Identification Of67kD Laminin Receptor As The Phamacologic Target Of Microglial Inhibition Of Epigallocatechin Gallate

Objective：Microglia are the primary immune cells of the brain and have been shownto be involved in the pathogenesis of numerous neurological disorders,including neurodegenerative diseases. Activation of microglial cells areclosely associated with the neuronal injury. Activation of microglia isbelieved to contribute to neurodegenerative processes through the release ofa host of proinflammatory factors, including IL-1, TNFα. Under physiologicalconditions, microglia are showed in immune surveillance. However, microgliabecome in response to injury or immunological challenges when they undego aprocess of activation. The principal polyphenolin greentea,(-)-epigallocatechin-3-gallate(EGCG), being the major component amongcore structure, is considered to be the main contributor to green tea’sbioactivity. Previous studies have been showed that green tea and its majorcomponents, green tea polyphenols may have neuroprotective property. We havefound that (-)-epigallocatechin gallate (EGCG), a monomer in green teapolyphenols, possesses the potent inhibitory effect on microglial activation,the mechanism, however, still need to be clearified.67-kD laminin receptor(67LR) has been showed as a cell surface receptor, and it was closelyassociated with the immigration, invasion, and metastasis in the malignanttumor. The aim of this study was to demonstrate that67kD laminin receptoron microglia is the pharmacologic target of EGCG which mediated the effectof microglial inhibition of EGCG. The study will value the green tea treatmentin Alzheimer’s disease and other neurodegenerative diseases.Methods：Microglial cells were prepared from Spraque-Dawley rat’s brains of day1to2. After the rat’s brains were dissected and the blood vessels and meninges were discarded, the tissues of brain cerebral cortices were mincedand digested with trypsin (0.25%). Dissociated primary mixed neuron-gliacells were maintained in DMEM supplemented with10%fetal bovine serum (FBS)and seeded in75cmflasks. The cells were cultured at37°C in an atmosphereof5%CO2and95%air. Twelve days after the seeding, when the culture cellswere grown completely confluence, the cells were treated with mildtrypsinization. When the mixed neuron-glia cultures was rinsed with PBS threetimes and was incubated with0.08%trypsin-EDTA for the upper layer detachmentin an incubator, the cells attached on flasks were removed to12-well platespreviously coated sections with poly-L-lysine and maintained for further use.To activate the microglia,500ng/ml LPS was added to the cultures. In somewells, EGCG was added30min before LPS treatment for the condition ofmicroglia inhibition. Microglia can be detected with CD11b antibody, thespecific antibody for microglial membrane antigen. Using specificity of theimmunocytochemical reaction, identify purity of cells. The purity ofmicroglia was identified with a monoclonal antibody to CD11b, showing a>98.8%purity. Using double labeling, after24h observe change of purifiedmicroglial morphology and67LR expression on microglial.Purified microglia at6-well plate cultured for indicated time and aftertreatment with EGCG for24h, EGCG at0.5~50μM was added30min before LPStreatment for the condition of microglia inhibition. The purified microgliawere collected and lysed for Western blot for67LR. After the measurement ofprotein concentration by BCA method with a kit,20μg proteins of homogenateswere separated to SDS-PAGE on12%gel for electrophoresis.The separatedprotein was then transferred to the nitrocellulose (NC). Membranes wereblocked with5%skim milk and incubated with polyclonal anti-67LR forovernight at4°C. Peroxidase-linked HRP and DAB color developing reagent andused β-actin antibody as internal control. Results：1．The addition of LPS induced a marked increase of microglial67LRexpression and morphological changes. Pretreatment of EGCG inhibitedLPS-induced morphological change. Resting microglia typically displayed aramified, either bipolar or unipolar profile. After challenged with LPS (500ng/ml) for24h, most of the microglia showed remarkably changes inmorphological appearances, appearing round with enlarged and“ameboid”appearance which was the characteristic of activated microglia.While pretreatment of EGCG made them remain most of “resting” appearance.At the same time, after incubated with500ng/ml LPS for24h,67LR expressionincreased of microglia and pretreatment of EGCG makes67LR expression remainmost of resting level.2. We examined inhibited effect of EGCG treatment on67LR expression ofmicroglia by using Western blot analysis. EGCG dose-dependently preventedLPS-induced67LR expression in the microglia. After exposed to LPS (500ng/ml)for24h,67LR levels were remarkably elevated in the cell plasma, which couldbe significantly inhibited in a dose-dependent manner by pretreatment withEGCG (0.5μM-50μM). Western blot analysis also indicated that EGCGdose-dependently suppressed67LR expression in the microglial cells.Conclusion：1. Our results confirmed that67LR is expressed on microglia.67LR is expressedin the cytoplasm but not in the nucleus. Expression67LR of resting microglialcells was less than that in activated microglia cells by LPS. Our observationssuggest that67LR plays important roles in mediating microgliosis.2. The inflammation can promote67LR expression of microglia. In microglialcells cultures, LPS induced microglia to become activated and to releasea lot of proinflammatory and cytotoxic factor, as indicated by a change inmorphology;3. The regulatory effects of EGCG on microglial67LR is in a dose-dependentcharacteristics, which suggest that67LR may be a molecular which mediatedthe inhibition of EGCG on the activation of microglia. The study will value the potential use of green tea reagents on the treatment in Alzheimer’s diseaseand other neurodegenerative diseases.