Liraglutide prevents beta-amyloid-induced neurotoxicity in SH-SY5Y cells via a PI3K-dependent signaling pathway.

OBJECTIVES: The aim of the study was to investigate the effects of the GLP-1 analog liraglutide on beta-amyloid (Aß)-induced neurotoxicity in the human neuroblastoma cell line SH-SY5Y and study the underlying mechanisms. METHODS: Cultured SH-SY5Y cells in vitro were randomly divided into normal control group, beta-amyloid (Aß) group (20, 40, and 80 uM), and liraglutide pre-treatment group (10, 100, and 200 nM). Cell viability was determined by CCK-8 and lactate dehydrogenase (LDH). Based on its higher protection potentials, the effect of the liraglutide (100 nM) and wortmannin (200 nM) on beta-amyloid (Aß) group (40 uM) damage in human SH-SY5Ycells was examined by DAPI fluorescence staining and flow cytometry. Caspase-3, Bcl-2, Bax, Cyt-C, Akt, and P-Akt expression were detected by western blotting. RESULTS: We found that exposure of SH-SY5Y to Aß (25-35)-induced cytotoxicity, increased lactate dehydrogenase (LDH) leakage, and cellular apoptosis. Interestingly, pre-treatment with liraglutide reversed these reactions. Liraglutide afforded protection against Aß (25-35)-induced toxicity by inhibiting apoptosis, which was also confirmed by the activated caspase-3 assay. P-Akt and Bcl-2/Bax expression increased after pre-treatment with liraglutide in SH-SY5Y cells exposed to Aß (25-35), whereas cytochrome-c release decreased. This effect could be reversed by wortmannin, an inhibitor of PI3K (phosphoinositide 3-kinase). DISCUSSION: These findings suggest that liraglutide prevented Aß (25-35)-induced neurotoxicity by inhibiting neuronal apoptosis and liraglutide may have a neuroprotective effect through activation of the PI3K/Akt signaling pathway. Thus, liraglutide may be a preventive or therapeutic agent for Alzheimer's disease.

[The Effect of recombinant adiponectin on apoptosis induced by t-BHP in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the protective effect and possible mechanism of recombinant adiponectin on apoptosis in Human Umbilical Vein Endothelial Cells (HUVECs) induced by tert-butyl hydroperoxide (t-BHP). METHODS: HUVECs were cultured in vitro and apoptosis was induced by t-BHP. On this basis, HUVECs were transfected with adenovirus carrying adiponectin prior to exposure to t-BHP, to further explore the protective effect of adiponectin on apoptosis induced by t-BHP. The percentage of cell viability was determined by MTT assay. The apoptotic rate was evaluated by fluorescence microsopic analysis with Hochest/PI staining. The protein levels of p-JNK, JNK and Caspase 3 were detected by Western blot. RESULTS: Following t-BHP 100 µmol/L administration for 8 h, the ratio of apoptotic cells was increased. Western blot revealed that the protein levels of p-JNK and active caspase 3 were increased(P<0.01) compared to the control group. When cells were pretreated by adenovirus with adiponectin, the apoptosis rate and protein levels of p-JNK and active caspase 3 were decreased significantly(P<0.01). CONCLUSIONS: Continuous exposure to t-BHP induced apoptosis in HUVECs. Recombinant adiponectin protected HUVECs from apoptosis induced by t-BHP, which was correlated with the downregulation of p-JNK and active Caspase 3.

Exendin-4 Protects MIN6 Cells from t-BHP-Induced Apoptosis via IRE1-JNK-Caspase-3 Signaling.

Objectives. This study aimed to explore the effect of exendin-4 on t-BHP-induced apoptosis in pancreatic ß cells and the mechanism of action. Methods. Murine MIN6 pancreatic ß cells were treated with exendin-4 in the presence or absence of tert-butyl hydroperoxide (t-BHP). Cell survival was assessed by MTT staining. The percentage of apoptotic cells was determined by fluorescence microscopy analysis after Hoechst/PI staining and flow cytometric assay after Annexin V-FITC/PI staining. The activity of caspase-3 was determined using a caspase-3 activity kit. Expression of P-IRE1α, IRE1α, C-Jun N-terminal kinase (JNK), P-JNK, C-JUN, and P-C-JUN was detected by western blotting. Results. Exendin-4 was found to inhibit t-BHP-induced apoptosis in pancreatic ß-cells by downregulating caspase-3 activity. Exendin-4 also inhibited the endoplasmic reticulum transmembrane protein IRE1, the apoptosis-related signaling molecule JNK, and c-Jun activation. Conclusions. Our findings suggest that exendin-4 ultimately reduces t-BHP-induced ß-cell apoptosis. IRE1-JNK-c-Jun signaling is involved in the exendin-4-mediated modulation of ß-cell apoptosis.

[Oxidative damage to the endoplasmic reticulum stress pathway of apoptosis-related molecules expression in MIN6 cell].

AIM: Through a third-butyl hydrogen peroxide (t-BHP) induced apoptosis in pancreatic islet ß-cells to study the oxidative damage induced endoplasmic reticulum stress-JNK pathway of apoptosis related molecules in vitro. METHODS: Mouse insulinoma(MIN6) cells was administered with t-BHP which were cultured in vitro. Choosing medicine with different concentrations(0-400 µmol/L)and time periods(0-8 h)to establish the cells apoptosis model. The percentage of cell viability was determined through CCK-8 assay. The percentage of apoptosis was determined through flow cytometric assay after Annexin-V-FITC-PI staining. The activity of caspase-3 was measured by the caspase-3 activity assay kit. The expression of Endoplasmic reticulum stress-related molecules and the apoptosis signal pathway IRE1, JNK, P-JNK, Caspase-3 were detected by Western blot. RESULTS: The percentage of MIN6 cell viability was reducing with the concentration of t-BHP increasing. The Caspase-3 significantly change the activity after exposured of t-BHP in a concentration ≥ 25 µmol/L when the role of ≥1 h, With t-BHP concentration was increased, the role of prolonged, endoplasmic reticulum stress transmembrane protein IRE1α, P-JNK, active caspase-3 expression was significantly increased. CONCLUSION: The study demonstrates that the percentage of MIN6 cell viability was reduced in a dose-dependent manner. Continuous exposuring of t-BHP induced oxidative damage in MIN6 cells to endoplasmic reticulum stress and apoptosis. The expression of Endoplasmic reticulum stress and apoptosis pathway-related molecules in cell apoptosis in a dose and time-dependent.

Exendin-4 protects murine pancreatic ß-cells from dexamethasone-induced apoptosis through PKA and PI-3K signaling.

AIMS: To explore the effect and mechanism of exendin-4 on dexamethasone-induced apoptosis in pancreatic ß-cells. METHODS: Murine MIN6 pancreatic ß-cells were treated with dexamethasone (100 nmol/l) over 48h following pretreatment with exendin-4 (100 nmol/l). Cell viability was determined using an MTT assay. The percentage of apoptotic cells was determined through fluorescence microscopy analysis after Hochest/PI staining and a flow cytometric assay after Annexin V-FITC/PI staining. Caspase 3 activity was measured using the caspase 3 activity assay kit. Expression of cyt-c, bcl-2, bax, AKT, and phosphorylated AKT was detected by western blot. RESULTS: Exendin-4 reduced the percentage of cells undergoing apoptosis when ß-cells were exposed to dexamethasone. Exendin-4 down-regulated caspase 3 activity, reduced cytochrome c levels in cytoplasm, and increased Bcl-2 protein levels and the Bcl-2 to Bax ratio in dexamethasone-treated ß-cells. These exendin-4 effects were blocked in the presence of an inhibitor of the phosphoinositide-3 kinase (PI-3K) pathway or of the protein kinase A (PKA) pathway. Exendin-4 reversed dexamethasone-mediated inhibition of Akt phosphorylation, which was abrogated by the PI-3K and PKA inhibitors. CONCLUSION: PI-3K and PKA signaling are involved in the exendin-4-mediated modulation of ß-cell apoptosis.

[The chronic effect of palmitic acid on apoptosis of pancreatic islet beta-cells and the mechanism].

AIM: To investigate the chronic effect of palmitic acid (PA) on apoptosis of pancreatic islet beta-cells and the possible mechanism. METHODS: Insulinoma cell line (MIN6 cells) were used in this study. After being incubated in PA (0.1 - 1.6 mml/L) for 24 and 48 hours, MTT method was used to evaluate the livability. After being incubated for 48 h, Hoechst-PI and Annexin-V-FTTC/PI FACS were used to estimate the apoptosis in each group, Western-blotting assay was used to estimate the protein level of p-Akt, Akt, Bax and Bcl-2. RESULTS: Chronic PA dose-dependently (1) decreased the availability and increased the apoptosis of MIN6 cells; (2) decreased the phosphorylation of Akt and Bcl-2, but had no significant effects on Akt and Bax. CONCLUSION: Chronic PA dose-dependently induced apoptosis of MIN6 cells, and this effect was possibly regulated by Akt/Bcl-2.

[Dexamethasone-induced apoptosis of murine MIN6 pancreatic beta-cells and its effect on AKT phosphorylation].

This study is to investigate the effect of dexamethasone on cell apoptosis of murine MIN6 pancreatic beta-cells, and to investigate the mechanism of dexamethasone-dependent cell apoptosis. The cell apoptosis model was established by choosing the murine MIN6 pancreatic beta-cells, which was cultured in vitro and induced by dexamethasone. The morphology of the cell apoptosis was observed through fluorescence microscopic analysis after Hochest/PI staining and flow cytometric assay after Annexin-V/PI staining. The expression of caspase-3 was detected with caspase-3 activity assay kit. The expressions of Cyt-c, Bcl-2, Bax, AKT and p-AKT were observed with Western blotting. The results indicated that after exposure to dexamethasone at a concentration ranging from 50-800 nmol x L(-1) for 48 h, the percentage of cell apoptosis was significantly increased with the concentration over 100 nmol x L(-1) of dexamethasone; after exposure to dexamethasone (100 nmol x L(-1)) for 72 h, the activity of caspase-3 increased significantly; after exposure to dexamethasone at a concentration ranging from 50-800 nmol x L(-1) for 48 h, the expression of Cyt-c increased, Bcl-2 and AKT phosphorylation decreased while Bax and T-AKT remained unchanged. It could be concluded that the effect of dexamethasone on murine MIN6 pancreatic beta-cells apoptosis is significant. The mechanism of dexamethasone-dependent cell apoptosis is probably related to down regulation of the Bcl-2 expression and reduction of AKT phosphorylation.