Effects of pioglitazone pre-treating on acinar cells apoptosis induced by caerulein in acute pancreatitis

Abstract

Objective To investigate the effects of pioglitazone pre-treating on pancreatic acinar cell (AR42J cells) apoptosis induced by caerulein. Methods AR42J cells were divided into blank control group (with normal culture), pioglitazone group (40 μmol/L), caerulein control group (1×10-8 mol/L), pioglitazone+ caerulein group (40 μmol/L pioglitazone+ 1×10-8 mol/L caerulein) and pioglitazone+ GW9662+ caerulein group (40 μmol/L pioglitazone+ 5 μmol/L GW9662+ 1×10-8 mol/L caerulein). Pioglitazone and GW9662 were added 30 minutes earlier than caerulein. Cell proliferation rate of each group was determined by MTT assay at three, six, 12 and 24 hour. The cell apoptosis rate was detected by flow cytometry with Annexin Ⅴ/PI staining and terminal dexynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining. The activity of Caspase 3, 8 and 9 of each group was measured. Mitochondrial membrane potential (MMP) was detected by flow cytometry with JC-1 staining. Single factor analysis of variance and LSD test were performed for data analysis. Results At six, 12 and 24 hour, the cell proliferation rate of pioglitazone group and pioglitazone+ caerulein group was 0.19±0.02, 0.22±0.02, 0.36±0.02 and 0.20±0.04, 0.23±0.02, 0.38±0.02, respectively, which were significantly lower than those of blank control group (0.25±0.04, 0.28±0.03 and 0.46±0.02) and caerulein group (0.23±0.02, 0.29±0.01 and 0.46±0.05, tcontrol group=-3.16, -4.61 and -6.25, tcaerulein group=-1.58, -4.61 and -6.15, all P 0.05), but cell apoptosis of these two groups were higher than those of control group ((5.52±0.64)%, (5.30±0.97)% and (5.47±0.88)%) and caerulein group ((5.98±1.21)%, (7.47±0.58)% and (8.11±1.32)%) respectively, and the differences were statistically significant (tcontrol group=9.81, 4.45 and 10.74, tcaerulein group=4.38, 7.62 and 6.98, all P <0.05). There was no significant difference in apoptosis rate between pioglitazone+ GW9662+ caerulein group ((5.82±0.26)%, (6.05±0.83)% and (9.23±0.90)%) and caerulein group; while significantly higher when compared with those of pioglitazone+ caerulein group (t=-4.63, -10.07 and -5.70, all P<0.05). At 12 hour, the apoptosis rate detected by TUNEL staining of pioglitazone group ((3.93±0.40)%) was significantly higher than that of control group ((2.73±0.68)%), the apoptosis rate of pioglitazone+ caerulein group ((8.43±1.65)%) was significantly higher than that of caerulein group ((2.80±0.56)%), the apoptosis rate of pioglitazone+ GW9662+ caerulein group ((3.87±0.35)%) was lower than that of pioglitazone+ caerulein group (t=7.93, 8.92, -5.35, all P<0.05). At 24 hour, the activity of Caspase 3, 8 and 9 of pioglitazone+ caerulein group (1.28±0.05, 1.38±0.04 and 1.53±0.09) significantly increased compared with those of caerulein group (1.12±0.88, 1.22±0.02 and 0.53±0.07, t=3.20, 8.62 and 1.29, all P<0.05). After treated with GW9662, part of activity of Caspase enzymes recovered. The number of cells with potential change of mitochondrial membrane in pioglitazone group and pioglitazone+ caerulein group was more than that of caerulein group (28.50±0.91)% and (28.20±2.56)% vs (15.00±3.67)%) and part of membrane potential recovered after GW9662 added ((20.67±2.20)%). Conclusions Pioglitazone might promote AR42J cell apoptosis through the activation of caspases enzymes and changing membrane potential. And the antagonist GW9662 would partially inhibit the apoptosis induced by pioglitazone. Key words: Pioglitazone; Caerulein; AR42J cells; Acute pancreatitis