| Objective:By observing the changes of Liraglutide intervention in the treatment of diabetes mellitus(DM) merger of nonalcoholic fatty liver disease(NAFLD) rat liver cell autophagy related markers LC3 ? m RNA and protein expression, and then observing the change of liver steatosis, and at the same time compared with adding chloroquine uesd blocking autophagy body fusion with lysosomes inhibit autophagy,then comparing the indicators of change, to explore the relationship between autophagy and liver lipid deposition and the effect of Liraglutide on fatty degeneration of liver cell by regulating autophagy.Methods:1.Diabetes mellitus merger of nonalcoholic fatty liver disease model preparation: 44 only 4 ~ 5 weeks, weight in l80 g ~ 200 g wistar male rats, adaptive feed l weeks, 6 per cage.44 rats serial numbe 1, 2,..., 44, and do the size, shape, number of the same sign, write down the number 44 respectively, will sign these Numbers together, stir evenly, then continuous extraction of no. 10, with the number and sign Numbers corresponding to the set to 10 rats in group A, the remaining 34 rats were set as group B. A group rats fed regular feed 13.4 k J/g, in which fat provides the heat accounts for 10.2%, protein 23.3%,carbohydrate 66.5%; B group rats fed a high sugar, high fat feed 21.8 k J/g, which provides the heat accounts for 56.0%, fat protein accounted for 7.0%, carbohydrate 37.0%. Two groups of rats were free drinking water, daily in the morning and hurl food once.In12 weeks taking blood from tail,measuring FBG of rats in both groups, group A rats FBG4.4-4.9 mmol/L, group B rats FBG5.3-5.9mmol/L. Aats in group B were injected chain urea with cephalosporins(STZ) 25 mg/kg, A group of rats were injected with equalvolume of saline.After72 h cut tail blood glucose meter fast measurement of FBG ?7.8 as building success as diabetes.FBG of group A rats is in the normal range, group B 1 rat death, the rest are successful. From group B rats, randomly take 8 rats executed and take line HE staining, the liver tissue were observed under light lobular?30% of the hepatocyte fatty change occurs, and given priority to big bubble sex fatty change is identified as NAFLD building successfully, all successful.2.Divided into gtoups and test index: The rats of group A is set to normal glucose tolerance group(NGT group, n = 10), the rats of group B were randomly divided into three groups: lalu peptide intervention group(LIR group, n = 8), to the subcutaneous injection with lalu peptide, 100 ug/Kg, the Bid; The lalu peptide + autophagy inhibitor Chloroquine(Chloroquine, CQ) group(LIR + CQ group, n = 8), to the subcutaneous injection with the lalu peptide, 100 ug/Kg, the Bid and Chloroquine, 50 mg/Kg, once every two days. DM with NAFLD group(DM/NAFLD group, n = 9), NGT and DM/NAFLD group to the same volume of physiological saline, ntervention 4 weeks totally. Injecting20% urethane anesthesia, open, take abdominal aortic blood, centrifugal, in the 4 ?refrigerator used the separation of serum with biochemical methods measuring TG, TC,HDL, LDL, ALT, AST. Take liver tissue weighting, calculate the liver index; Visceral fat,calculate the relative contents of visceral fat(VF/W); Cut another take 100 mg of liver tissue cryopreserved tubes, after put into liquid nitrogen preservation, for PCR method to detect LC3 ? m RNA. On the edge of the right liver lobe from 5 mm take 1 cm× 1 cm ×0.5cm liver tissue block after 48 hours soaked in 10% formalin fixed paraffin embedding,prepared to do pathological, used in the immunohistochemical method to detect LC3 ?protein and hepatocyte fatty degeneration observed under optical microscope.Using SPSS13.0 statistical software analysis, measurement data with mean + /-standard deviation(`x±s) said, between each two comparison using single factor analysis of variance(ANOVA), LSD test, P? 0.05 or less for the difference was statistically significant.Results:1.General situation : At the period of adaptability feeding, each rat has no difference inthe diet content and weight, has good mental state, the colour luster. During starting building moeld the group B(high sugar and high fat feed) rats compared with group A(normal feed) diet content, weight of group B rats increased, less activity. After the success of building moeld, during the period of drug intervention, NGT group rats has no change in ateing than before, weight increase steadily, has good mental state, color luster.DM/NAFLD rats compared with the same period NGT rats,DM/NAFLD group weight gain, less move; LIR group rats compared with the same period DM/NAFLD rats diet content, weight decreased, hasgood mental state.LIR + CQ group rats depression, body hair fleeciness and lose luster, have depilation, compared with DM group/NAFLD rats, diet content, weight decreased, there was no difference in the diet content, weight compared with the same period LIR group rats.2.Changes of hepatocyte fatty degeneration of each rat and comparition of index:HE dyeing shows liver tissue of NGT group complete structure, clear and has normal lobular architecture, cytoplasm has a small amount of lipid droplets. Liver cell volume of DM/NAFLD group rats was obviously edema, can be seen in the cytoplasm of lipid droplets size, nuclei were pushed aside. LIR group rats structure of hepatic lobule is clear,cell order, only a small amount of lipid droplets in the cell. LIR + CQ group rats has normal lobular architecture disappear, liver cells also increased obviously, the liver cell is covered by lipid drops.Compared with NGT group, DM/NAFLD and LIR + CQ group liver wet weight, liver index, VF/W increased, there was statistically significant difference( P < 0.05),there was no statistically significant difference compared with LIR group(P > 0.05).Compared with DM/NAFLD group, LIR group liver wet weight, liver index, VF/W fell(P < 0.05), LIR + CQ group liver wet weight and liver index increases(P < 0.05), the difference of VF/W has no statistical significance(P > 0.05). The liver wet weight, liver index, VF/W of LIR + CQgroup is higher than LIR group(P < 0.05).3.Comparition of the liver LC3?m RNA, LC3?protein:Compared with NGT group,DM/NAFLDand L IR group LC3 ? m RNA, LC3 ? protein expression increase, there was statistically significant difference(P < 0.05). Compared with DM /NAFLDgroup, LC3 ?m RNA, LC3 ? protein expression of L IR group increased(P < 0.05), LIR + CQ groupLC3 ? m RNA, LC3 ? protein expression decreased(P < 0.05). LIR + CQ compared with L IR group, LC3 ? m RNA, LC3 ? protein expression of L IR+ CQ group decreased(P <0.05).4.The biochemical index changes :Compared with NGT group, DM/NAFLD and LIR+CQ group FBG, TG, TC, LDL,ALT,AST increased,HDL decreased, there was statistically significant difference(P < 0.05), there was no statistically significant difference compared with LIR group(P > 0.05). Compared with DM /NAFLD group, TG, TC, LDL,ALT,AST,of LIR group decreased, HDL increased(P < 0.05), FBG, TG, TC, LDL,ALT of LIR+ CQ group increased, HDL decreased, the difference was statistically significant(P <0.05). FBG, TG, TC, LDL,ALT,ASTof LIR+ CQ group is higher than LIR group, HDL decreased(P < 0.05). |
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