Disorders of proteolytic homeostasis in the liver of rats with hyperhomocysteinemia

Abstract

Introduction: The liver is a key organ of the body that is responsible for maintaining homeostasis. It helps regulate almost all biochemical pathways associated with metabolism, nutrition, energy supply, and the formation of immunity. Consequently, impaired liver function can contribute to the progression of hyperhomocysteinemia and the development of associated complications. The present study aims to investigate the level and activity of metal-dependent and serine proteases in the liver of rats with hyperhomocysteinemia. Methods: A total of 60 albino nonlinear male rats were used in this study. Hyperhomocysteinemia was induced by intragastric administration of DL-homocysteine thiolactone. Total proteolytic activity was measured using casein as a substrate. To determine the activity of metal-dependent and serine proteases, ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride were used. The levels of MMP-1, MMP-2, and MMP-3 were studied by enzyme-linked immunosorbent assay. The fraction of serine proteases was isolated by affinity chromatography on the benzamidine sepharose column. The composition of serine protease fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results: The pathogenesis of hyperhomocysteinemia was found to be accompanied by a proteolytic imbalance manifested in the increase in the total proteolytic activity, the activity of serine proteases and especially the activity of metal-dependent enzymes. The increase in the levels of MMP-1, MMP-2, and MMP-3 indicates the involvement of these enzymes in the amplification of proteolysis mediated by metal-dependent enzymes. Despite a significant increase in the content of the serine proteases, their activity did not increase significantly. This may be because of the proteolytic degradation of serine proteases, which was confirmed by changes in their qualitative composition. The accumulation of low-molecular-weight proteins and the decrease in the part of proteins with a molecular weight of greater than 50 kDa in the fraction of serine proteases were revealed. Conclusion: Collectively, our observations indicate that impaired proteolysis in the liver may be an important determinant in the development of hyperhomocysteinemia-associated disorders.