| Objective1. To investigate the relationship between the plasma visfatin and type 2 diabetes mellitus(T2DM) with nonalcoholic fatty liver disease(NAFLD). Provid an experiment basis and theory evidence for the diagnosis and therapy of NAFLD.2. Cultivate normal human hepatocyte in vitro. Observe visfatin on the proliferation of normal human hepatocyte. Study the mechanism of the action of visfatin on nomal human hepotocye. Providing a primary theoretical basis for the clinical therapy of visfatin related effect, such as fall of blood glucose, release insulin resistance and improvement of nonalcoholic fatty liver disease.Methods1. The level of plasma visfatin were assayed by enzyme-linked immunosorbent asssy(ELISA) in all subjects, patients of T2DM with NAFLD(group A); T2DM without NAFLD(group B) and nomal control group(group NC). Blood glucose, blood lipids, glycosylated homeostasis(HbAlc), fasting insulin(FIns) were also assayed in all objects. Waist-to-hip(WHR), bosy mass index(BMI) and homeostasis model assessment for insulin resistance(HOMA-IR) were calculationed by correspondence formula. The relationship was analyzed between plasma visfatin and other indexes; 2. Cultivate normal human hepatocyte HL-7702 cell line in vitro, then do morphological observation through inverted microscope; 3. The cultivate normal human hepaocyte is randomly divided into five groups:control group, visfatin (50ng/mL) group, visfatin (100ng/mL) group, visfatin (250ng/mL) group, visfatin (500ng/mL) group, and give them intervention respectively in 24 and 48 hours. Observe the morphological changes of hepatocyte in each group.Then use MTT to detect the effects of visfatin on the number of survival and vitality of the normal human hepatocyte,and to check whether there is different effect on the growth of normal human hepatocyte by use visfatin of different concentrations; 4. The cultivate normal human hepaocyte is randomly divided into five groups:control group, visfatin (50ng/mL) group, visfatin (100ng/mL) group, visfatin (250ng/mL) group, visfatin (500ng/mL) group, and give them intervention respectively in 1 hour,6 hours,12 hours,24 hours,48 hours. Use ELISA to measure the expressions of phosphatidylinositol 3 kinase (PI3K), phosphate adenosine monphosphate activated protein kinase (pAMPK), phosphoenolpyruvate carboxykinase (PEPCK), acetyl coa carboxylase (ACC).Results1. The level of systolic blood pressure, weight, BMI, waist circumference, WHR, TC, TG, LDL, HDL, HbAlc didn't have statistics difference in group A and B, but they were higher compared with the group NC(P<0.01); 2. The correlation analysis showed that the level of plasma visfatin was positively correlated with HOMA-IR, Fins,2 h postprandial blood glucose (P2hBG), BMI, LDL in group A and group B (P＜0.05), meanwhile, it was negatively correlated with HDL(P<0.05). Logistic regression analysis showed that visfatin and FBG were the factors that can influence the NAFLD. Through the ROC curve we get the cut off value of visfatin for predictioning NAFLD, the sensitivity was 98.2%, the specificity was 39.1%, the value of OR was 36.84; 3. The normal human hepatocytes were well subcultured. The growth velocity of hepatocyte of visfatin groups were not faster than control group. The result of MTT showed that the OD values of visfatin groups were not have significant difference with control group; this indicated that visfatin can not accelerate the proliferation of normal human hepatocyte; 4. The results of PI3K, pAMPK, PEPCK, ACC:(1) Concentration trends:visfatin can increase the expression of PI3K, pAMPK, reduce the expression of PEPCK. After higher concentration visfatin invention, the expression of ACC did not have difference with control group. While the lower concentration visfatin increased the expression of ACC. (2) Time trends:The expression of PI3K and pAMPK had consistent trend: increased in lh, decreased in 6h, then increased again until 24h, down again in 48h. The expression of PEPCK was opposite with PI3K and pAMPK. All intervention times, the expression of PEPCK were significant decreased compaired with Ctrl. The expression of ACC due to the different intervention levels showed different trends. 50ng/ml visfatin intervention group, 100ng/ml visfatin intervention group at all time points are to promote the expression of ACC. While the expression of ACC in 250ng/ml visfatin intervention group and 500ng/ml visfatin intervention group over time to fluctuations in the control group as baseline. Conclusion1. Through assay the plasma vifatin level in T2DM, T2DM with NAFLD and nomal person, we discovered that the plasma visfatin were higher inT2DM, highest in T2DM with NAFLD; 2. This study confirmed in vitro that visfatin may act directly on normal human hepatocyte and concentration-dependent increase the expression of pAMPK and PI3K, decreased expression of PEPCK, stimulate the expression of ACC after lower concentration intervention. The expression of PI3K, pAMPK, PEPCK, ACC all changes with times prolong. But the overall trend is still PI3K, pAMPK, ACC increased expression, PEPCK was decreased. That visfatin may be through PI3K, AMPK reduced gluconeogenesis pathway to increase fatty acid synthesis, and then adjust the liver glucose and lipid metabolism. |
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