ALPHA-1-ANTITRYPSIN REDUCE LEVELS OF TUMOR NECROSIS FACTOR ALPHA BOTH IN VITRO AND IN VIVO DIABETES MODELS.

Abstract

Purpose : Diabetic retinopathy (DR) is in part consequence of a chronic hyperglycemic condition. Early stages of DR are characterized by inflammatory and microvascular changes. It is known that Muller cells are responsible for secrete several molecules involved in angiogenesis and inflammation. The role of several endogenous anti-inflammatory molecules and their interactions with endothelial cells in DR is of intense study. The aim of our work was to evaluate the levels of Tumor Necrosis factor alpha (TNF-α) in the retina of diabetic animals and in vitro culture of Muller cells exposed to hyperglycemia with and without treatment of AAT.

Methods : Muller cells were obtained from retinas of c57/BL6 mice. In order to characterize the isolated cells were used to perform an immunofluorescence assay with antibodies against CRALBP, GFAP and Vimentin. Cells were growth in DMEM Medium supplemented with 30mM glucose, and standard conditions. The cells were exposed to 0, 1.5, 3 and 4.5 mg/ml of AAT and incubated 16h. For metabolic control we perform MTT assay. The mRNA and protein levels of TNF-α were measured by real time PCR and Enzyme Linked Immunoassay (ELISA).
In the in vivo study Mice c57BL6J of 8 weeks old were injected with 2 doses of 100mg/kg of streptozotocin (STZ) spaced 48 hours. A week after the 2nd injection of STZ the blood glucose test was performed on fasting 8 hours. Animals with blood glucose levels of 200 mg/dl or higher were considered diabetic. Animals were injected one week after becoming diabetics with a single dose of 60mg/kg of AAT, weekly administered until sacrifice.

Results : We observed a decrease gene and protein expression of TNF-α in Muller cells treated with AAT (1.5, 3, 4.5mg/ml). Besides, a lower expression of TNF- α was found in the retina of animals treated with AAT compared to those without treatment

Conclusions : These findings might help us to better understand the inflammatory process associated with stress induced by chronic hiperglycemia, as occur in DR. The use of AAT as anti-inflammatory agent could be a promising treatment for early diabetic retinopathy.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.