Study On The Neuroprotective Effects Of Pterostilbene Using Cell-based Model

Estrogen has neuroprotective activity against a variety of injury in the brain. Many in vivo and in vitro studies have demonstrated the important role of estrogen in the neuronal growth and differentiation, neuroprotection, cognition, and regulation of mood. Because of the side effects of estrogen including tumor and cardiovascular diseases, researchers were searching for effective substitutes of estrogen for decades.Phytoestrogens are naturally plant-derived compounds and can be converted into weak estrogen-like substances in the gastrointestinal tract which can exert biological activity by interacting with the hormone system and mimicking the effects of estrogen. Pterostilbene, a dimethylated anolog of resveratrol, is a natural product found in grapes and berries. Till now, most of the researches focus on the anticancer and anti-arthritis activity of pterostilbene. Recently, it is reported that pterostilbene also has neuroprotective effect.However, little is known about the biochemical and molecular mechanisms associated with pterostilbene’s effects on neuronal cells. The goal of our study was to determine and evaluate the neuroprotection of pterostilbene and the involved molecular mechanisms on SH-SY5Y cells injured by H2O2. Our study may provide some theoretical basis and experimental evidence for phytoestrogen, the potential substitute of estrogen.[Background]Estrogen is one of the most important endocrine hormones for female reproduction. It also plays essential roles in some systems and organs including cardiovascular system, liver, bones and neural system. Because of the side effects of estrogen including tumor and cardiovascular diseases, researchers were searching for effective substitutes of estrogen for decades. Phytoestrogens are naturally plant-derived compounds which can mimick the effects of estrogen after being converted into estrogen-like substances in gastrointestinal tract. Several kinds of phytoestrogens have been proposed as the "natural alternatives" to estrogen replacement therapy for postmenopausal women or women who have undergone a hysterectomy. Phytoestrogens are commonly ocurred in a wide range of foodstuff, while has much less side effect compared to estrogen. Resveratrol (3,5,4-trihydroxystilbene) is a natural polyphenol that has anti-cancer, anti-inflammation and neuroprotection effect. However, resveratrol is metabolized quickly which leading to short life and limiting its clinical trial. Pterostilbene (3,5-Dimethoxy-4’-hydroxy-trans-stilbene), a dimethylated anolog of resveratrol, is a natural product found in grapes and berries. Compared to resveratrol, pterostilbene exhibits a higher bioavailability and longer half-life. Recently, it is reported that pterostilbene also has neuroprotective effect. Several studies proposed low dose of pterostibene treatment can inhibit cellular stress, inflammation, and Alzheimer disease (AD) pathology in AD animal models which suggested that pterostilbene be a more potent modulator of cognition and cellular stress than resveratrol. Another superiority of pterostilben is its low toxicity, as animal and clinical trial has indicated.However, little is known about the biochemical and molecular mechanisms associated with pterostilbene’s effects on neuronal function.[Objective]The goal of our study was to determine and evaluate the neuroprotection of pterostilbene and its molecular mechanisms on SH-SY5Y cells in the model of H2O2injury.[Methods]H2O2was added to the medium for SH-SY5Y cell culture to construct a neural cell model of H2O2injury, then the cell model was treated with different concentrations of pterostilbene. The following data was observed to analyze the protective effects of pterostilbene. Cell survival rate was determined by MTT assay, and Cell apoptosis was quantified by staining cell with annexin-VFITC/PI, and flow cytometric analysis was used to measure the relative amounts of Annexin-V and propidium iodide (PI) stained cells. The phosphorylation of Akt or ERK molecule was examined by western blotting to determine the signaling pathways involved. We also used two types of antiestrogens:a specific antagonist of ERa,1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy) Phenol-lN-pyrozole dihydrochloride (MPP); and a specific antagonist of ERβ,4-[2-phenyo-5,7-bis(trifluoromrthyl)pyrazolo(1,5-a)pyrimidin-3-yl]phenol (PHTPP) to address which type of ER was involved in pterostilbene’s effects on neural cells.[Results](1) MTT results demonstrated that pterostilbene can attenuate cell loss induced by H2O2in cultured SH-SY5Y cells.(2) Flow cytometric analysis showed pterostilbene can reduce the number of apoptotic and necrotic cells induced by H2O2in cultured SH-SY5Y cells. Western blotting results showed that pterostilbene pretreatment increases Bcl-2protein levels in SH-SY5Y cells.(3) Western blotting results indicated that pterostilbene(?) protective effect of H2O2treatment was mediated by MAPK/ERK and PI3K/Akt pathways.(4) MTT results indicated that addition of MPP inhibited the protection of pterostilbene on the cells treated by H2O2. Western blotting showed that MPP ean also abolish the activation of Akt and ERK signal induced by pterostilbene. However, PHTPP can inhibit neither the neuroprotection nor the activation of signal pathway induced by pterostilbene.[Conclusion]Our results demonstrated that pterostilbene has neuroprotective activity against the injury induced by H2O2in cultured SH-SY5Y cells. We proposed that pterostilbene may act on the membrane composition of ERa, through which to activate the quick signals including MAPK/ERK and PI3K/Akt pathways.