Защитное действие карнозина в условиях токсического действия акролеина на клетки PC-12

Проведены исследования на клетках линии PC-12. Изучен высокотоксичный альдегид-акролеин как индуктор окислительного стресса, который проявлял дозозависимый токсический эффект на клетках, выражающийся в накоплении активных форм кислорода (АФК) и гибели клеток. Показано, что природный нейропептид карнозин предотвращал акролеин-индуцированную гибель клеток культуры РС-12 и рост АФК. Установлено, что эффективность карнозина находилась в зависимости от токсичной дозы акролеина, времени инкубации в его присутствии, а также способа его введения.

Protective effect of carnosine on PC-12 cells in the conditions of acrolein toxic action

Oxidative stress (OS), characterized by the accumulation of reactive oxigen species and decrease in efficiency of endogenous antioxidant protection, accompanies development and progressing of many diseases. Currently acrolein is now seen as one of the most toxic aldehydes and it is one of the most pathogenetically significant biomarkers of ischemic stroke and neurodegenerative diseases. Acrolein-induced cell death proceeding mainly by necrosis, accompanied by accumulation reactive oxygen species in cells. In these conditions there is expedient an application of antioxidants as «traps» of the free radicals one of which is the natural neuropeptide carnosine. PC-12 pheochromocytoma cell line is used as in vitro model experiments of various biological processes studying. Acrolein was used for OS induction in two final concentrations: 10 mkM or 100 mkM. Cells were incubated with acrolein within 1, 3 or 24 hours. Carnosine was added in 1мМ concentration to incubation medium in 1 hour prior to introduction with acrolein or after an hour, three-hour or 24-hour incubation with the acrolein. The following parameters were analyzed: level of reactive oxygen species and the number of dead cells in studied PC12 cellular population. When cells were incubated in the presence of 10 mkM of acrolein for 1 h, 3 h and 24 h the gradual growth of ROS proportional to time of an incubation was noted: in 1.7 times, 3 times and 15,2 times respectively, relatively to intact cells. The incubation of cells in the presence of acrolein 100 mkM during 1 h, 3 h and 24 h also revealed temporary dependence: in 4.2 times, 13.4 and 15.3 times respectively. And also noted a marked increase in the number of dead cells – in 6 ; 6.8 times and 8.7 times. Thus the maximum death of cells (to 90 %) is noted in the conditions of 100 mkM acrolein influence during 24 hours. The obtained data specify that acrolein is a factor factor that triggers the development of oxidative stress; they they are consistent with results of the previous works. In our research of carnosine influence on ROS levels and PC-12 cell death exposed to oxidative stress under the action of acrolein, two experimental approaches were taken. In one case, carnosine was introduced into the cell culture before exposure to acrolein, the so-called «Preventive» introduction in the other – carnosine was added after incubation of cells with acrolein for 1.3 and 24 h, the so-called «Therapeutic» effect. Preliminary carnosine introduction in incubation medium before acroleine, irrespective of its concentration, promoted decrease in growth of ROS, the most significant after the 24th hour of an incubation, and also prevented cell death, induced by acrolein, regardless of its concentration and time of incubation. The effect of the introduction of carnosine in the incubation medium after exposure to acrolein were dose dependent on acrolein: it is most clearly expressed by incubation with acrolein 10 mkM within 24 hours and with acrolein 100 mkM for 3 hours. Also, there is a tendency to reduce the ROS during the first three hours of incubation with 10 mkM acrolein. Effect of carnosine on cell death is also dependent on the dose and time of incubation. Significant reduction in cellular death occurs during incubation with acrolein 10 mkM for 24 hours and during the incubation with acrolein 100 mkM for 1 hour. In all other cases, the reduction in mortality is negligible.