Discussion On Mechanism And Effect Of Intestinal Endotoxemia In Rats With Nonalcoholic Steatohepatitis

Objective：To established animal model of rats with nonalcoholic steatohepatitis（NASH）induced by high-fat diet, detect endotoxin levels and intervent withoral administration of Saccharomyces boulardii and observe changes ofhepatic steatosis and inflammation by improving intestinal flora and reducingthe level of endotoxin, to disscuss whether this change acts by the expressionof scavenger receptor A（SR-A）, to provide new theoretical basis in thepathogenesis and treatment of NASH.Methods：1Animal modelForty-two male Sprague-Dawley（SD） rats weighing180±20g weredivided randomly into normal control group （NG, n=14）fed with normal diet，model group （MG, n=14） and intervention group（TB，n=14）both fed withhigh-fat diet （88%common food+10%lard+2%cholesterol） after normalfeeding seven days. After16weeks, four rats selected randomly per groupwere sacrificed，then histological pathology by liver HE staining showedNASH was established. From17th week，Rats in intervention group wereadministrated with oral Saccharomyces boulardii (78*10⁸CFU/kg·d^-1) and fedwith high-fat diet consecutively，rats in normal group and model group wereadministrated with oral equal voloum Distilled Water daily respectively. Atthe end of24th week，all rats were sacrificed. Animals had access to food andwater ad libitum and were housed in the animal laboratory conditions fortemperature（25±2℃）,light and shade controlled in each12h.2Observing general conditions of rats, weighting body weight and liverwet weight by electronic balance and calculating liver index as follow: liverindex=liver wet weight/body weight. 3After all rats were sacrificed by intraperitoneal injection10%hydrationchlorine aldehyde, portal blood was collected into an apyrogenic tube andserum was collected by centrifugation at3000r/min for15minutes, thenendotoxin levels were measured using the limulus amebocyte lysate test;Serum taken from the heart was used to measure the levels of alanine aminotransaminase （ALT）, nmda amino transaminase （AST） and triglycerides （TG）with automatic biochemical analyzer.4The histology of liver was examined microscopically followinghematoxylin eosin （HE） staining.5The protein expression levels of hepatic SR-A were detected byimmunofluorescence.6The mRNA levels of hepatic SR-A and TNF-αwere detected by realtime PCR.Results：1Body weight, liver weight and liver index of rats in each group:At the end16th week, histological pathology by liver HE stainingshowed NASH was established.The levels of body weight, liver wet weight and liver index in modelgroup rats were significantly higher than those of normal control group （bodyweight:567.60±18.9νs460.71±14.71, P<0.05; liver wet weight:19.68±2.68νs11.53±0.98, P<0.05; liver index:3.47±0.43νs2.50±0.12, P<0.05）.Compared with model group, the levels of body weight, liver wet weight andliver index significantly decreased in intervention group （body weight:521.50±19.4νs567.60±18.9, P<0.05; liver wet weight:15.06±1.32νs19.68±2.68, P<0.05; liver index:2.89±0.20νs3.47±0.43, P<0.05）.2Serum biochemical index:Compared with normal group, the levels of AST, ALT and TG in modelgroup increased significantly（AST:276.95U/L±25.06U/L νs145.37U/L±15.19U/L, P<0.05; ALT:103.44U/L±8.41U/L νs39.10U/L±6.01U/L, P<0.05; TG:0.93mmol/L±0.14mmol/L νs0.54mmol/L±0.10mmol/L,P<0.05）; the levels of AST, ALT and TG decreased in intervention group compared with those of the model group（AST:191.68U/L±14.62U/L νs276.95U/L±25.06U/L,P<0.05; ALT:71.02U/L±5.04U/L νs103.44U/L±8.41U/L,P<0.05; TG:0.73mmol/L±0.11mmol/L νs0.93mmol/L±0.14mmol/L, P<0.05）.3Serum endotoxin level:The level of serum endotoxin in the model group significantly increasedcompared with that of the normal control group （0.36EU/ml±0.02EU/ml νs0.17EU/ml±0.01EU/ml, P <0.05）. The level of endotoxin decreased inintervention group compared with that of model group （0.22EU/ml±0.01EU/ml νs0.36EU/ml±0.02EU/ml, P <0.05）.4Expressions of SR-A protein:The result of immunofluorescence：SR-A is mainly expressed in Kupffercells （KCs） and sinusoidal endothelial cells and green fluorescence representspositive expression. The expression of SR-A in normal group was diffused,which decreased significantly in model group. Hepatic SR-A proteinexpression increased slightly in intervention group.5Levels of SR-A mRNA and TNF-αmRNA:The levels of SR-A mRNA in liver tissues significantly reduced in modelgroup compared with that of normal group （0.52±0.32νs1.43±0.46, P <0.05）;while it increased in intervention group compared with that of model group（0.87±0.34νs0.52±0.32, P <0.05）.Compared with normal group, The levels of TNF-αmRNA in modelgroup increased significantly （1.56±0.35νs0.57±0.23, P <0.05）; the levels ofTNF-αmRNA decreased in intervention group compared with that of themodel group（1.23±0.24νs1.56±0.35, P <0.05）.6Liver histological pathology：Naked eye：The rats’ liver surface in normal group was smooth andbright red with sharp edge and soft quality; The rats’ liver in model groupwas khaki and diffuse enlargement, capsular was tensed with blunt and thickedge, greasy feeling obviously and hard quality; in intervention group, the ratliver is a little yellow with medium quality and the edge is a little blunt. Light microscope: The hepatic lobule structure in normal group rats wasclear and complete, hepatic cords arranged radically around the central vein.Liver cell volume was normal and boundaries was clear with large and roundnucleus and uniform cytoplasm, there wasn’t steatosis and inflammatoryinfiltration; While the lobular in model group was disorder with diffusehepatic steatosis and varying sizes fat vacuoles in the cytoplasm, whichaccompanied by varying degrees of focal necrosis and inflammatory cellinfiltration within portal area and lobular, compare with normal group, therewas statistically significant （NAS:6.90±0.57νs0.70±0.48, P <0.05）；Inintervention group,the liver cell structure still existed around the central veinwith mild steatosis of liver cells away from the central vein and a smallamount of inflammatory cell infiltration, compare with model group, therewas statistically significant （3.60±0.71νs6.90±0.57, P <0.05）.7Pearson’s correlation analysis between the indicators:The level of serum endotoxin which was positively correlated with scoreof liver pathology and the level of TNF-α mRNA（r=0.645，r=0.696，P<0.05），was negatively correlated with the level of SR-A mRNA（r=-0.632，P <0.05）.Conclusions：1The rats model of NASH could be replicated successfully by high-fatdiet, gut-associated endotoxemia might play a role in the pathogenesis ofNASH.2In the process of NASH, endotoxin might aggravated hepatic injurythrough the decreased level of SR-A in liver tissue，which provided newtheoretical basis for the role of gut-associated endotoxemia on the impact ofNASH.3Saccharomyces boulardii could reduce gut-associated endotoxin and the degree of hepatic steatosis and inflammation by increasing level of SR-Ain liver tissue, which provided new target for the treatment of NASH.