Digested raspberries polyphenols for brain health
Digested raspberries polyphenols for brain health: attenuation of neuroinflamation
CLOSED
Background
Consumption of berries is associated with neurological benefits in aging/neurological diseases. Neuroinflammation is closely associated with the pathogenesis of neurodegenerative diseases being microglial activation the hallmark. Once activated, microglia display functional plasticity and ultimately increases cytokine production (IL-1b, TNFa), inducible NO synthase and NO and NADPH oxidase activation, all contributing to glial-induced neuronal death. Consequently, BBB may become more permeable further enhancing the recruitment of immune cells to the lesion site. Also, peripheral increase of pro-inflammatory mediators triggers cytokines activation. Neurons are vulnerable to oxidative stress and exposure to agents causing redox imbalances, affect mitochondrial function, causing neuronal damage and release of several factors as neuromelanin, protein laminin and MMP-3 that together with multiple modifications of the extracellular matrix, can mediate microglial activation. Subsequently, a feed-forward cycle of glial-activation and neurodegeneration can lead to degeneration of the nigrostriatal dopaminergic pathway. Previous results from host lab reveal that chemically different wild Rubus, submitted to digestion, promoted differential cytoprotective effects inducing adaptative cellular processes in a neurodegeneration cell model. Based on that, we propose to evaluate neuroprotective effects of near-isogenic Rubus idaeus (raspberry) cultivars. Serum available polyphenols obtained after digestion, at physiological concentrations, will be evaluated for: cytoprotection in a neuroinflammation cell models. Cellular processes related with inflammation and oxidative stress like cell death mechanisms, microglial reactivity, inflammatory-related parameters and redox cell response, as well as their cross-talk will be evaluated.
Working plan
The work will encompass the following tasks:
TASK 1: Establishment of neuroinflammation model
A neuroinflammation cell model will be implemented by using an immortalized human microglial cell line (CHME-5 cell line). The cells will be stimulated with 1 microg/mL lipopolysaccharide , and 10U/mL IL1-alpha.
Activation of CHME-5 cells will be assessed by immunocytochemistry with antibodies for two specific markers for human microglia: glucose transporter Glut-5, whose expression is increased upon activation, and CR3/43 (or HLA-DR/DP/DQ, a class II major histocompatibility complex), which is specifically expressed in activated microglia. Secretion of pro-inflamatory markers will be assessed by detection of TNFa and IL-6 in the culture media by ELISA.
TASK 2: Digested raspberries metabolites safety in microglia cells
To screen the raspberry fractions for eventual toxicity to the human microglia, cell cultures will be treated with the metabolites at levels up to 4 microM (physiological levels reported for individual polyphenols) for 1-24h. Cell viability will be determined using the resazurin-based colorimetric method.
Task 3: Raspberry metabolites-mediated cytoprotection after pro-inflammatory and oxidant insults
Digested raspberries metabolites cytoprotective effect will be evaluated after pro-inflammatory and oxidant insults in microglia. In parallel to inflammation an oxidative stress will be performed since in a neurodegeneration process microglia environment also have increased ROS. Cell viability will be evaluated by flow cytometry using PI. Intracellular ROS formation will be assayed by fluorimetry using dichlorodihydrofluoresceindiacetate for hydrogen peroxide and hydroethidine for superoxide anion. Other redox metabolic alterations such as a) total thiols content (GSH/GSSG levels) will be evaluated by HPLC.
Supervisors
Paula Pinto (paulapinto@itqb.unl.pt)
Cláudia Nunes dos Santos (csantos@itqb.unl.pt)
Laboratory/Institution
Disease and Stress Biology Laboratory @ ITQB
