| Objective:1. To research the effect of invigorating spleen and soothing liver prescriptions on lipid metabolism of the livers of rats with non-alcoholic fatty liver disease (NAFLD).2. To study the mechanism of the method of invigorating spleen and soothing liver and relevant prescriptions in treating NAFLD by observing the effect of invigorating spleen and soothing liver prescriptions on AMPK protein's activity of NAFLD rats'liver tissues.3. To enrich the Traditional Chinese Medicine's knowledge on the etiology and pathogenesis of NAFLD.Methods:1. Grouping:55 SD rats were randomly divided into five groups:normal group(group A), model group (group B), soothing liver group (Bupleurum Liver-Soothing Powder group, also named group C), invigorating spleen group (Shenling Baizhu Powder group, also named group D), integrated Group (Bupleurum Liver-Soothing Powder and Shenling Baizhu Powder group, also named group E). There were 15 rats in group B and 10 in the other 4 groups.2. Modelling and Drug Administration:Group A were fed with general feed, others with general feed and high-fat emulsion (10mL/kg.BW) once a day, meanwhile, group C, D and E were perfused with corresponding prescriptions and group A and B with distilled water. At the end of the 8th week, two rats were killed to examine the pathologic change to verify the success of modeling.3. Indexes testing:At the end of the 8th week, the content of TC, TG in rats'liver tissues and TC, TG, AST, ALT, FFA and insulin in serum were tested; the changes of pathogenesis of liver tissues were observed by H&E staining and Oil red O staining. The expression of AMPK in the liver tissue was detected by Western Blot.Results:1. Liver functionCompared with group A, serum ALT, AST of group B increased significantly (P<0.05 or P<0.01). Compared with group B, serum AST of group C, D and E all decreased significantly (P<0.01). Compared with group A, the ratio of AST to ALT of group B had significantly increased (P<0.01). Compared with group B, the ratio of AST to ALT of group C, D and E all decreased (P<0.01). The results above showed that the prescriptions of invigorating spleen and soothing liver could reduce liver damage.2. Serum lipidsCompared with group A, serum TC and TG significantly increased (P<0.05). Compared with group B, serum TC (P<0.01) and TG (P<0.05) of group C, D and E all decreased, and TC and TG group C were the lowest.3. Hepatic fatCompared with group A, TC (P<0.01) and TG (P<0.05) content of liver tissues in group B significantly increased. Compared with group B, the TC content of group C, D and E all remarkably decreased(P<0.05), and the TG content of group D and group C decreased(P<0.05). It showed that the TC and TG content in liver tissues of NAFLD rats perfuse with high fat emulsion. The prescriptions of soothing liver and invigorating spleen worked well in lipid-lowering, which could improve lipid metabolism of rats'liver tissues. 4. FFACompared with group A, the FFA content of serum and liver tissues significantly increased (P<0.01). Compared with group B, the FFA content of serum of group C significantly decreased, and the FFA content of liver tissues of group C, D and E all decreased(P<0.01 or P <0.05).5. InsulinCompared with group A, the insulin content of group B increased significantly (P<0.01). Compared with group B, serum insulin of group C and group D decreased significantly(P<0.01).6. Pathological observingOf the normal group, the rats had integrated structure of liver tissues, clear structure of hepatic lobules, neatly arranged cells and no obvious lesions. Of the model group, the rats'liver cells had fattily degenerated, cells arranged disorderly, liver cells swelled and rounded and enlarged volume, there were fat vacuoles with different sizes in cytoplasm, cell nuclei pressed against the membrane in some cells, and there were a little inflammatory cell infiltration. The drug intervention groups all improved and appeared low-grade fatty degeneration. The effect of group C was the most significant. Oil red O staining:there were a few orange lipid droplets in normal group, but revealed numerous large intracellular lipid droplets in model group, even has the part of lipid droplets to fuse chain. In the each treatment group, lipid droplets were significantly decreased compared with model group. It showed that the modeling of NAFLD was succeeded and the prescriptions of soothing liver and invigorating spleen could improve fatty degeneration of NAFLD rats'liver cells, reduce inflammation.7. The expression of AMPKThere was no obvious difference of total level of the expression of AMPK. Compared with group A, the expression of pAMPK protein of group B decreased significantly (P<0.01). Compared with group B, the expression of pAMPK protein of group C and D increased significantly (P<0.01 or P<0.05). It showed that the mechanism of improving the fatty degeneration of rats'liver tissues with prescriptions of invigorating spleen and soothing liver might be relevant to the expression of pAMPK protein.Conclusions:1. NAFLD rat model could be successfully established by perfusing high-fat emulsion.2. High-fat diet is a major cause of NAFLD, and the dysfunction of liver and spleen may be the main pathogenesis of NAFLD.3. The prescriptions of invigorating spleen and soothing liver can adjust the content of NAFLD rats'blood lipid, liver function, and liver fatty, improve lipid metabolic disturbance and fatty degeneration of liver tissues, reduce livers damage, and have good effect against NAFLD.4. The expression of pAMPK of liver tissues may be one of the mechanisms of the formation of NAFLD. The mechanism of the prescriptions of soothing liver and invigorating spleen treating NAFLD may be relevant to the increasing of the expression of pAMPK protein of liver tissues. AMPK may be an important target to prevent and treat NAFLD with prescriptions of soothing liver and invigorating spleen.
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