| Background and ObjectiveNonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disorder charaterized with excessive lipids in hepatocytes, in addition to alcohol and other specific factors.Nowadays the incidence of NAFLD has increased rapidly and it has become one of the serious threats to the health of our people.NAFLD includes simple fatty liver (nonalcoholic fatty liver, NAFL), nonalcoholic steatohepatitis (NASH) and fatty liver cirrhosis.Among them, the existence of NASH is an important factor affecting the prognosis of patients with NAFLD.A great number of evidences suggest that the hepatocyte apoptosis plays an important role in the development of NASH.Meanwhile, endoplasmic reticulum stress (ERS) is closely related to hepatocyte apoptosis.The definite mechanism responsible for the regulation of ERS on hepatocyte apoptosis of NASH needs to be fully investigated.In previous studies, we found that the saturated fatty acid could induce hepatic steatosis, followed by hepatocyte apoptosis, which consistents with previous studies of NASH.We also found that saturated fatty acids could iuduce lipoapoptosis of hepatocytes by unfolded protein response (UPR) and non-unfolded protein response (non-UPR) signaling pathways.C/EBP-homologous protein (CHOP) and Bcl-2-associated X protein (Bax) are signaling molecules of ERS UPR and non-UPR pathways respectively in hepatocytes induced by saturated fatty acids .Recent studies have reported that phosphatidylinositide-3-OH kinase (PI3K)/protein kinase B (PKB/Akt) pathway could suppress the expression of CHOP and Bax in the ERS induced by thapsigargin [9-10]. There were few studies on whether PI3K/Akt involved in the regulation of CHOP and Bax of ERS UPR and non-UPR signaling pathways in the hepatocytes induced by saturated fatty acid.In this study, normal human hepatocytes (L02) and Hepatoma (HepG2) are induced by saturated fatty acids for establishing the steatosis model.We will observe the expressive change of PI3K/Akt signaling pathway and ERS-related molecules, in order to explore the roles of PI3K/Akt pathway in the UPR and non-UPR signaling pathways of ERS and apoptosis in hepatocytes under conditions of saturated fatty acids-induced steaosis. Methods1. Cell culture:L02 cell and HepG2 cell was respectively cultured in RPMI 1640 and DMEM containing 10% fetsal bovine serum,0.1 g/L streptomycin and 0.0625g/L penicillin.2. Goups division:there were control group (with no addition), model group (experimental group, treated with the optimal concentrated sodium palmitate) and intervention group (treated with sodium palmitate and LY294002, a PI3K/Akt inhibitor).3. MTT was used for the determination of cell viability, lipids deposition in the hepatocytes was observed by Triglycerides (TG) kits and oil red O staining.4. CCK8 was used for the cell viability.Cell apoptosis was detected by flow cytometry with Annexin V/PI double-staining.Western blot analysis was used to examine the expression of glucose regulated protein78(GRP78), PI3K, p-PI3K, Aktl, p-Akt1, CHOP and Bax.ResultsPart one1. Screening of the optimal concentration of sodium palmitate:The viability of L02 and HepG2 cells treated with various doses of sodium palmitate (54,108,126,144,162 and 216umol/L) for 24h and 48h was evaluated by MTT.The results showed that sodium palmitate with higher doses (higher than 108μmol/L) inhibited the growth of cells significantly more than that in the control group.The cells viability was less than 50% when the concentration was higher than 144μmol/L at 48h.Combining with the results of previous research we adopted 108μmol/L sodium palmitate to establish the model.2. The detection of hepatocellular lipid:(1) For the model and the control group, the TG contents of L02 cells were 50.2±3.3 vs 20.3±3.0 after 24 hours of treatment, and 67.4±3.8 vs 20.8±3.4 after 48hours of treatment, and of HepG2 cells were 42.1±3.3 vs 18.1±2.7 after 24 hours of treatment, and 64.6±2.9 vs 20.4±2.8 after 48 hours of treatment.The TG contents in sodium palmitate-induced hepatocytes gradually increased with significantly defferences (P<0.05).(2) Oil Red O staining:Compared with thre control group, much more visible orange lipids accumulatiosns and increased integration were observed in hepatocytes of the model group.3. The detection of apoptosis ratio of hepatocytes:Flow cytometry with Annexin V/PI double-staining analysis showed that, for the control and the model group, the apoptosis ratios of L02 cells were 4.41±0.78 vs 6.01±1.49 after 24 hours of treatment, and 12.56±2.78 vs 29.72±6.39 after 48 hours of treatment, and of HepG2 cells were 11.16±1.15 vs 17.50±6.83 after 24 hours of treatmen, and 22.37±1.24 vs 33.85±5.79 for 48 hours of treatment.The apoptosis ratios significantly increased in the model group compared with the control group at 48h (P<0.05).4. Western blot analysis of the relative protein expression levels of GRP78, p-PI3K, p-Aktl, CHOP and Bax:(1) The GRP78 expression gradually increased in a time-dependent manner in L02 cells induced by sodium palmitate(0,12,24,48h). While in HepG2 cells, it reached a peak at 24h and decline slightly 24h after treatment with sodium palmitate (P<0.05).(2) The expression of p-PI3K and p-Aktl protein in HepG2 and L02 cells induced by sodium palmitate(0,12,24,48h) increased in a time-dependent manner within 24h and decreased after then (P<0.05).In contrast, the expression of PI3K and Aktl made no difference (P> 0.05).(3) The data shows that the levels of CHOP and Bax protein increased in a time-dependent manner in L02 and HepG2 cells treated by sodium palmitate for 0,12,24,48h (P<0.05). Part two1. Screening of the optimal concentration of LY294002:The hepatocytes were pretreated with different concentration of LY294002CL02 cells:0.10,0.25,0.50,1.00,2.00μmol/L; HepG2 cells:4,5,6,8, 10μmol/L) before treated with sodium palmitate for 24h or 48h.CCK8 was used to detect the cell viability.Combining the results of Western blot with the related study, the intervention concentrations of LY294002 were 0.50μmol/L (L02 cells) and 5μmol/L (HepG2 cells) for the following experiments.2. The detection of apoptosis ratio of hepatocytes:The results of flow cytometry with Annexin V/PI double-staining demonstrated that, for the control, model and intervention group, the apoptosis ratios of L02 cells were 4.41±0.78 vs 6.01±1.49 vs 19.50±2.53 after 24 hours of treatmend, and 12.56±2.78 vs 29.72±6.39 vs 47.60±4.17 after 48 hours of treatment, and of HepG2 cells were 11.16±1.15 vs 18.17±3.16 vs 30.41±3.62 after 24 hours of treatmend, and 22.37±1.24 vs 33.85±1.20 vs 58.56±4.21 after 48 hours of treatment.The apoptosis ratios was significantly increased in the intervention group compared with the model group at 24h and 48h (P <0.05).3. Western blot analysis of the relative protein expression levels of GRP78, p-PI3K, p-Aktl, CHOP and Bax:(1) The expression of p-PI3K and p-Aktl protein increased in a time-dependent manner within 24h and decreased after then in intervention group, which statistically decreased compared with those of experimental group at corresponding time (P<0.05).The expression of PI3K and Aktl protein among control, model and intervention group made no difference (P>0.05).(2) The GRP78 expression gradually increased in a time-dependent manner in L02 cells of intervention group.While it reached a peak at 24h and decline slightly then in HepG2 cells.However, that in the two intervention groups were statistically greater than that in model groups at corresponding time (P<0.05).(3) The levels of CHOP and Bax protein increased in a time-dependent manner in L02 and HepG2 cells of each group.CHOP and Bax protein expression in the intervention group increased comparing with model group at corresponding time respectively (P<0.05).Conclusion1. Saturated fatty acid induces the lipoapoptosis as well as activates PI3K/Akt pathway of L02 and HepG2 cells.2. PI3K/Akt pathway may be involed in the regulation of the UPR and non-UPR signaling pathways of endoplasmic reticulum stress and may promote apoptosis of hepatocytes by enhancing the expression of CHOP and Bax protein in saturated fatty acid-induced steatotic hepatocytes. |
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