Abstract
Introduction: The NLRP3 inflammasome can be activated by host-derived molecules including reactive oxygen species (ROS). Oxidative stress induces the movement of thioredoxin interacting protein (TXNIP) from the nucleus to the mitochondria, where it forms a bond with TRX2 (thioredoxin 2). Then, this interaction hinders TRX2s ability to regulate ROS levels ultimately leading to inflammasome activation. Suppressing the initiation of inflammasome activation could have a significant therapeutic impact on various clinical disorders.
Objectives: To investigate whether the sirtuin1 activator (SIRT1 aptamer) can be used to treat inflammation by inhibiting TXNIP and ROS.
Materials and Methods: In this in vitro experimental study, the RAW 264.7 cell line served as the model system for macrophages. The cells were cultured in RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and then incubated at 37 °C in a humidified 5% CO2 environment. Variable concentrations of the SIRT1(silent information regulator 2 homolog 1) aptamer were applied to the cells. The viability of the RAW 264.7 cell line was assessed for cytotoxic effects of the SIRT1 aptamer using the tetrazolium bromide (MTT) assay analyzed by nonlinear regression analysis using GraphPad Prism software 9.2. The expression of SIRT1 and TXNIP proteins was examined using the western blot technique and analyzed with GraphPad Prism software 9.2. Additionally, ROS levels were determined using a fluorometric assay kit and analyzed by one-way ANOVA with Tukey test.
Results: The SIRT1 aptamer was able to inhibit the growth rate of RAW 264.7 cells after 72 hours with an IC50= 1.98 µM. Additionally, it significantly increased the expression level of SIRT1 protein (P<0.05). The expression of TXNIP protein decreased when treated with SIRT1 aptamer at concentrations of 0.99 µM and 3.9 µM, and completely disappeared in samples treated with the IC50=1.98 µM. Furthermore, the SIRT1 aptamer dramatically reduced lipopolysaccharide (LPS)-stimulated ROS levels at concentrations of 1.9 µM and 4 µM by 55% and 75% respectively, compared to the ROS induced by LPS (ROS=195%).
Conclusion: There is a crosstalk between the SIRT1 protein and TXNIP, as this interaction results in the reduction of TXNIP expression, thereby protecting the cell from the damaging effects of ROS after treatment with a SIRT1 activator. The discovery highlights the potential of a novel anti-inflammatory biotherapy.