厚朴叶兰（Anoectochilus roxburghii (Wall.) Lindl. ，简称AR）作为一种非常有价值的中药，已被广泛用于治疗肝炎、癌症、糖尿病等。然而，AR对抗衰老及与衰老相关的学习记忆减退的效果和主要作用成分尚未被探索。本研究旨在探究AR水提取物（AEAR）对衰老及其相关的学习记忆损伤的保护作用，并进一步研究AEAR的主要活性成分和作用机制。D-半乳糖（D-gal）诱导的衰老小鼠和暴露于L-谷氨酸（Glu）的HT22细胞分别用作体内和体外模型。通过使用Morris水迷宫测试、免疫组化染色、生物化学分析等方法，探究了AEAR对衰老及其相关的学习记忆减退的影响。通过细胞活力试验、生物化学分析、qRT-PCR、Western blot和分子对接研究，探讨了AEAR和金码苷（Kin）在体外的抗氧化作用及其机制。用含有69.52±0.85% Kin的AEAR进行为期63天的治疗（内服），缓解了衰老小鼠生长速度下降、脑、肝和胸腺指数异常以及学习记忆能力下降。同时，AEAR抑制了衰老小鼠血清和脑组织中SOD和GSH-PX活性的下降，GSH/GSSG比值的降低以及MDA的增加，并促进了D-gal诱导的衰老小鼠脑组织中Nrf2核转运。AEAR在缓解衰老小鼠异常生理特征、减轻学习记忆损伤和抑制氧化应激方面的效果与维生素C（Vc）相似甚至更好。在暴露于Glu的HT22细胞中，Kin增加了细胞活力，上调了SOD和GSH-PX的活性，增强了GSH/GSSG比值，并下调了MDA，这一点优于AEAR。Kin上调了p-ERK1/2与ERK1/2的比值，促进了Nrf2的核转运和其下游靶基因（HO-1，NQO-1，GCLC和GCLM）在mRNA和蛋白水平上的表达，这与AEAR的结果一致。进一步的分子对接结果也证实了Kin与ERK1和ERK2具有较强的结合能力。本研究表明，作为AR的主要活性成分，Kin可通过激活ERK/Nrf2信号通路来减轻衰老小鼠中的氧化应激，从而减缓衰老和与衰老相关的学习记忆损伤。版权所有 © 2023. Elsevier B.V. 发表。

Anoectochilusroxburghii (Wall.) Lindl. (AR), as an exceptionally valuable traditional Chinese medicine, has been widely used to treat hepatitis, cancer, diabetes, etc. But, the effects and the primary functioning element of AR on attenuating aging and aging-related learning and memory degradation has not yet been explored.This study aimed at exploring the protective property of aqueous extract of AR (AEAR) on alleviation of aging and aging-related learning and memory impairment in vivo, and further investigating the main active ingredient and mechanism of AEAR.D-galactose(D-gal) induced aging mice and HT22 cells exposed with L-Glutamic acid (Glu) were used as in vivo and in vitro model, separately. The effects of AEAR on aging and aging-related learning and memory degradation were explored by using morris water maze test, immunohistochemistry staining, biochemistry assay, etc. The effects and mechanism of AEAR and Kinsenoside (Kin) on antioxidation in vitro were investigated by cell viability assay, biochemistry assay, qRT-PCR, western blotting and molecular docking studies.Treatment with AEAR (containing 69.52 ± 0.85% Kin, i.g.) for 63 days, alleviated low growth rate, abnormal brain, liver and thymus index, and decline in learning and memory capability of aging mice. Meanwhile, AEAR inhibited the decreased activities of SOD and GSH-PX, the decline in the ratio of GSH to GSSG, and the increase of MDA in both serum and brain, and also promoted the Nrf2 nuclear translocation in brain of aging mice induced by D-gal. The effects of AEAR on alleviating abnormal physiological characteristics, attenuating learning and memory impairment, and inhibiting oxidative stress in aging mice was similar to or even better than that of Vc. In HT22 cells exposed with Glu, Kin increased the cell viability, up-regulated the activities of SOD and GSH-PX, enhanced the ratio of GSH to GSSG, and down-regulated MDA, which was superior to AEAR. Kin up-regulated the ratio of p-ERK1/2 to ERK1/2, promoted the Nrf2 nuclear translocation and its downstream target genes, i.e. HO-1, NQO-1, GCLC and GCLM expression at the mRNA and protein levels, which were consistent with AEAR. Further, molecular docking results also confirmed that Kin had strong binding energy with ERK1 and ERK2.The present study indicated that Kin could alleviate the oxidative stress in aging mice via activating the ERK/Nrf2 signaling pathway, in order to attenuate aging and aging-related learning and memory impairment, as the main active ingredient of AR.Copyright © 2023. Published by Elsevier B.V.