| Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by liver parenchymal cells steatosis and fat piling up, and without excessive alcohol consumption history. ANFLD has became one of the common liver diseases, the pectrum of disease, including nonalcoholic simple fatty liver(NAFL), nonalcoholic steatohepatitis(NASH), and its related cirrhosis and liver cancer,were mixed with the progression. During the long course of disease in NAFLD, NASH is a necessary stage of NAFL occur cirrhosis. Obesity, type2diabetes, hyperlipidemia, alone or together, constitute the predisposing factors of NAFLD. But its pathogenesis has not yet been elucidated and there is no effective drug treatment.The study find that the most of patients with NAFLD are asspciated with the small intestine bacterial overgrowth(SIBO). SIBO can affect lipid metabolism, increas liver lipid deposition, play a major role in the development of non-alcoholic fatty liver. Although the study found that non-alxoholic fatty liver disease in patients with small intestinal bacterial overgrowth and enterogenous endotoxemia, but the specific mechanism in NAFLD and lipid metabolism of the body mechanism has not yet fully elucidated.The peroxisome proliferator-activated receptors(PPARs) signal transduction pathway plays an important role in the process of lipid metabolism in the liver and its subtype PPARα mainly regulate the metabolism of hepatic fatty acid and triglyceride. So whether endotoxin interfere the liver lipid metabolism through the PPARαmRNA expression needs to be validated, and the study of the relationships between them may provide a new direction with prevention of non-alcoholic fatty liver.This study investigates the change of endotoxin level in serum and the expression of PPARα mRNA in liver tissue. We give probiotics to intervene in the rat NAFLD, this drug has the role of regulating the intestinal flora. Then we observe that the probiotics on liver steatosis and intestinal endotoxemia in-depth understanding of the pathogenesis of NAFLD, and provide new therapeutic targets.Objective:1To establish non-alcoholic steatohepatitis rat model by high-fat diet.2To explore the role of intestinal endotoxemia in the pathogenesis of NAFLD and its relationship with PPARa.3To observe the effect of probiotics treatment on rats with NAFLD, and explore its possible mechanism of action.Methods:1Animal model and designs experiment:Thirty-two male Sprague Dawley rats weighing180±20g were randomly divided into three groups after normal feeding seven days:control group(CG, n=12)were fed with common diet; model group(MG, n=12)and treatment group(TG, n=8)were fed with high fat diet(88%common food+5%egg yolk powder+1%cholesterol+10%lard). The experimental animals (4per cage) housed at animal laboratory with25±2℃, light and shade of each12h, and eating and drinking at free. After12weeks,4rats of the control group and model group were killed, and we took liver HE staining to determine if model was successed. Then the treatment group were given Bifid. Given treatment group the bifidobacterium triple viable enteric-coated capsules (0.113g·kg-1·d-1) by intragastric administration since the13weeks, control group and model group were given equal volume of distilled water by intragastric administration.Recorded the body weight of rats weekly, and adjusted the intragastric admimistration dose according to the change of body weight. Progressed continuouly to the end of the dosing period of16weeks, all rats were killed.2Index detection(1)The general situation of the liver were observed. Then we completely resected the liver, and weighed the liver.(2) Liver tissue sections with HE staining and the changes in liver pathology were observed.(3) Determined the serum alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglycerides, high density lipoprotein levels using automatic biochemical analyzer.(4) Determined endotoxin levels in serum using Limulus Amebocy Lysate(LAL).(5) Detected the changes of liver tissue PPARa mRNA gene expression usig Real-time PCR.Results:1High-fat diet rats appear liver steatosis and inflammatory cell infiltration at the end of12weeks, showed that high-fat diet successfully produced NASH rats model.2The condition of the body weight, liver weight and liver index(liver weight/body weight×100%)HE staining of part of the liver tissue at the end of12weeks were determined the success of the rat NASH model.The remaining rats weighed again at the end of16weeks, the body weight(570.85±13.41), liver weight (19.81±2.47), and liver index (2.55±0.16%) of model group rats were significantly higher than the body weight(507.43±11.65)、liver weight (12.91±0.78) and liver index (3.48±0.46) of the normal control group,there are significant differences(P<0.01). Compared with model group, body weight (238.58±15.24), liver weight (15.37±1.23) and liver index (2.93±0.22%) of treatment group were significantly lower (P<0.01),there are significant differences.3Serum biochemical index:(1) AST and ALT:AST level of model group(204.88±18.22)was increased significantly compared with normal group(157.88±17.60)(p<0.01), the treatment group AST(183.00±18.47) decreased compared with the model group (P<0.05). The model group ALT(71.00±8.73) was increased significantly compared with normal group(41.00±7.43)(P<0.01), the treatment group ALT(45.88±15.06) significantly decreased compared with the model group (P<0.01)(2) TG, TC, HDL:Serum TG level of model group(0.60±0.08)was increased significantly compared with normal group(0.31±0.08)(p<0.01), the treatment group TG(0.50±0.09) significantly decreased compared with the model group (P<0.05). The model group TC(1.20±0.18) was increased significantly compared with normal group(0.68±0.16)(P<0.01), the treatment group TC(1.02±0.17) was no significant difference compared with the model group (P<0.01). The model group HDL (0.65±0.06) was decreased significantly compared with normal group(0.82±0.08)(P<0.01), the treatment group HDL(0.73±0.06)was increased compared with the model group (P<0.05)4endotoxin and expression of PPARa mRNASerum endotoxin level of the model group (0.24±0.02) lower significantly than the normal control group0.12±0.01)(P<0.01). The treatment group endotoxin level (0.17±0.02) was decreased significantly compared with model group (P<0.01)The liver is expressed with high PPARamRNA (1.62±0.35) in the normal control group.PPARamRNA expression in model group liver tissue(0.66±0.25) significantly reduced compared with normal group(P<0.01). Treatment group PPARamRNA expression (0.10±0.23) compared with the model group has been enhangced (P<0.05),but reduced compared with the normal group(P <0.01), there are significant differences.5Histological analysisAt the end of the experiment,rat liver were stained with HE, the liver cells of normal rats without steatosis, and there was no inflammatory cell infiltration. Observed under light microscope,the liver tissue of model group after12weeks can be seen diffuse hepatic steatosis, accompanied by a small amount of inflammatory cell infiltration.Then the degree of steatosis gradually increased. Severe hepatic steatosis, espacially around central vein, were found at16th week.Lobular inflammation, periportal inflammation and degeneration, focal necrosis were found in model group.There are significant differences compared with the normal group (P<0.01). Compared with model group, the degree of liver steatosis of the treatment group inproved significantly compared with the model group(P<0.01); Compared with the model group there are no significant improvement in the inflammation grade of the treatment group.Conclusion:1High fat diet can successfully establish NASH rat model.2Small intestinal bacterial overgrowth and intestinal endotoxe-mia possibly play an important role in the pathogenesis NAFLD, and endotoxin may regulate liver lipid metabolism by PPARa mRNA expression.This findings have important significance in-depth understanding of the pathogenesis of NAFLD.3Probiotics can significantly reduce the hepatic steatosis, improve liver function and dyslipidemia, increase expression of PPARamRNA, it may play a therapeutic effect on NASH. |
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