Sorption of apoptosis-inducing factor on a polyvinylidene fluoride membrane to determine its content in the heart of rats with diclofenac-induced myocardial injury

Abstract

. In the course of this study, the level of apoptosis-inducing factor (AIF), sorbed on a polyvinylidene fluoride (PVDF) membrane, was analyzed from the heart of rats (Rattus norvegicus of the Wistar line) with myocardial damage caused by diclofenac administration. Laboratory animals were randomly assigned to two experimental groups, with 10 animals in each group. From the 15th day of the study, a saline solution was administered intraperitoneally to the rats in the control group for a period of 7 days. Animals in the second group received an injection of 100 μl of Freund's Complete Adjuvant subcutaneously into the plantar surface of their hind paw. Fifteen days later, rats in this group were administered diclofenac sodium intraperitoneally at a dose of 10 mg/kg, once daily for 7 consecutive days. 24 hours following the final injection, the rats were removed from the study, and blood and cardiac tissue samples were collected for analysis. Commercial kits were used to determine marker enzymes of cardiomyocyte cytolysis in serum. The oxidative state was analysed by biochemiluminescence induced with hydrogen peroxide and iron sulfate. The Western blotting technique was employed to evaluate the level of AIF. The proteins in the lysate of heart tissue were separated by electrophoresis on a polyacrylamide gel containing sodium dodecyl sulfate. Following this, they were adsorbed onto PVDF membranes for one hour at 4°C at a voltage of 35V. After adsorption, the membranes were incubated with specific antibodies. The target proteins were then detected using secondary antibodies linked to horseradish peroxidase and a chemiluminescent substrate. The mRNA level of the AIF gene was measured using real-time polymerase chain reaction. The results showed that the levels of biochemiluminescence in the hearts and blood serum of rats with pathology were increased, indicating an intensification of free radical-induced oxidation. During Western blotting, rat heart proteins were successfully separated by molecular weight and sorbed onto a PVDF membrane. Detection revealed a significant increase in AIF levels. In addition, administration of diclofenac to rats led to an increase in mRNA levels of the AIF gene in heart tissue. The observed changes may be due to an increase in the production of reactive oxygen species as a result of drug action and indicate a significant role for AIF in triggering caspase-independent apoptosis in the pathogenesis of myocardial damage caused by diclofenac.