The Role Of Gut Barrier And High Fiber Diet In Acetaminophen-induced Hepatotoxicity In Mice

Background and objectiveAcetaminophen(APAP)is a common over-the-counter drug used for analgesic and pain relief.APAP hepatotoxicity can be directly induced.It is generally safe at recommended therapeutic doses.However,APAP overdose is a predominant cause of acute liver failure.APAP overdose also induces organ injury including kidney and lung.Recently,gut was reported to play an essential role in disease progression and development,such as the fatty liver disease and sepsis.The gut intestine as an important absorption organ mediates drug efficiency,but the role of gut in APAP-induced liver injury is unknown.Moreover,whether the diet effects modulate APAP-induced liver injury through the gut is also unclear.The present work aims to explore the role of gut barrier and high fiber diet in APAP-induced acute liver injury in mice.Research contents and methods1.APAP hepatotoxicity and APAP-induced gut injury.(1)C57BL/6J mice were injected intraperitoneally with 300 mg/kg APAP,and detected the liver injury after 24 hours.(2)Gut barrier was detected with western blotting and immunofluorescence;Fecal albumin concentration and plasma LPS levels were detected with ELISA according to the manufacturer’s instruction;Plasma penetration of FD-4 detection;Bacterial DNA,cytokines and chemokines of colon tissue and colonocytes were conducted with Quantitative-PCR to confirm the "leaky gut" and bacterial translocation.2.Mice was genetically modified with CCL7 specifically overexpressed in intestinal epithelial cells(CCL7tgIECmice)and APAP hepatotoxicity mouse model was conducted.(1)CCL7tgIEC mice were injected intraperitoneally with 300 mg/kg APAP,and detected liver injury after 24 hours.(2)Hepatic GSH depletion,CYP2E1 expression levels and APAP-adducts production were detected with biochemical kit,Quantitative-PCR and western blotting to confirm whether the APAP metabolic process was altered with the CCL7 specifically overexpressed in epithelial cells.3.The mechanisms about the higher susceptibility of APAP hepatotoxicity in CCL7tgIEC mice.(1)Western blotting,immunofluorescence and transmission electron microscopy were used to detect the tight junction expression and structure in WT and CCL7tgIEC mice.(2)Fecal albumin concentration and plasma LPS levels were detected with ELISA according to the manufacturer’s instruction;Plasma penetration of FD-4 detection;Bacterial DNA,the expression of inflammatory factors and receptors in liver of CCL7tgIEC mice were conducted to confirm the "leaky gut" and bacterial translocation.(3)Determined the gene expression pattern in liver of WT and CCL7tgIEC mice with RNA-sequencing.4.The effects of high fiber diet in APAP-induced liver injury in mice.(1)BABL/C mice were fed with high fiber diet for 2 weeks,intraperitoneally injected with 300 mg/kg APAP,and detected liver injury after 24 hours.(2)Hepatic GSH depletion and CYP2E1 expression levels were detected with biochemical kit and western blotting to confirm whether the APAP metabolic process was altered in high fiber diet group mice.(3)Using 16s rRNA sequencing detected the component alteration of gut bacteria between the high fiber diet and normal chow diet groups.Result1.APAP not only induced acute liver injury,but also the gut barrier dysfunction.Importantly,APAP induced the higher mRNA expression levels of CCL7 in isolated colon epithelial cells.2.Mice with CCL7 specifically overexpressed in epithelial cells triggered more severe liver injury after APAP treatment.And the metabolic process of APAP was not altered in CCL7tgIEC mice,including GSH depletion,CYP2E1 levels,APAP-adducts formation in the liver.3.At baseline,CCL7tgIEC mice displayed gut barrier dysfunction and bacterial translocation.Moreover,RNA-sequencing data showed that the signaling pathways related with inflammation activation were activated in CCL7tgIEC mice.4.High fiber diet alleviated APAP-induced liver injury with antioxidative effects,and mediated the microbiome composition alteration.ConclusionAPAP induced liver failure and gut barrier dysfunction.CCL7 specifically overexpressed in intestinal epithelial cells destroyed gut integrity,and high permeability of intestine increased the susceptibility of APAP hepatotoxicity in mice.Supplementation with fiber protected mice against APAP-induced liver injury through the antioxidative effects,also modulated the microbiome composition.Targeting the gut would provide strategies for APAP hepatotoxicity treatment in clinic.