT alleviating the toxicity of Pb,provided a reference for the study of toxic effect of Pb in aquatic ecosystem,provided a new theoretical basis for the development of Pb detoxification drugs,and provided a reference for the healthy aquaculture of aquatic organisms and the development of aquatic feed.