Effects Of High Fat And NanoDEN Co-exposure On Liver Injury

Liver is an important metabolic site as well as an essential detoxification organ.With the development of people’s living standards,especially the change of dietary habits,the liver bears a considerable load,even suffering serious damage.Liver injuries can be divided into acute liver injury and chronic liver injury.There are many treatment methods with a high cure rate towards acute liver injury.Comparatively,curing chronic liver injury is much more difficult because of many inducements,unconspicuous symptoms and unknown mechanisms.It is too difficult to prescribe the right medicine that we need more research on chronic liver damage and provide a scientific basis for clinical treatment.Based on our previous research findings about liver injury in mice induced by a single chemical toxic nitrosamine and it’s nano preparations,this study was designed to explore the hazardous effects of exposure with high-fat diets on liver damage and the corresponding pathogenesis.Objective:The aim was to investigate the effects of high fat and nano-denosamine(Nano DEN)co-exposure on the liver.This could provide scientific evidence for the prevention and treatment of chronic liver damage in the clinic.Methods:1)SPF Kunming mice were randomly divided into four groups-the normal,high-fat diet group,Nano DEN group,and high-fat diet + Nano DEN co-exposure groups.Among them,the normal and Nano DEN groups were fed with ordinary feed;the high-fat and co-exposure groups were fed with high-fat feed.During the experiment,the animals of the Nano DEN and co-exposure groups received an intraperitoneal injection of the Nano DEN weekly.Meanwhile the animals of the normal and high-fat diet groups were injected the same amount of normal saline.At10 weeks,20 weeks,and 25 weeks,some mice in each group were sacrificed,to obtain blood and liver samples of each mouse.While recording the gross view of the liver,the biochemical analyzer was used to detect the biochemical characteristics of liver function in mouse serum indicators and lipid levels in liver tissue.2)H&E staining technology was used to detect the pathological changes of the liver and intestine of mice;and to score the inflammation.At the same time,16 s r RNA gene sequencing technology was used to detect intestinal bacterial flora.3)Real-time quantitative PCR was used to examine and analyze inflammatory factors(COX-2,TNF-α),fatty factors(PPAR-γ,AP-2),and fibrotic factors(TGF-β1,Smad-2)m RNA expression.4)Detection of inflammatory factors(COX-2),proliferation factors(β-catenin),aging factors(p16,p21)and hypoxia factor(HIF-1)related proteins in liver tissue samples of mice by immunohistochemical staining.Results:1.High fat can induce fatty liver and its malignant changes1)Compared with the animals in the normal group,mice that had long-term intake of high-fat diets were obese overall.Their livers showed multiple white tumor nodules,and a large number of fatty vacuoles appeared in the liver tissue.At the same time,the increase of triglyceride content in liver tissue also showed that the fatty group had severe fat.2)The results of immunohistochemical staining showed that compared with the normal group,high-fat feeding could increase the protein expression of the inflammatory factor COX-2 in mouse liver tissues(up 210%;p <0.01).Cell aging-related factors of p21 protein(up 210%,p <0.01)and hypoxia-inducible factor HIF-1 protein(up 158%,p <0.05)also increased significantly.3)Based on 16 s r RNA gene sequencing technology,gram-negative bacteria were up-regulated in the samples of the high-fat group.Whereas the colonies that regulated the intestinal microenvironment and maintained a steady downward trend.2.High-fat diet + Nano DEN co-exposure can promote fatty liver and its malignant process1)Compared with the normal group,the mice in the high-fat group and the Nano DEN group showed significant liver injury at week 25.The mice in the co-exposure group had corresponding liver injury lesions about 5 weeks earlier.This was accompanied by more severe inflammatory infiltration,fibrosis,and multiple white tumor nodules such as liver pathological disorders.In addition,triglyceride results in serum and liver tissue also suggested that liver lipids were most severe in the co-exposed animals.2)The results of real-time quantitative PCR showed that:(1)from the 20 th week,inflammatory factors of both the high-fat group(COX-2: up 194%,p <0.05;TNF-α:up 49%,p <0.05)and the Nano DEN group(TNF-α: up 288%,p <0.05)were significantly over-expressed.These were not as good as those in the co-exposure group(COX-2: up 367%,p <0.01;p <0.01),which was significant,especially in the late stage.The expression of inflammatory factors reached the highest value in the total exposure group(COX-2: up 648%,p <0.05;TNF-α: up 298%,p <0.001)at week25.(2)The changes of fatty factors were mainly concentrated in the high-fat group and the co-exposure group.The high-fat group expression level increased(AP-2: up452%,p <0.01)at 20 weeks;(increased by 716%,p <0.01;AP-2: increased by 575%,p <0.01).This growth trend peaked at week 25,and the total exposure group(PPAR-γ:up 924%,p <0.01;AP-2: up 935%,p <0.05)was particularly prominent with a ten-fold increase.(3)In the early stage,the expression of fibrosis in the liver tissue of each group of animals was not obvious.Until the 25 th week,the degree of fibrosis of liver had a significant difference among all the groups.The m RNA expression of fibrotic factors of high-fat group(Smad-2: up 164%,p <0.01;TGF-β1: up 77%,p<0.05)and Nano DEN group(Smad-2: up 144%,p <0.001;TGF-β1: up 101 %,P<0.05)with the m RNA expression of fibrotic factors increased,but not as significantly as the co-exposed group(Smad-2: up 331%,p <0.01;TGF-β1: up 169%,p <0.05).3)The results of immunohistochemical staining showed that:(1)compared with the normal group,high-fat group and Nano DEN group,the expression of COX-2protein and β-catenin protein in the liver tissue of mice in the co-exposure group was abnormal and increased.The most obvious performance(COX-2: up 178%,p <0.001;β-catenin: up 178%,p <0.001),and the nucleation rate of β-catenin also increased as well(up 213%,p <0.001).(2)In the hypoxia-inducible factor HIF-1 protein,the Nano DEN group(up 115%,p <0.001)was not significantly higher than the co-exposed group(up 141%;p <0.01).(3)As for cell aging,the expression of aging-related factors p16 and p21 proteins in liver tissues in the co-exposure group were obviously up-regulated(p16: up 139%,p <0.05;p21: up 133%,p <0.01)and the nucleation rate of p16 also changed greatly(up 213%;p <0.01).Conclusion:After continuous exposure to a high-fat diet and the chemical poison Nano DEN ingestion,the liver and intestine can be injured,while the inflammatory factors COX-2 and β-catenin-mediated signals can be activated,which disturbs the metabolism of the liver.The lipids can not break down so they excessively deposit in the liver.As a result,it promoted a hypoxic environment in cells.This in turn stimulated the oxidative stress response of the liver tissue,which damaged the cell mitochondria,triggered cell aging,inflammatory delay,and resulted with a fatty liver or kidney cancer.