ROLE OF ANNEXIN A1 IN THE EXPRESSION OF THE NRLP3 INFLAMMASOME AND IN THE INTEGRITY OF THE EPITHELIAL BARRIER: STUDY IN A MURINE DIABETES MODEL

Abstract

The complex interaction between intestinal microbiota and inflammation is related to intestinal permeability, indicating that endothelial dysfunction is a primary feature of type 1 diabetes (T1D). The annexin A1 (AnxA1) protein, found in inflamed tissues of the intestinal mucosa, acts in the inflammatory response and in the regulation of intercellular junction proteins, such as ZO-1 and cadherin. Furthermore, it is important for the regulation and activation of NLRP3. Therefore, the general objective of the work will be to investigate the role of the AnxA1 protein in regulating the intestinal epithelial barrier and NLRP3 activity in DM1. To investigate the impact of the lack of AnxA1 on the dysregulation of the intestinal epithelial barrier, we used C57BL/6 wild-type AnxA1+/+ and AnxA1 knockout mice (AnxA1-/-). The animals will be distributed into four experimental groups: AnxA1+/+ control, AnxA1+/+ diabetic, AnxA1-/- control and AnxA1-/- diabetic. DM1 induction will be performed through intraperitoneal injection of strepzotocin (STZ) at 65mg/kg once a day, for five consecutive days. On days 30, 60 and 90 after the initial STZ induction, animals will be weighed and glucose measured to check for diabetes. Animals in control groups receive vehicle only. Small intestines will be collected and processed for histological and molecular analyses. The expression of AnxA1, ZO1 and cadherin proteins will also be evaluated, as well as the inflammasome components NLRP3, ASC and cleaved caspase 1 by Western Blotting. By immunohistochemistry, we evaluated the immunoreactivity of ZO-1, cadherin, NLRP3 and AnxA1. Furthermore, we will evaluate the levels of cytokines and chemokines by multiplex assay based on Luminex beads, using intestine homogenates in different experimental conditions. Above all, the study may contribute to a better understanding of the pathophysiology of DM1 and, technically, identify therapeutic targets for the treatment of intestinal complications associated with the disease.