| Objective To investigate the effect as well as the potential mechanism of high glucose on the induction of endoplasmic reticulum stress (ERS) and apoptosis in HepG2 cells, and also to explore the protective effect of melatonin and sitagliptin on the liver of T2DM rats with high-fat diet. Methods HepG2 cells exposed to high glucose as the cell model. Hoechst 33258 staining was used to detect cell apoptosis. Caspase-3 activity assay was performed to reveal the catalytic activity of caspase-3. Western blot was to measure the expression levels of apoptosis-, ER stress-, oxidative stress-associated proteins (procaspase-9, GRP78, CHOP, PERK and p47phox) and MAPKs. To further characterize the molecular mechanisms, the influence of antioxidant alpha-lipoic acid (ALA) and selective inhibitors for JNK and p38 (SP600125 and SB203580, respectively) were examined by Hoechst 33258 staining, western blot and immunofluorescence. The model of type 2 diabetes. Thirty-seven male SD rats of SPF level were randomly distributed into 6 groups:normal control (NC) group, high fat (HF) group, type 2 diabetes mellitus (T2DM) group, insulin (INS) group, melatonin (MEL) group and sitagliptin (SIT) group. The model of T2DM rats was set up by feeding of high-fat food combining intraperitoneal injection of STZ (27 mg/kg) and fasting blood glucose (FBG) was measured to decide the rat model of T2DM was successfully established. Then insulin 4 U/(kg·d), melatonin 10 mg/(kg·d) and sitagliptinl 0 mg/(kg-d) were given to the rats of INS, MEL, and SIT group respectively for 12 weeks. Then FBG, fasting serum insulin (FINS) and other related parameters were measured; the hepatic pathology changes of rats were observed with HE staining; the expression level of caspase-3 was detected by IHC; the levels of ERS-associated proteins and melatonin receptors were measured with western blot. Results After HepG2 cells were treated with high glucose for 48 h, the apoptosis of HepG2 cells, the activity of caspase-3 and the degradation of procaspase-9 were all presented an concentration-dependent enhancement. Western blot showed a decrease in GRP78 expression, increases in CHOP and p47phox as well as enhancements of ASK1 expression and p38 and JNK phosphorylation. In the case of the contributions of oxidative stress and the MAPK signaling pathways, all ALA, SP600125 and SB203580 were able to largely reverse high glucose-triggered apoptosis, the decreased level of Bcl-2, the increased level of Griml9 and the expression and sub-localization to the nuclei of CHOP. The rat model of T2DM was established successfully, with evidences of increased FBG ((16.91±1.79) mM) and decreased lnISI (-6.60±0.21) indicating occurrence of insulin resistance (IR). HE staining displayed the pathology changes of liver being existed in both HF and T2DM rats, which was more serious in diabetic rats. IHC showed an elevation in expression and translocation to the nuclei of caspase-3 in diabetic liver. Western blot revealed that CHOP was up-regulated while GRP78 was down-regulated notably in the liver of both HF and T2DM and the change was more significant in diabetic rats. Neither antioxidant Mel nor SIT (DPP-4 inhibitor) could less the serum concentrations of TG, Tch, LDL-c, however, both of them were able to decrease FINS and increase ISI, and to reverse the pathology changes of liver, the up-regulation of CHOP and caspase-3 as well as the down-regulation of GRP78 in T2DM rats. Conclusions Apoptotic events were induced by high concentration of glucose in HepG2 cells, which may be mediated by the activation of ASK1-p38/JNK pathway via ERS and oxidative stress and the modulation of its downstream apoptosis-associated genes. Mel and SIT could protect the hepatocyte of T2DM rats from glucotoxicity and lipotoxicity probably by reducing IR and alleviating ERS and apoptosis in the diabetic liver. |
