The effect of bile acid TLC-S vitality of pantacric acinary cells and level of mitochondrial membrane potential

Abstract

To investigate the mechanism of action of bile acids, TLC-S, the viability of pancreatic acinar cells, and the level of mitochondrial membrane potential were studied using various oxidation substrates. The experiments were conducted on male Wistar rats weighing 250–300 g. A suspension of isolated pancreatic acinar cells was obtained using collagenase (type IV, 0.2 mg/ml). The trypan blue exclusion test assessed cell viability after isolation and the amount was >90 %. The survival of acinar cells after prolonged incubation in different environments was determined by fluorescent dyes propidium iodide (0.5 mg/ml) and Hoechst 33258 (5 mg/ml) dyes. Mitochondrial membrane potential was recorded by adding the dye TMRM (50 nM). Cells were photographed using an inverted microscope Olympus IX73 with a digital camera DP-74. Cell counting and fluorescence intensity of TMRM were analyzed using ImageJ software. It was found that the presence of TLC-S in the medium at a concentration of 0.5 mmol/L, when used as an oxidation substrate for glucose (10 mmol/L), pyruvate (2 mmol/L), or alanine (2 mmol/L), did not significantly affect the proportion of live cells after 2 and 4 hours of incubation. However, when TLC-S was added to the medium at a concentration of 2 mmol/L, the proportion of live cells significantly decreased. This decrease was least pronounced during pyruvate oxidation. Under the action of TLC-S (after 25 min) in the presence of glucose alone, the mitochondrial membrane potential decreased by 9.8 % (P<0.05, n=3), in the presence of alanine (in the presence of glucose) – by 23.5 % (P<0.05, n=4). When pyruvate was added to the medium (in the presence of glucose), the mitochondrial membrane potential likely did not change significantly. These results suggest the presence of several different mechanisms of TLC-S’s negative effects on pancreatic acinar cells: at low concentrations (or at early stages), it disrupts alanine transamination reactions, leading to the disruption of mitochondrial membrane potential generation, while at high concentrations, it affects the integrity of plasma and/or intracellular membranes.