| When lipid metabolic balance is destroyed, a large amount of lipid deposition will appear in liver and result in hepatic adipose infiltration. Sirtl plays an important role in regulation of lipid metabolism, but the specific regulatory mechanism is not clear; PI3K-Akt-mTOR signaling pathway is involved in regulation of lipid and glucose metabolism, and its specific mechanism is also a the hot spot in recent studies; At present, there are few reports indicate whether there is an interaction'between Sirtl and PI3K-Akt-mTOR pathway in lipid regulation. So goose primary hepatocytes were isolated and cultured in vitro; After cultured 12h in serum-free medium, hepatocytes were incubated for 24 h in either no addition as a control or 50,100,250 and 500 ?mol/L of nicotinamide; To choose the optimal concentration of nicotinamide and reveal the change of lipid metabolism when Sirtl was inhibited, the expressiom of Sirtl and the key factor of lipid metabolism were determined; Then, different PI3K-Akt-mTOR signaling pathway inhibitors LY294002, NVP-BEZ235, Rapamycin were added in Sirtl-inhibited cell model. The expression of genes involved in lipid metabolism and PI3K-Akt-mTOR signaling pathway were determined by RT-PCR, Elisa kit and Western blot; Lipid droplets were determined by oil red O staining method; Cell vitality was determined by CCK-8 kit. At last, according to the results of this study, regulating mechanism of Sirtl involved in lipid deposition via PI3K-Akt-mTOR pathway was analyzed.The main results:1. The mRNA and protein expression levels of Sirtl were decreased as a concentration-dependent issue in different concentrations of nicotinamide treatments in goose primary hepatocytes; when nicotinamide concentration was 100?mol/L, protein concentration of Sirtl was 51.50% of the control group:2. Different concentrations of nicotinamide treated goose primary hepatocytes could promote fatty acid synthesis key factor SREBP1, FAS, ACCa expression (P<0.05): inhibit expression of fatty acid oxidation key factor PPARa. PPARy, ACOX1, CPT1 (P<0.05); reduced expression levels of lipid transit critical factor FoxO1, MTTP. DGAT1, DGAT2 (P<0.05); nicotinamide treatment could also increase intracellular TG levels (P<0.05), reduce VLDL and extracellular TG levels (P<0.05), induced intracellular lipid droplets enlarged and lipid content increased (P<0.05), result in lipid deposition;3. Added 100?ol/L Nicotinamide in goose primary hepatocytes, the expression of PI3K was inhibited (P<0.05). mTOR expression was upregulated (P<0.05), expression of Akt1 showed a no significant change (P>0.05); when added PI3K-Akt-mTOR pathway inhibitor LY294002, expression of mTOR and Aktl were downregulated, however, the expression of PI3K showed a no significant difference compared with 100?mol/L nicotinamide treatment alone; when added pathway inhibitor Rapamycin, expression of mTOR and Akt were decreased, while PI3K significantly higher than Nicotinamide individual treatment group (P<0.05); when added NVP-BEZ235. Akt showed no significant change compared with control group, mTOR was lowered than control group, expression of PI3K had no significant difference compared with Nicotinamide treatment alone. When add NVP-BEZ235 inhibitors, Sirt1 showed a no significant difference compared with Nicotinamide individual treatment group, but when added the two other pathway inhibitors, expression of Sirtl was significantly reduced (P<0.05);4. The optimum concentration 100?mol/L of Nicotinamide treated goose primary hepatocytes, inhibited the expression of Sirtl, leading to fatty acid synthesis key factor expression increased; when added pathway inhibitor LY29002. NVP-BEZ235 and Rapamycin, the fatty acid synthetic key factor expression was significantly reduced (P<0.05). and the effect of NVP-BEZ235 was strongest:5. Pathway inhibitor LY294002 or NVP-BEZ235 added in Sirtl-inhibited cell model, could reduce the inhibition effects of Nicotinamide on expression of the key factors involved in fatty acid oxidation, and the effect of NVP-BEZ235 was strongest:but protein expression of CPT1 showed that the reduced-effect of LY294002 was strongest(P<0.05);6. Lipid transport key factors were inhibited in 100?mol/L Nicotinamide group, when added inhibitor LY294002, NVP-BEZ235 and Rapamycin, the lipid transport key factor expression were further reduced; however, mRNA expression levels of DGATl was significant raised when added LY294002 (P<0.05); the mRNA expression and protein concentration of FoxO1 were significantly increased when added NVP-BEZ235 (P<0.05);7. 100?mol/L of Nicotinamide could significantly increase the activity of hepatocytes, the content of intracellular TG, but reduce the content of VLDL and extracellular TG; when added pathway inhibitor LY294002, NVP-BEZ235, the content of intracellular TG was reduced, but the content of VLDL and extracellular TG were significantly higher than Nicotinamide separate treatment group (P<0.05); however, when added pathway inhibitor Rapamycin, the content of VLDL and extracellular TG achieved the minimum content; Oil red 0 staining found that after adding pathway inhibitors, lipid droplets in hepatocytes became smaller and lipid content was reduced (P<0.05), lipid deposition of hepatocytes was weakened;... |
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