Non-alcoholic Simple Fatty Liver Mice Model Building With MCDD And Dynamic Observation Of Probiotics Therapy

[Objective] Non-alcoholic fatty liver disease is a chronic liver disease with highprevalence in our country, the incidence increasing year by year. The mechanism ofNAFLD has not been exact, and currently considered as a metabolic stress-inducedliver damage with insulin resistance (IR) and genetic susceptibility closely related.The disease spectrum of NAFLD including nonalcoholic simple fatty liver(NAFL),Non-alcoholic steatohepatitis(NASH), Cirrhosis and eventually progress tohepatocellular carcinoma. Therefore, early intervention of NAFLD means importantfor the prognosis improvement of patients, and there is no sufficient evidence basedon medicine drug treatment. And also, the golden standard of diagnosis is pathologicaldiagnosis which is traumatic, diagnosis of the disease is mainly dependent on theclinical diagnosis currently, the accuracy and sensitivity are still need to be improved.Especially simple non-alcoholic fatty liver has no typical symptom, thus difficult tobe found. Therefore, the establishment of a suitable animal model is significant for thestudy of law, and iprovement the diagnosis and treatment of disease.[Methods] The rearch is divided into two parts. Part I:1. According to the principle ofrandomization,20mice were divided into observation group n=12and normalcontrol group n=12;2.mice in the observation group were feeding with Methionine-choline-deficient diet MCDD for3weeks, the control group feeding normally;3.observe the general condition of the mice dynamic, regularly weighed, and culture4kinds of bacteria (including Escherichia coli, Enterococcus faecalis, bifidobacteria andlactobacilli) with fresh stool specimens;4.kill the mice at8,15,22d, and then collectingthe liver specimens and serum samples;5.the serum samples were used to detectalanine aminotransferase ALT, aspartate aminotransferase AST, triglyceridesTG, and tumor necrosis factor-a TNF-a, interleukin-6IL-6, andlipopolysaccharide LPS level detected by enzyme-linked immunosorbent assay. PartII:1.322-week modified mice were randomly divided into drug treatment group(Group D n=16) and food recovery group (Group D n=16), and the established8mice as normal control(Group C n=8) with same age;2. Bifico was given to the drugtreatment group through a gastric tube twice a day for6weeks;3. observe the generalcondition of the mice dynamic, regularly weighed, and culture4kinds of bacteria;4.kill the mice at43d, and then collecting the liver specimens and serum samples;5.Liver specimens obtained for pathological diagnosis and grade quantified;6.detect thelevel of ALT AST TG TNF-a IL-6LPS in the serum;7. statistical analysis the results, the group paired t-test was used within a group, and independent sample t-testwas used between groups, Pearson correlation test was used for correlation analysis,one-way ANOVA was used for ntervention effect, all P <0.05was consideredstatistically significant.[Results]Part I:1.After three weeks MCDD induced mice appeared apathetic and hair disorders,pathological analysis defined that hepatic fatty change at8d, and the NAFLD scorewere always between3and4at8d,15d, having diffience with22d>5and theobservation group=0, prompted success of NAFLD mouse model, and control thestage in NAFL.2.There was a significant weight deceasing of MCDD mice compared with controlgroup from the4d P=0.02,with apathetic and hair disorders; the result of bacteriaculturing made sense that MCDD mice had a intestinal dysbiosis compared with thecontrol group.3.The levels of ALT,AST were increasing with the time, and the average numbershave upward trends, but there is no statistically significant differences, the level ofTG was increasing also, and had statistically significant differences with controlgroup in all the time.4.The level of TNF-a IL-6LPS had statistically significant differences with controlgroup in all the time, and the level of TNF-a IL-6have a significantly positivecorrelation with modifying time.5. At the level P <0.05, TNF-a, IL-6, LPS, ALT was positively correlated with thepathological score, including TNF-a, IL-6, LPS correlated highly (r=0.843,0.839,0.884)ALT correlated moderately (r=0.574); the intestinal bacterial culture B/E valuewas negatively correlated with pathological score strongly (r=-0.662).Part II:1.Recovery of mice in the drug treatment group (D) was significantly better than thethe diet recovery group (B). B group had a31.25%mortality rate, considered infectionas three causes of death, and two unknown. The weight of both two group recoveredto normal level in3weeks, group D make faster, but there was no significant statisticaldifference.2. The intestinal dysbiosis of Group D recovery obviously, there had been nosignificant difference with normal control group from11d, and it keep stable. The intestinal dysbiosis of Group B could recovery only through normal diet recovery.3. The liver fatty lesions of Group B were still evident, and lobular inflammation evenincreased; The liver fatty lesions of Group D declined over time, and there is almost noaggravation of lobular inflammation. The treatment make an improvement of groupD’s pathological manifestations, and has significant statistical difference with GroupB P=0.032.4Correlation analysis showed that pathological score with the B/E values whichrepresenting colonization resistance in the P=0.05level was moderate-intensitynegative correlation (r=-0.526), Lactobacillus and Enterococcus faecalis andpathological score in the P=0.01level was stronger intensity negative correlation (r=-0.648,-0.632). Prompt that correlation between the severity of abnormal intestinalflora and NAFLD.5. The TNF-a, IL-6and LPS levels of Group D decreased after treatment with drug,while the TNF-a and LPS levels of Group B increased, and the IL-6level decreasedcompared with before. The impact of intervention on the levels of TNF-a, IL-6, LPSin the two groups showed a statistically significant difference (P=0.0120.0040026). But Group D still had a statistically significant difference with the same agedcontrol group(P<0.05).6. After the intervention, the ALT, AST and TG levels of Group B increased a littlecomapared with before, while Group D decreased significantly. The impact ofintervention in the two groups showed a statistically significant difference(P=0.0560.0130007).[Conclusion]Two weeks of MCDD induced stable NAFLD animal models the severity controllingin NAFL. Levels of endotoxin, lipids and inflammatory factors in the animal modelsignificantly elevated, related to the liver injury severity, and ALT, AST had nosignificant exaltation. NAFLD animal model had definitely intestinal dysbiosis, andsupplement of Bifico could correct it effectively, only diet recovery could not makeany sense. Drug treatment could delay the development of the disease, improve theprognosis of the animal, improve the liver lesions, effectively reduce theinflammatory cytokines and endotoxin levels, and decrease the levels of triglycerideand liver enzymes. The situation of intestinal dysbiosis was related with the diseaseseverity.