Sun Binmei, Tan Deguan, Pan Dongjin, Baker Margaret R, Liang Zhibin, Wang Zhizheng, Lei Jianjun, Liu Shaoqun, Hu Ching Yuan, Li Qing X
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI, USA.
College of Horticulture, South China Agricultural University, Guangzhou, China.
J Nutr. 2021 Jul 1;151(7):1717-1725. doi: 10.1093/jn/nxab057.
Obesity is among the most serious public health problems worldwide, with few safe pharmaceutical interventions. Natural products have become an important source of potential anti-obesity therapeutics. Dihydromyricetin (DHM) exerts antidiabetic effects. The biochemical target of DHM, however, has been unknown. It is crucial to identify the biochemical target of DHM for elucidating its physiological function and therapeutic value.
The objective of this study was to identify the biochemical target of DHM.
An abundant antiadipogenic flavanonol was extracted from the herbal plant Ampelopsis grossedentata through bioassay-guided fractionation and characterized with high-resolution LC-MS and 1H and 13C nuclear magnetic resonance. Antiadipogenic experiments were done with mouse 3T3-L1 preadipocytes. A biochemical target of the chemical of interest was identified with drug affinity responsive target stability assay. Direct interactions between the chemical of interest and the protein target in vitro were predicted with molecular docking and subsequently confirmed with surface plasmon resonance. Expression levels of peroxisome proliferator-activated receptor γ (PPARγ), which is associated with 78-kDa glucose-regulated protein (GRP78), were measured with real-time qPCR.
DHM was isolated, purified, and structurally characterized. Cellular studies showed that DHM notably reduced intracellular oil droplet formation in 3T3-L1 cells with a median effective concentration of 294 μM (i.e., 94 μg/mL). DHM targeted the ATP binding site of GRP78, which is associated with adipogenesis. An equilibrium dissociation constant between DHM and GRP78 was 21.8 μM. In 3T3-L1 cells upon treatment with DHM at 50 μM (i.e., 16 μg/mL), the expression level of PPARγ was downregulated to 53.9% of the solvent vehicle control's level.
DHM targets GRP78 in vitro. DHM is able to reduce lipid droplet formation in 3T3-L1 cells through a mode of action that is plausibly associated with direct interactions between GRP78 and DHM, which is a step forward in determining potential applications of DHM as an anti-obesity agent.
肥胖是全球最严重的公共卫生问题之一，安全的药物干预措施很少。天然产物已成为潜在抗肥胖疗法的重要来源。二氢杨梅素（DHM）具有抗糖尿病作用。然而，DHM的生化靶点尚不清楚。确定DHM的生化靶点对于阐明其生理功能和治疗价值至关重要。
本研究的目的是确定DHM的生化靶点。
通过生物测定指导的分级分离从草药植物显齿蛇葡萄中提取一种丰富的抗脂肪生成黄酮醇，并用高分辨率液相色谱-质谱以及1H和13C核磁共振进行表征。用小鼠3T3-L1前脂肪细胞进行抗脂肪生成实验。用药物亲和力响应靶点稳定性分析确定感兴趣化学物质的生化靶点。用分子对接预测感兴趣化学物质与蛋白质靶点在体外的直接相互作用，随后用表面等离子体共振进行确认。用实时定量聚合酶链反应测量与78 kDa葡萄糖调节蛋白（GRP78）相关的过氧化物酶体增殖物激活受体γ（PPARγ）的表达水平。
分离、纯化并对DHM进行了结构表征。细胞研究表明，DHM显著减少3T3-L1细胞内油滴的形成，中位有效浓度为294 μM（即94 μg/mL）。DHM靶向与脂肪生成相关的GRP78的ATP结合位点。DHM与GRP78之间的平衡解离常数为21.8 μM。在3T3-L1细胞中用50 μM（即16 μg/mL）的DHM处理后，PPARγ的表达水平下调至溶剂对照水平的53.9%。
DHM在体外靶向GRP78。DHM能够通过一种可能与GRP78和DHM之间的直接相互作用相关的作用模式减少3T3-L1细胞中的脂滴形成，这在确定DHM作为抗肥胖剂的潜在应用方面向前迈进了一步。
