Effects of Curcumin on the Expression of Flotillin-1 in the Brains of APP/PS1 Double Transgenic Mice

Alzheimer’s Disease (AD) is a chronic and progressive neurodegenerative disease. Flotillin-1 is a marker protein of lipid raft in nervous tissue, which is significantly increased in AD. To investigate the changes of flotillin-1 expression in the brains of APP/PS1 double transgenic mice treated with curcumin, thirty six-month-old APP/PS1 double transgenic mice were randomly divided into model group (MOD), low-dose curcumin group (LCUR) and high-dose curcumin group (HCUR). LCUR group mice and HCUR group mice were fed with curcumin (0.16 g·kg −1 and 1.0 g·kg −1 , respectively) every day for 6 months. Western blot and real-time PCR were used to detect the expression of flotillin-1 in brain. Results showed that compared with MOD group, the expression of flotillin-1 protein (P < 0.05) and mRNA (P < 0.01) in brains of HCUR group were statistically decreased. Thus, high-dose curcumin may play a neuroprotective role in AD through downregulating the expression of flotillin-1 in APP/PS1 double transgenic mice.

Journal of Biosciences and Medicines ration of the individual's personality and failure to communicate or perform routine tasks. Flotillin-1 is a marker protein of lipid raft in nervous tissue, which is significantly increased in AD nerve cells. It was reported that flotillin-1 accumulated in lysosomes of tangle bearing neurones in the course of AD [2]. Flotillins 1 and 2 increased in pyramidal neurons and astrocytes in the white matter during the formation of senile plaque in Alzheimer's disease [3]. These findings suggest that flotillins are associated with progression of Alzheimer pathology.
Curcumin possesses anti-carcinogenic, anti-oxidant and anti-inflammatory properties and may help delay or prevent neurodegenerative diseases, including AD [4]. In this study, flotillin-1expression levels were studied in curcumin treated APPswe/PSEN1dE9 (APP/PS1) transgenic mice to explore the mechanism of curcumin in AD prevention and treatment. 3) Western blot Total protein samples from each group of brain tissue were lysed with RIPA lysis buffer following the manufacturer's instructions. Protein concentrations were measured using the BCA method. A total of 20 µg proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Nonspecific binding was blocked with 5% skimmed milk at room temperature for 1 h, the membranes were incubated with primary antibody (flotillin-1, 1:500; β-actin, 1:500) at 4˚C overnight. The next day, the membranes were washed three times with 0.1% Tween-20/TBST (pH 7.6) and incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies Journal of Biosciences and Medicines for 2 h at room temperature. An enhanced chemiluminescence detection reagent was used for enhanced blot detection. The band intensity was assessed with Image Lab Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed. (mean ± SD). Differences between multiple groups were analyzed by One-way analysis of variance (ANOVA). Pairwise comparison was analyzed by SNK-q test. p-value less than 0.05 was considered statistically significant.

Curcumin Downregulated the Expression of Flotillin-1 in APP/PS1 Transgenic Mice
In order to assess the expression of flotillin-1, western blot was performed and the relative level of flotillin-1 expression was calculated as the ratio to the inter-    tillin-1, as a specific lipid rafts protein, could recruit amyloid precursor protein (APP) into lipid rafts, thus providing a platform for cleavage of APP to release amyloidogenic amyloid beta (Aβ) peptide [9]. The enhanced expression of flotillin-1 in brain tissue of AD patients was positively correlated with the Aβ levels.

Discussion
Chen TY et al. reported that flotillin-1 interacted with APP intracellular domain (AICD) and may recruit APP to lipid rafts, therefore participated in the localiza-tion and processing of APP [10]. Hattori et al. found that in BACE1-HEK cells, overexpressed flotillin-1 could recruit β-site APP cleaving enzyme-1 (BACE1) into lipid rafts and regulate the function of BACE [11]. Nishikawa et al. also detected the abnormally enhanced expression of flotillin-1 in NFTs, suggesting that flotillin-1 may be involved in the pathological changes of tau/NFTs in AD [12].
Curcumin is a chemical component extracted from the rhizome of turmeric.
In recent years, curcumin has been reported to possess anti-amyloidogenic, anti-inflammatory, anti-oxidative, and metal chelating properties that may result in potential neuroprotective effects, but the mechanism is still not clear. In this study, we observed that the protein and gene expression of flotillin-1 decreased