Research On The Correlation Between Endotoxemia And Intestinal Epithelial Cell Apoptosis In NASH Rats

Non-alcoholic fatty liver disease is a spectrum of disease which incharacter with lipid accumulation in hepatocytes, excluding ethanol intake,drugs and other definite reasons inducing hepatic injury. It includes simplefatty liver, hepatic steatohepatitis, hepatic fibrosis, liver cirrhosis, hepaticcellular carcinoma, several severe complications for example hepatic failureare also involved in. It has become the major type of chronic liver disease inwestern developed countries and origin of most cryptogenic cirrhosis withgradually increased morbidity and mortality. Simple fatty liver is benigndisease that its progress is reversible, however hepatic steatohepatitis as theadvanced and severe state is more easily progressing to hepatic fibrosis andliver cirrhosis. Understanding the mechanisms that lead to progression fromsimple steatosis to NASH is critical to the design of rational treatment methodto block the progression.Resent years more and more studies indicate that hepatic inflammatoryinjury induced by intestinal endotoxemia (IETM) is involved in thepathegenesis of simple fatty liver progressing to NASH. Variety of factors areinvolved in the evoking of intestinal endotoxemia. Intestinal dysbacteriosis,bacteria overgrowth, accompanined with the injury of intestinal barrier willresult in bacterial translocation(BT), and finally levels of endotoxin areincreased in the portal or system circulation. Lipopolysaccharide, a componentof the cell wall of gram-negative bacteria, is the major composition ofendotoxemia, it can be recognized by pattern recognition receptors on Kuffercells in liver mediating LPS-CD14-TLR4signalling and trigger stimulation ofinnate immune system with following increased release of proinflammatorycytokines, those cytokines including TNF-α, IL-6then induce hepatic injury. Initial stage of injury of intestinal barrier function represents increasedintestinal permeability.Intestinal permeability is fundamental for connectingintestinal toxin and liver, and increased intestinal permeability is participativein kinds of disease, including alcoholic fatty liver disease(ALD), inflammatorybowel disease(IBD), acute pancreatitis, chronic renal failure and pulmonaryinterstitial fibrosis, NAFLD was also mentioned ever. Alcoholic direct injury,oxidative stress, intestinal ischemia and reperfusion and many other factorscan broke the balance of intestinal epithelium cell apoptosis and proliferation,accompanined with alteration in tight junction or not. Either of them willdisrupt intestinal epithelial integrity leading to increased permeability. Theintestinal epithelial cell apoptosis has been referred in alcoholic fatty liverdisease and inflammatory bowel disease for years, but rarely been researchedon NAFLD. We hypothesize that NASH rats present intestinal endotoxemia iscorrelated with increased intestinal permeability and increased epithelial cellapoptosis.Objectives: Trying to establish NASH rats model feeding with High fatdiet to investigate the epithelial cell apoptosis in terminal ileum, gutpermeability and the level of serum endotoxin and their correlation.Materials and methods:20male Sprague Dawley rats weighing180±20g were randomly divided into2groups after normal feeding one week:①C ontrol group(n=10)rats were fed with standard chow diet;②Modelgroup(n=10) rats were fed with high fat diet(88%commonfood+2%cholesterol+10%lard). The animals were raised in the animallaboratory conditions for25±2℃, light and shade in each12h, and free to foodand water. Record the body weight each week. Until the end of the24thweeksacrifice all the rats.Completely remove the entire liver tissue and observe the generalcondition, weight the liver’s wet weight. Automatic biochemical analyzer forthe determination of serum ALT, AST and TG levels of serum. Limulustesting for endotoxin level of serum. Hematoxylin and Eosin staining for liver tissue and terminal ileum tissue and calculate the NAFLD activity score ofliver paraffin section. Spectrophotometric method for D-Lactate of serum.TUNEL(terminal deoxynucleoitidyl transferase mediated dUTP nick endlabeling) for testing the epithelial cell apoptosis in terminal ileum paraffinsection.Result:①High-fat diet for24weeks, apparent hepatic steatosis,hepatocellular ballooning and inflammatory cell infiltration were observed inliver HE staining. Validating that high fat diet successfully establish NASHrats model.②C omparison ofbody weight, liver wet weight and liverindex(liver weight/body weight×100%) in two groups at the end of the24thweek: the model group and the control group had apparent difference in bodyweight, liver wet weight and liver index, the model group were moreincreased(567.60±18.89vs460.71±14.71,19.68±2.68vs11.53±0.98,3.47±0.43vs2.5±0.12, P<0.05).③Serum biochemical index: the level ofserum ALT, AST and TG in model group significantly increased compared tonormal group(103.44±8.41vs39.10±9.37,276.95±25.06vs145.37±15.19,0.93±0.14vs0.54±0.10, P<0.05).④t he model group and the control grouphad apparent difference in the level of serum endotoxin, and the model groupwas more increased(0.3600±0.0293vs0.1747±0.0121, P<0.05).⑤The levelof serum D-Lactate: the model group and the control group had apparentdifference in The level of serum D-Lactate, and the model group was moreincreased(0.375±0.0353vs0.182±0.0107, P<0.05).⑥L iverhistologicalanalysis: HE staining showed that the control group present normal cell shape,without hepatic steatosis or hepatocellular ballooning, few inflammatory cellinfiltration observed. However the model group present diffused hepaticsteatosis, hepatocellular ballooning, accompanied with lobular and periportalinflammation, some focal necrosis was also observed. The model group andthe control group had apparent difference in NAFLD activity score(6.90±0.57vs0.70±0.48, P<0.05).⑦the histological changes in the ileum mucosa:the control group present complete and an orderly fashion in the villi, with noabnormal morphology in the epithelial cells, as well as no manifestation of congestion, edema or infiltration of inflammatory cells. However the intestinalmucosal villi of model rats were loosened and disrupted while the epithelialcells were necrotic and the mucosa was edematous and infiltrated withinflammatory cells.⑧The model group and the control group had apparentdifference in the intestinal epithelial cell apoptotic index, the model grouppresented increased apoptosis (0.609±0.071vs0.407±0.037, P<0.05).⑨Pearson’s analysis among intestinal epithelial cell apoptotic index, level ofserum D-Lactate and endotoxin show that the intestinal epithelial cellapoptotic index was positively correlated with the level of serum D-Lactate toa certain degree, r=0.488; The intestinal epithelial cell apoptotic index waspositively correlated with the level of serum endotoxin, r=0.397; The level ofserum endotoxin and D-Lactate had significant positive correlation. r=0.954,P<0.05.Conclusion:1NASH rats represent intestinal endotoxemia and injury of intestinalbarrier function.2Increased epithelial cell apoptosis may be involved in the injury ofintestinal barrier function,aggravating intestinal endotoxemia and thereforeinducing progression of NASH.