Evaluation of expression of GRP78 protein in liver from mice submitted to chronic intoxication by fluoride and different types of diet

Abstract

In the previous work conducted by our research group (Process FAPESP: 2013 / 25756-0) it was observed that fluoride (F) when administered in animals with non-alcoholic fatty liver disease (NAFLD) previously induced by a hyperlipidic diet exerts different effects when compared with animals that consumed normocaloric diet (absent NAFLD), which may be related to protein alterations, since F exerts diverse effects on several cellular functions. Among these has been reported the disturbance that this ion causes in the antioxidant defense. An indication of oxidative stress is given by the increase in Glucose-Regulation Protein 78 expression (GRP78). Thus, the objective of this work is to use the western blot technique to verify if there are alterations in the GRP78 protein when administered F in animals with previously induced NAFLD compared to animals that consumed normocaloric diet. Thirty-two Swiss male mice with a body weight range of 26g to 44g will be used, which will be carefully divided in order to balance the groups. Initially the animals will be divided into 2 groups (n = 16 per group), who will receive for 30 days hyperlipid ration and drinking water without F addition, for induction of NAFLD (Group I) or control diet and drinking water without addition F (Group II). After this period, group I will be subdivided into two groups (n = 8 per group), according to the type of drinking water administered, namely: hyperlipid diet and deionized drinking water (group A), hyperlipid diet and water of drinking at 50 ppm F (group B). Group II will be subdivided into two groups (n = 8 per group), according to the type of drinking water administered, namely: control diet and deionized drinking water (group C) and control diet and drinking water with 50 ppm F (group D). At the end of the experimental period (20 days), the animals will be anesthetized by intraperitoneal injection of 0.5 mL / kg body weight of Anasedan (Agribrands, USA) + 1.5 mL / kg of ketamine hydrochloride (Vetaset, Fort Dodge, Iowa, USA), liver and plasma will be collected. F analysis in plasma and liver with specific ion electrode after diffusion facilitated by hexamethyldisiloxane will be performed and a portion of the liver will be stored in a freezer at -80 ° C for Western blot analysis. Statistical analysis will be performed after verifying the homogeneity of the data and then the most appropriate test for analysis will be chosen, using Graph Pad Software InStat3 and Graph Pad Prism6 (p <0,05).