Study On The Mechanism Of Chronic Exposure To Thiacloprid-Induced Liver Injury In Japanese Quails Based On Microbiota-Gut-Liver Crosstalk

Thiacloprid(THI)is a broad-spectrum neonicotinoid insecticide widely used in agricultural pest control around the world.With the continuous and extensive use of such insecticides in agriculture,environmental pollution caused by neonicotinoids is becoming increasingly prominent,posing health hazards to animals and humans.The intestine is an important barrier that protects the body from harmful external substances;an imbalance in gut microbiota can cause intestinal mucosal damage,leading to inflammatory bowel disease and various diseases such as liver and kidney disease.The bidirectional interaction between the intestine and liver is a key influencing factor in liver disease.However,whether gut microbiota is involved in THI-induced liver damage remains to be confirmed.Therefore,elucidating the effects of THI on the microbiota-gut-liver axis is essential for assessing the risk of liver damage after exposure to THI.This study proposed the concept and research methods and content of veterinary toxicology based on multi-omics technologies to investigate the mechanisms underlying THI-induced liver damage and gut microbiota changes comprehensively and systematically in Japanese quails.This will provide a theoretical basis and practical foundation for using gut microbiota as a target for the prevention and treatment of liver damage induced by chronic exposure to THI.In this study,27 healthy male Japanese quails aged three weeks were randomly divided into three groups,9 in each group: the control group,low-dose THI group,and high-dose THI group,and were given 0,2,and 4 mg/kg/day THI solution by gavage,respectively.After continuous administration for 42 days,blood,ileal contents,ileal tissues,and liver tissues samples were collected from the quails.Techniques such as hematoxylin-eosin(H&E)staining,Sirius Red staining,Masson staining,Oil Red O staining,transmission electron microscopy,blood biochemical analysis,enzyme-linked immunosorbent assay(ELISA),16 S r RNA gene sequencing,metabolomics,transcriptomics,Western blot,and real-time quantitative PCR(q PCR)were used to observe the pathological changes in the ileum and liver tissues of Japanese quails exposed to THI,detect differences in blood biochemical indicators and LPS content,mine the data of different omics,clarify the correlation between microbiota,metabolites,and genes,and how they interact to participate in THI-induced liver injury in Japanese quails.The results of the study showed that:(1)Compared with the Control group,the quails in the low and high doses of THI group showed varying degrees of depression,while some quails in the low dose of THI group exhibited increased aggressiveness.(2)Compared with the control group,THI exposure significantly increased the serum activities of AST and ALP,and the serum ALT activity in the THI high-dose group was significantly increased.THI also dosedependently increased the levels of serum triglycerides,total cholesterol,low density lipoprotein,and total bile acids,and decreased the levels of high-density lipoprotein.(3)Liver H&E staining,Sirius Red staining,Masson staining,and Oil Red O staining showed that THI caused fat deposition and fibrosis in the liver of Japanese quails,inflammatory cell infiltration in the central zone of the lobule,and transmission electron microscopy results showed that THI caused a large accumulation of lipid droplets in liver cells.(4)H&E staining of ileal tissue showed that THI exposure led to a decrease in the depth of the ileal crypt and an increase in the distance between intestinal villi in Japanese quails.(5)16S r RNA gene sequencing results showed that after THI exposure,the composition and diversity of gut microbiota in Japanese quails were significantly changed,and there were significant differences in the relative abundances of certain bacteria at the phylum and genus levels.(6)ELISA results showed that THI induction increased the level of LPS in the serum.(7)Transcriptome data from liver tissue of quails exposed to THI revealed significant changes in 420 genes,including 241 upregulated and 179 downregulated genes.Enrichment analysis indicated that differentially expressed genes were mainly involved in energy metabolism,such as steroid biosynthesis,unsaturated fatty acid biosynthesis,and fatty acid elongation.Gene expression analysis of cholesterol and bile acid synthesis pathways indicated that THI exposure promoted cholesterol and bile acid synthesis in quail liver,with upregulated expression of proteins encoded by ATP binding cassette subfamily B(ABCB)11 and ABCB4,which are involved in bile acid transport.Additionally,the transcription level of the proto-oncogene MYC was increased in quail liver.(8)THI caused a disturbance in serum metabolic homeostasis,as evidenced by serum metabolomics results.Specifically,THI induced increased levels of long-chain fatty acids like arachidonic acid and docosatrienoic acid,and decreased levels of short-chain fatty acids like2-hydroxybutyric acid and isocitric acid.Furthermore,harmful metabolic products related to gut microbiota,such as benzoic acid,were also increased.(9)Analysis of gene expression and protein levels related to intestinal permeability and inflammation showed that THI induced an increase in intestinal permeability and inflammation in the ileum tissue.Specifically,the expression and protein levels of tight junction genes in the tissue were significantly downregulated,while those of inflammatory factors like NF-κB were significantly upregulated.In summary,this study elucidates how THI exposure disrupts the gut microbiota of quails,leading to intestinal inflammation and increased intestinal permeability,which promotes harmful substances such as LPS and benzoic acid to enter the bloodstream and cause liver damage.Additionally,THI exposure leads to changes in bile acid and cholesterol metabolism in quail livers,resulting in abnormal lipid metabolism,severe liver damage,fibrosis,and steatosis.This study provides the first evidence that THI exposure aggravates liver damage through the regulation of the microbiota-gut-liver axis,which enriches and expands the understanding of the metabolic toxicology of organic pesticides in the environment.