Department of Pharmacology, Pharmacognosy and Botany, Faculty of Pharmacy, Complutense University of Madrid, Plaza Ramón y Cajal s/n, 28040 Madrid, Spain.
Int J Mol Sci. 2023 Mar 2;24(5):4863. doi: 10.3390/ijms24054863.
Quercetin has been studied extensively for its anti-Alzheimer's disease (AD) and anti-aging effects. Our previous studies have found that quercetin and in its glycoside form, rutin, can modulate the proteasome function in neuroblastoma cells. We aimed to explore the effects of quercetin and rutin on intracellular redox homeostasis of the brain (reduced glutathione/oxidized glutathione, GSH/GSSG), its correlation with β-site APP cleaving enzyme 1 (BACE1) activity, and amyloid precursor protein (APP) expression in transgenic TgAPP mice (bearing human Swedish mutation APP transgene, APPswe). On the basis that BACE1 protein and APP processing are regulated by the ubiquitin-proteasome pathway and that supplementation with GSH protects neurons from proteasome inhibition, we investigated whether a diet containing quercetin or rutin (30 mg/kg/day, 4 weeks) diminishes several early signs of AD. Genotyping analyses of animals were carried out by PCR. In order to determine intracellular redox homeostasis, spectrofluorometric methods were adopted to quantify GSH and GSSG levels using o-phthalaldehyde and the GSH/GSSG ratio was ascertained. Levels of TBARS were determined as a marker of lipid peroxidation. Enzyme activities of SOD, CAT, GR, and GPx were determined in the cortex and hippocampus. ΒACE1 activity was measured by a secretase-specific substrate conjugated to two reporter molecules (EDANS and DABCYL). Gene expression of the main antioxidant enzymes: APP, BACE1, a Disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), caspase-3, caspase-6, and inflammatory cytokines were determined by RT-PCR. First, overexpression of APPswe in TgAPP mice decreased GSH/GSSG ratio, increased malonaldehyde (MDA) levels, and, overall, decreased the main antioxidant enzyme activities in comparison to wild-type (WT) mice. Treatment of TgAPP mice with quercetin or rutin increased GSH/GSSG, diminished MDA levels, and favored the enzyme antioxidant capacity, particularly with rutin. Secondly, both APP expression and BACE1 activity were diminished with quercetin or rutin in TgAPP mice. Regarding ADAM10, it tended to increase in TgAPP mice with rutin treatment. As for caspase-3 expression, TgAPP displayed an increase which was the opposite with rutin. Finally, the increase in expression of the inflammatory markers IL-1β and IFN-γ in TgAPP mice was lowered by both quercetin and rutin. Collectively, these findings suggest that, of the two flavonoids, rutin may be included in a day-to-day diet as a form of adjuvant therapy in AD.
槲皮素因其抗老年痴呆症 (AD) 和抗衰老作用而受到广泛研究。我们之前的研究发现，槲皮素及其糖苷芦丁可调节神经母细胞瘤细胞的蛋白酶体功能。我们旨在探索槲皮素和芦丁对转基因 TgAPP 小鼠（携带人瑞典突变 APP 转基因，APPswe）脑内细胞内氧化还原稳态（还原型谷胱甘肽/氧化型谷胱甘肽，GSH/GSSG）的影响，及其与β-位点 APP 切割酶 1（BACE1）活性和淀粉样前体蛋白（APP）表达的关系。由于 BACE1 蛋白和 APP 加工受泛素蛋白酶体途径调节，并且 GSH 补充可保护神经元免受蛋白酶体抑制，我们研究了含有槲皮素或芦丁（30mg/kg/天，4 周）的饮食是否会减少 AD 的几个早期迹象。通过 PCR 对动物进行基因分型分析。为了确定细胞内氧化还原稳态，采用荧光光谱法使用邻苯二醛定量测定 GSH 和 GSSG 水平，并确定 GSH/GSSG 比值。TBARS 水平被确定为脂质过氧化的标志物。在皮质和海马中测定 SOD、CAT、GR 和 GPx 的酶活性。通过与两个报告分子（EDANS 和 DABCYL）偶联的特异性酶切底物测量 BACE1 活性。通过 RT-PCR 测定主要抗氧化酶的基因表达：APP、BACE1、一种含有解整合素和金属蛋白酶结构域的蛋白 10（ADAM10）、caspase-3、caspase-6 和炎性细胞因子。首先，与野生型（WT）小鼠相比，TgAPP 小鼠中 APPswe 的过度表达降低了 GSH/GSSG 比值，增加了丙二醛（MDA）水平，并且总体上降低了主要抗氧化酶的活性。用槲皮素或芦丁处理 TgAPP 小鼠增加了 GSH/GSSG，降低了 MDA 水平，并有利于酶抗氧化能力，特别是芦丁。其次，在 TgAPP 小鼠中，槲皮素或芦丁均降低了 APP 表达和 BACE1 活性。关于 ADAM10，它在 TgAPP 小鼠用芦丁治疗时趋于增加。至于 caspase-3 表达，TgAPP 显示出增加，而芦丁则相反。最后，在 TgAPP 小鼠中，炎性标志物 IL-1β 和 IFN-γ 的表达增加被槲皮素和芦丁降低。总的来说，这些发现表明，在这两种类黄酮中，芦丁可能被包含在日常饮食中，作为 AD 的辅助治疗形式。
