In vitro evaluation of the inhibitory activity of cystatins on cathepsins and proinflammatory cytokines expressed by murine macrophages RAW 264.7 after inflammatory stimuli

Abstract

Periodontitis is an infectious disease of inflammatory nature which results in the destruction of the supporting tissues of the teeth. This destruction is induced by the host response against the aggression caused by bacterial biofilms. In this process, several active proteinases including cathepsins B and K are involved. They play an important role in the onset and progression of inflammation and has cystatins as inhibitors. These recombinantly produced inhibitor are shown to be effective in inhibiting human cathepsins, cysteine proteases and others. The understanding of the involvement of cystein proteinases in the progression of periodontal disease may lead to new therapies for the control of periodontal disease. Thus, the aim of this study is to evaluate the effect of the inhibitory activity of phytocystatins (Cane Cane CPI-1 and CPI-4 derived from sugar cane and citrus derived from orange) on cathepsins B and K and proinflammatory cytokines (IL-1², IL-6, and TNF-±) in murine macrophage cells (RAW 264.7) stimulated with lipopolysaccharide (LPS) from Escherichia coli (E. coli) and inactivated bacteria Porphyromonas gingivalis (P. gingivalis). Therefore, a time-response test will be performed to evaluate the gene expression of cathepsins B, K and proinflammatory cytokines (IL-1², IL-6 and TNF-±) after 6, 12, and 24 hours, using real time RT-PCR. In addition, an assay will be performed to assess the cytotoxicity of phytocystatins on RAW 264.7 macrophages after 6, 12, 24, and 48 hours using the Alamar Blue Cytotoxicity Test. And finally, based on both previous experiments, the best inflammatory stimulus will be used to evaluate the inhibitory effect of phytocystatins on gene expression of cathepsins B, K and proinflammatory cytokines (IL-1², IL-6 and TNF-±) in RAW 264.7 macrophages 6, 12, and 24 hours after stimulus using RT-PCR in real time.