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Objective To investigate the effect of inhibition of ghrelin O-acyltransferase (GOAT) by small interfering RNA (siRNA) on hepatocyte fatty degeneration and related mechanism of action.Methods Human LO2 hepatocytes were treated with free fatty acid (FFA)to induce hepatocyte fatty degeneration.LO2 hepatocytes were treated with FFA and siRNA-GOAT alone or in combination and then divided into normal control (NC) group (treated with phosphate buffered saline alone),siRNA-GOAT group (treated with siRNA-GOAT at a final concentration of 10 nm),FFA group (treated with FFA at a final concentration of 1 mm),and FFA + siRNA-GOAT group (treated with FFA at a final concentration of 1 mm and siRNA-GOAT at a final concentration of 10 nm).Oil red O staining was performed for hepatocytes to identify lipid droplets;the triglyceride (TG) test kit was used to measure the lipid level in LO2 hepatocytes;Western blot,qRT-PCR,immunofluorescent staining,and electron microscopy were used to measure autophagy;ELISA and RT-PCR were used to measure the levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6);ELISA was used to measure the changes in the levels of mammalian target of rapamycin (mTOR),phosphorylated mTOR (p-mTOR),AMP-activated protein kinase (AMPK),and phosphorylated AMPK.A one-way analysis of variance was used for comparison between multiple groups,and the least significant difference t-test was used for further comparison between any two groups.Results Compared with the FFA group,the FFA + siRNA-GOAT group had a significant reduction in the formation of lipid droplets and a significantly lower TG level (P ＜0.001).Compared with the FFA group,the FFA + siRNA-GOAT group had significant reductions in the protein and mRNA expression of TNFα and IL-6 (all P ＜ 0.005).The siRNA + GOAT group had significantly higher mRNA expression of LC3-Ⅱ and Beclin-1 than the NC group (all P ＜0.001).The FFA + siRNA-GOAT group had significantly higher mRNA expression of LC3-Ⅱ and Beclin-1 than the FFA group (all P ＜0.001).The siRNA + GOAT group had significantly higher protein expression of LC3-Ⅱ and Beclin-1 than the NC group (all P ＜ 0.05).The FFA + siRNA-GOAT group had significantly higher protein expression of LC3-Ⅱ and Beclin-1 than the FFA group (all P ＜ 0.05).Immunofluorescent staining showed that compared with the FFA group and the siRNA-GOAT group,the FFA + siRNA-GOAT group had a significant increase in the expression of endogenous LC3-Ⅱ in LO2 hepatocytes.Electron microscopy showed that compared with the FFA group,the FFA + siRNA-GOAT group had a significant increase in the expression of autophagosome.After the LO2 hepatocytes were treated by autophagy inhibitors siRNA-ATG5 and 3-MA or an autophagy stimulant,rapamycin,there was a significant difference in TG level between the FFA + siRNA-ATG5 group and the FFA + siRNA-GOAT group (P ＜ 0.001),as well as between the FFA + 3-MA group and the FFA + rapamycin group (P ＜ 0.001).The FFA + siRNA-GOAT group had a significantly higher level of LC3-Ⅰ/Ⅱ than the FFA + siRNA-ATG5 group (P ＜0.05),and the FFA + rapamycin group had a significantly higher level of LC3-Ⅰ/Ⅱ than the FFA + 3-MA group (P ＜ 0.05).Compared with the FFA group,the FFA + siRNA-GOAT group had significantly higher protein expression of p-AMPK (P ＜ 0.05) and significantly lower protein expression of p-rmTOR (P ＜ 0.05).Conclusion GOAT inhibition by siRNA can upregnlate autophagy and alleviate hepatocyte fatty degeneration,possibly by regulating the AMPK/mTOR pathway.
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Background:Prostaglandin E2(PGE2 )could promote the proliferation of tumor cells,microRNA-21(miR-21)could inhibit the proliferation of tumor cells,but its signal pathway is still unclear. Aims:To investigate the mechanism of PGE2 on promoting proliferation of gastric cancer cells potentially mediated by miR-21. Methods:Gastric cancer AGS cells were cultured and divided into control group,PGE2 group,anti-miR-21 group and PGE2 + anti-miR-21 group. Cell proliferation was determined by WST-1 chromatometry. Cell apoptosis rate was detected by flow cytometry. The expression of miR-21 mRNA was detected by RT-PCR. After AGS cells were intervened by Akt specific inhibitor perifosine,cell proliferation was assessed,and expression of PTEN/ Akt protein was detected by Western blotting. Results:Compared with control group, survival rate of AGS cells was significantly increased(P < 0. 05),apoptosis rate was significantly decreased(P < 0. 05), and expression of miR-21 mRNA was significantly increased in PGE2 group(P <0. 05). Compared with PGE2 group, survival rate of AGS cells was significantly decreased(P < 0. 05),apoptosis rate was significantly increased(P < 0. 05), and expression of miR-21 mRNA was significantly decreased in anti-miR-21 group and PGE2 + anti-miR-21 group(P <0. 05). After intervention with perifosine,survival rate of AGS cells was significantly decreased(P < 0. 05),apoptosis rate was significantly increased(P < 0. 05),expression of PTEN protein was significantly increased,and expression of p-Akt protein was significantly decreased. Conclusions:MiR-21 mediates the promoting of proliferation of gastric cancer cells by PGE2 through PTEN/ Akt pathway,which might become a new target for the prevention and treatment of gastric cancer.
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Objective To observe the effect of ghrelin on the expression of procollagen type Ⅰ and alpha smooth muscle actin (α-SMA) synthesis in vitro cultured human hepatic stellate cell (HSC-LX2) stimulated by Platelet-derived growth factor-BB (PDGF-BB).Besides,the effect of PI3K-AKT pathway was studied.Methods Cultured LX2 were intervented and jointing intervented according to the different ghrelin concentration by ghrelin and PDGF:control group,0.1 μmol/L Ghrelin group,10 μg/L PDGF group,0.05 μmol/L Ghrelin +10 μg/L PDGF group,0.1 μmol/L Ghrelin + 10 μg/L PDGF group,0.15 μmol/L Ghrelin + 10 μg/L PDGF group.Culture HSC-LX2 in vitro,joint intervention cells with different concentrations.Procollagen Ⅰ mRNA expression were detected by Polymerase chain reaction (PCR),besides,α-SMA and AKT expression were detected by Western blot in each groups.After treatment by PI3K specific inhibitor LY294002 in LX2,three groups were divided into PDGF,Ghrelin + PDGF and LY294002 + Ghrelin +PDGF.Procollagen Ⅰ mRNA expression were detected by PCR,and α-SMA was detected by Western blot.Results PCR results showed that procollagen Ⅰ expression in PDGF treated group was significantly higher than the control group ((6.91 ± 0.46) vs.(1.00 ± 0.08),P ＜ 0.05),so PDGF can promote the expression of procollagen type Ⅰ.Procollagen Ⅰ mRNA expression between ghrelin group(0.60±0.13) and blank control group had no significant change(P＞0.05).Procollagen Ⅰ mRNA expression between different concentrations of Ghrelin and PDGF (3.11 ± 0.28,2.03 ±0.23,0.70 ± 0.06) was significantly reduced than PDGF group.The difference was statistically significant (P ＜0.05),and with the increase of the ghrelin concentration of the inhibition,the effect was more obvious in a concentration dependent manner.Western blot showed that α-SMA expression was lower in Ghrelin +PDGF group than PDGF group.AKT expression was higher in Ghrelin +PDGF group than PDGF group,indicating that PI3K-AKT may participate in the anti-fibrosis effect of ghrelin in LX2.After treatment of PI3K specific inhibitor,procollagen Ⅰ expression in LY294002+Ghrelin +PDGF group was significantly higher than Ghrelin +PDGF group((4.13±0.21) vs.(2.34±0.25),P＜0.05).Western blot also showed that α-SMA expression was higher in LY294002 + Ghrelin + PDGF group than Ghrelin + PDGF group.It was suggested that after inhibitation of PI3K,the anti-fibrosis effect of ghrelin in LX2 was attenuated.Conclusion After stimulated by PDGF in hepatic stellate cell,ghrelin can inhibit procollagen type Ⅰ and alpha-SMA synthesis in the process of hepatic fibrosis via PI3K-AKT pathway,thus,ghrelin may become one of the new ways of prevention and treatment of liver fibrosis.