| Objective:Induced by high-fat diet with nonalcoholic fatty liver disease(NAFLD) in animal models, by observing berberine on AMPK / SIRT1 / UCP2 signaling pathway-related genes, regulation of protein from the liver tissue to explore the pathogenesis of NAFLD and small Bo alkali prevention mechanisms underlying NAFLD.Methods:The SPF 30 healthy adult male SD rats were randomly divided into three groups,namely: normal group, model group, berberine groups of 10. NAFLD build high-fat diet in rats, in addition to the normal group, other groups with high-fat diet. Modeling for drug intervention while the rats given 10 m L / kg ? BW gavage doses corresponding prescription or experimental animals purified water, administered continuously for 16 weeks. After 16 weeks, the rats fasted water 12 h, abdominal aortic blood, serum detection of TC, TG, LDL-C, HDL-C, ALT, AST routine biochemical markers to detect liver tissue TC, TG, and HE staining and oil red O staining pathological changes. ELISA method for detecting MDA, SOD, GSH-px, NO expression levels of oxidative stress factor; AMPK,SIRT1, UCP2 m RNA Q-PCR to detect the expression of liver tissue; Western Blot method to detect the liver tissue was detected in liver tissue AMPK, p-AMPK, SIRT1, UCP2 protein levels..Results:1. Histopathological observation of live(1) Oil red O stainingHepatocyte nucleuses were blue and had no orange in cytoplasm in normal group. In the model group, Hepatocytes had diffused of red lipid droplets, narrowing of hepatic sinusoidal, disorganized liver rope. The nucleuses were more pressed to one side by lipid droplets.. Berberine group showed a large slug of red stained lipid droplets, hepaticsinusoid structure cord disorder, lipid droplets red dye was significantly less than the model group.(2) HE staining. Hepatocyte nucleuses were blue, and cytoplasm had uniform red-stained, less or no adipose hollow space. The hepatic lobule and liver rope both had clear structure and regular arrangement. It found no point necrosis, soakage of inflammation cell. In the model group, the central vein and portal area appeared diffuse adipose hollow space.Hepatocytes had obvious tumefaction or enlargement, even ballooning. A great of adipose hollow space were seen in cytoplasm. Some small hollow spaces were converged to the big one. The narrow hepatic sinusoidal and disorder liver rope were observed. It is difficult to found the normal Hepatocyte. Berberine group of sinusoidal arrangement of hepatic cords, hepatocyte morphology, bullous lipid droplets, ballooning degeneration,inflammatory infiltration and punctate necrosis, to improve compared with the model group.2. Comparison of liver lipids, serum lipids, ALT, AST levels(1) Levels of TC, TG in liver tissueCompared with the normal group, the liver tissue of rats in the model group TC, TG levels were significantly higher(P <0.01); berberine intervention group TC, TG content than the untreated group had reduced to different extents, these results indicate that,NAFLD rat model serious liver fat deposition, berberine can reduce the fat content of rat liver NAFLD, improve liver lipid metabolism.(2) Levels of serum TC, TG, HDL-C, LDL-CThe levels of serum TC, TG, LDL-C were higher than that of normal group obviously(P<0.01), while HDL-C was significantly lower(P <0.01); compared with the model group, drug intervention group serum TC, TG, HDL-C, LDL-C levels have varying degrees of improvement trend. These results suggest that the presence of NAFLD rats with severe lipid metabolism, berberine can reduce rat TC NAFLD, TG, HDL-C,LDL-C content, thus improving liver lipid metabolism and fat accumulation.(3) Levels of ALT, ASTCompared with the normal group, serum AST content rats were significantly increased, the difference was statistically significant(P <0.01 Compared with the model group, the intervention group AST berberine content decreased(P <0.05). Compared with the normal group, serum ALT levels in the model group was no significant increase(P>0.05), berberine group compared with the model group was no statistically significant difference(P> 0.05). The results show that high-fat diet for 16 weeks rat hepatocytes NAFLD model severely damaged. Berberine group can be improved to varying degrees of liver damage, delaying the progression of NAFLD..3. Expression of SOD, MDA, GSH-Px,NOCompared with the normal group, model group rat liver tissue SOD, GSH-Px were significantly lower(P <0.01), and significantly increased levels of MDA,NO in rats.Compared with the model group, berberine group hepatocytes SOD, GSH-Px mean different degrees of water increased(P <0.05). Berberine intervention group were reduced MDA,NO levels in liver tissue(P <0.05). These results indicate that prompts liver tissue of rats NAFLD obvious oxidative stress phenomena, while berberine can be adjusted oxidant / antioxidant imbalance effect.4. AMPK, SIRT1, UCP2 m RNA relative expression in liver tissueCompared with the normal group AMPK, SIRT1 expression in model group than the normal group, lower(P <0.05, P <0.01); compared with the model group, AMPK,significantly increased the expression of SIRT1 berberine group(P <0.01). UCP2 expression in the model group was significantly increased compared with the normal group(P <0.01); the expression of berberine group compared with the model group down,but the difference was not statistically significant(P> 0.05). These results indicate that NAFLD rat liver tissue, AMPK, SIRT1 low expression, berberine groups can play on the expression activation; UCP2 is overexpressed in NAFLD rats, and berberine perhaps by inhibiting its expression, thus play a role in anti-NAFLD.5. The relative expression of AMPK, p-AMPK, SIRT1, UCP2 protein in liver tissuein liver tissue, protein AMPK model group than in the normal group down, the difference was not statistically significant(P> 0.05); p-AMPK, SIRT1 model group thannormal histone downregulated(P <0.05, P <0.01); AMPK, p-AMPK, compared to a marked increase in the expression of SIRT1 protein berberine group and model group(P<0.01). UCP2 protein in model group was significantly increased compared with the normal group(P <0.01); berberine group compared with the model group reduced protein expression, but the difference was not statistically significant(P> 0.05). These results suggest that NAFLD in rat liver tissue, AMPK, p-AMPK, SIRT1 low expression,berberine group can upregulate its expression; UCP2 in NAFLD rats was highly expressed and berberine perhaps through Since inhibition of the expression of protecting the liver.Conclusions:1. Rats fed high-fat diet continued 16 w SD, NAFLD can lead to pathological changes in liver tissue, can be successfully constructed an experimental model NAFLD rats.2. Compared with the normal group, model group rats AMPK NAFLD / SIRT1 / UCP2 signaling pathway activation, AMPK, SIRT1 downregulation, UCP2 upregulation induced lipid metabolism, lipid peroxidation, which may be important in the pathogenesis of NAFLD.3. Berberine can regulate liver tissue AMPK / SIRT1 / UCP2 gene and protein expression related signal pathways, improve lipid metabolism, inhibition of hepatic steatosis, adjust oxidation and antioxidant imbalance. This may be one mechanism of action of berberine anti NAFLD. |
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