Total polyphenol of Anemarrhena asphodeloides ameliorates advanced glycation end products-induced endothelial dysfunction by regulation of AMP-Kinase (知母多酚通过激活AMP-激酶来缓解糖基化终末产物引起的内皮损伤)

Objective Anemarrhena asphodeloides Bunge is widely used in China for the treatment of diabetes and the polyphenol components are responsible for its anti-diabetic action. This study aimed to investigate the effect of total polyphenol of Anemarrhena asphodeloides (TPAA) on endothelial dysfunction and to elucidate underlying mechanisms. Methods We stimulated endothelial cells with advanced glycation end products (AGEs) to establish the model of endothelial dysfunction in vitro and observed the effect of TPAA (10, 30, or 100 μg/mL) on AMP-Kinase (AMPK) activation implicated in regulation of nitric oxide (NO) and endothelin-1 (ET-1) production. Meanwhile, nuclear factor-κB (NF-κB) activation, intracellular reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) and eNOS expression were investigated by western blot, fluorescence microscopy and real time-quantitative PCR analysis, respectively. Results Total polyphenol of Anemarrhena asphodeloides enhanced AMPK phosphorylation and promoted the basal NO production along with the inhibition of ET-1 secretion in endothelial cells. TPAA inhibited NF-κB activation by attenuating p65 phosphorylation and suppressed ROS production, well demonstrating its action in inhibition of ROS-associated inflammation in the endothelium. Meanwhile, TPAA protected mitochondrial function and endothelial homeostasis against AGEs insult by restoring ΔΨm and mRNA expression of eNOS. AGEs stimulation inhibited AMPK activation and induced the loss of NO production together with increased secretion of ET-1, but these changes were reversed by TPAA in a concentration-dependent manner. Compound C, an AMPK inhibitor, attenuated the effects of TPAA mentioned above, indicating the involvement of AMPK. Conclusions Total polyphenol of Anemarrhena asphodeloides inhibited AGEs-induced ROS-associated inflammation and ameliorated endothelial dysfunction through beneficial regulation of AMPK activation. 摘要 背景 在中国，知母多酚被广泛用于治疗糖尿病，其中的多酚成分是治疗糖尿病的主要成分。本研究目的旨在探究知母多酚在改善内皮损伤方面的作用及其可能的作用机制。 方法 我们用糖基化终末产物（advanced glycation end products，AGEs）来刺激内皮细胞并以此建立内皮损伤体外模型，观察三个剂量组的知母多酚（10、30或100 µg/mL）对AMP-激酶（AMP-Kinase，AMPK）的活化、NO的释放以及ET-1分泌方面的影响。以此同时，我们通过Western Blot法检测炎症通路NF-κB信号通路的活化，通过荧光法检测ROS、线粒体膜电位的变化，通过实时定量PCR法检测eNOS的表达。 结果 知母多酚可以促进AMPK磷酸化的表达以及增加NO的释放，同时抑制内皮细胞ET-1的分泌。知母多酚通过减少p65磷酸化以及抑制ROS的产生来减少炎症通路NF-κB的活化，这些结果证明了知母多酚对内皮细胞的保护作用是通过抑制ROS相关的炎症通路而产生的。同时，知母多酚通过重建线粒体膜电位以及促进eNOS的mRNA表达来抵抗AGEs所致的损害，保护线粒体功能和内皮细胞内稳态。AGEs可抑制AMPK的磷酸化，减少NO的产生，增加ET-1的分泌，但是这些现象可以被知母多酚呈剂量依赖性的逆转。Compound C作为一种AMPK的抑制剂，可以减弱知母多酚的上述作用，提示知母多酚改善内皮紊乱的作用是通过激活AMPK产生的。 结论 知母多酚通过激活AMPK通路来抑制AGEs引起的ROS相关的炎症表达并缓解内皮损伤。