Ginsenoside Rg1 Attenautes Amyloid-β(Aβ) Generation By Regulating β Secretase Through PPARγ And NF-κB In N2a/APP695 Cells

Alzheimer's disease (AD) is an age-related neurodegenerative disorder resulting in progressive neuronal death and memory loss. The disease is neuropathologically characterized by the presence of both neurofibrillary tangles and neuritic plaques composed of aggregates of amyloid-β(Aβ) protein, as well as synapse and neuronal loss. Aβis a 40-43 aa proteolytic product of the amyloid precursor protein (APP) and is generated through sequential cleavages by enzymes calledβ- andγ-secretase. Increasing numerous studies demonstrate that Aβ1-40 and Aβ1-42 levels are increased in postmortem AD brains, which is correlated with cognitive memory deficits. Mutated APP may result in changes in efficiency and outcome of enzymatic cleavage byβ- andγ-secretase, which may contribute to pathological increase in toxic fragments of Aβin the brain.Physiologically, most of the APP is cleavaged byα-secretase, which do not produce Aβfragment and therefore it is called as non-amyloidgenesis pathway. In contrast, some toxic Aβfragments are produced by amyloidgenesis pathway when APP is enzymatically cleavaged byβ- andγ-secretase.β-secretase is considered as rate-limiting enzyme during this process.β-secretase is composed byβ-site amyloid precursor protein cleaving enzyme 1 (BACE1) and 2 (BACE2) with a predominant role of BACE1 in Aβgeneration. The transcription and translation of BACE1 is modified by various physiological and pathological stimuli. Importantly, BACE1 promoter contains some pivotal transcription factor binding sites such as PPARγand NF-κB, which may resulted in PPARγor NF-κB-mediated inhibition of BACE1 expression. Ginsenoside Rg1 is a representative effective component of the extracts of Ginseng. Recent domestic studies have demonstrated that ginsenoside Rg1 consumption prevented memory loss and improved cognitive function in a variety of animal models. Therefore, this study was designed to investigate whether ginsenoside Rg1 can potentially delay or reverse neuropathological process in AD by modifying APP metabolism and Aβgeneration. In the present study, N2a/APP695 cells, an mutated APP overexpressed neuronal cell line, was applied to model APP metabolism and Aβgeneration in vitro. Firstly, soluble Aβwas measured by ELISA and BACE1 mRNA and protein levels were detected by RT-PCR and Western blot, respectively. The enzymatically cleavaged APP fragment such asβ-CTF andα-CTF were determined simultaneously. Finally, the expression levels and subcellular localization of PPARγand NF-κB were observed by Western blot and confocal laser scanning microscope, respectively. The results are as follows:1 Ginsenoside Rg1 treatment significantly reduced the extracellular levels of Aβ1-40 and Aβ1-42 in N2a/APP695 cells in a dose- and time-dependent manner. Additionally, there was no significantly change in APP level after Rg1 treatment when compared with vehicle-treated alone.2 Treatment of ginsenoside Rg1 at dose of 2.5μM for 6-12 h dramatically down-regulated the mRNA and protein expression levels of BACE1. Moreover, Rg1 treatment resulted in an reduction of protein level ofβ-CTF, a APP fragment cleavaged byβ-secretase. In contrast,α-CTF, a APP fragment cleavaged byα-secretase, was markedly increased after Rg1 treatment.3 Treatment of ginsenoside Rg1 at dose of 2.5μM increased the protein level of PPARγand NF-κB p65 in N2a/APP695 cells, and consequently, resulted in translocation of PPARγand NF-κB from cytoplasm to nuclear in a time-dependent manner.In summary, these results suggest that ginsenoside Rg1 may modify the APP metabolism by enhancing nuclear PPARγand NF-κB binding into BACE1 promoter, and thereby inhibiting the transcription and translation of BACE1, which may ultimately attenuate Aβgeneration by amyloidgenesis pathway. Therefore, ginsenoside Rg1 is potentially a highly effective therapeutic agent in treating AD.