基于RAGE靶标研究牡丹皮萜苷组分调控食源性AGEs诱导肾系膜代谢紊乱的自噬作用与机制

Advanced glycation end products (AGEs) can improve flavor and appearance of food, but its potential harmful effects have attaracted serious concern. The abnormal increase of foodborne AGEs can cause metabolic disorders of kidney mesangial cells, leading to thickening of glomerular basement membrane, which is closely related to the ability of RAGE mediated-autophagy. Our previous study found that the terpene glycoside component of Moutan Cortex could down-regulate RAGE-expression and autophagy levels in mesangial cells induced by foodborne AGEs, however, its underlying mechanism is unclear. We believed that: "The terpene glycoside component from Moutan Cortex may regulate foodborne AGEs-induced metabolic disorders and autophagy of mesangial cells through targeting RAGE, to maintain metabolic homeostasis of mesangial matrix". In this study, we take RAGE as a key target and then utilize dietary AGEs -induced DN mice and mesangial cells HBZY-1 models, combined with transmission electron microscopy, siRNA, immunofluorescence, and other methods, to explore the regulation of the terpene glycoside component of Moutan Cortex on matrix metabolism and autophagy of mesangial cells, and to reveal its regulation on RAGE and RAGE-mediated signaling. This project provides potential targets for the prevention and treatment of foodborne AGEs-induced injury, and also provides scientific evidence to develop health products and food for preventing and treating diabetic nephropathy.

糖基化终末产物 (AGEs)可改善食品的风味和外观，但其潜在的危害作用已引起关注。食源性AGEs的异常增多可引起肾系膜细胞的代谢紊乱，导致肾小球基底膜增厚，这与RAGE介导的自噬能力密切相关。我们前期研究发现牡丹皮萜苷组分可下调食源性AGEs诱导的系膜细胞RAGE表达及自噬水平，但是其作用机制不明确。我们认为：“牡丹皮萜苷类组分可通过RAGE发挥调控食源性AGEs诱导的肾系膜代谢紊乱及自噬作用，以维持系膜基质代谢稳态”。本课题拟以RAGE为靶标，采用含AGEs饲料诱导DN小鼠模型和肾系膜细胞HBZY-1模型，结合透射电镜、siRNA、免疫荧光等方法，探索牡丹皮萜苷类组分对食源性AGEs诱导的系膜细胞基质代谢、自噬的调节作用，揭示其对RAGE的调控作用及信号机制。本项目为防治食源性糖基化产物损伤研究提供潜在的靶点，也为开发防治糖尿病肾病的保健品和食品提供科学依据。

食品中过量的糖基化终末产物 (AGEs)可引起肾系膜细胞的代谢紊乱，与RAGE介导的自噬能力密切相关。本课题基于RAGE靶标，探讨牡丹皮萜苷组分(MCTG)对RAGE介导的系膜代谢紊乱和自噬的调控作用，并揭示其内在作用机制。本课题考察了MCTG保护外源性AGE刺激后HBZY-1细胞系膜损伤的作用，发现其通过调节caspase3蛋白表达抑制AGEs诱导的细胞凋亡，但对细胞周期无影响，另外可显著降低HBZY-1细胞内FN、ColⅣ蛋白及mRNA表达。研究发现在AGEs刺激后系膜细胞后，自噬标志物LC3Ⅱ和Beclin-1的水平分别在4h和8h达到峰值，而p62在4h达到峰值。GFP-RFP-LC3腺病毒转染结果显示AGEs组自噬下游阻滞无法正常融合成自噬溶酶体，而MCTG及芍药苷(PF)组自噬溶酶体数量多于模型组。MCTG及PF还可以下调AGEs刺激后HBZY-1细胞中RAGE和LC3Ⅱ蛋白增加的情况，但均对p62的表达调节无显著性差异。透射电镜观察结果显示，MCTG及PF能降低AGEs刺激的HBZY-1细胞内自噬体数量，并出现典型的自噬小体及自噬溶酶体，此结果与p62蛋白表达结果一致，表明PF及MCTG能降低自噬潮。给药MCTG及PF 12周后，能显著改善DN大鼠血糖、血肌酐、尿蛋白及肾重体重比及肾脏病理特征，并降低肾皮质中FN，ColⅣ及RAGE蛋白表达，上调LC3Ⅱ水平，增加自噬活性，并且抑制mTOR蛋白表达。PF能降低AGEs引起的HBZY-1内LDH的释放，并且下调HBZY-1内RAGE/自噬通路，上调mTOR的表达，从而缓解AGEs引起的ColIV过度产生及细胞损伤。自噬激活剂雷帕霉素干预对RAGE的表达无显著影响，而PF能逆转雷帕霉素对mTOR的抑制作用，抑制雷帕霉素对LC3Ⅱ的促进作用。分子对接研究表明RAGE AC1与芍药苷、丹皮酚结合活性较好，而芍药苷对于RAGE的活性更优。综上所述，MCTG通过降低肾皮质RAGE及抑制mTOR通路来调控食源性AGEs诱导肾系膜代谢紊乱，从而保护和改善糖尿病肾病。目前已在国内外核心期刊上发表论文5篇，其中中文核心期刊论文3篇，SCI刊源文章2篇；培养硕士生研究生4名，本科生2名。本研究为临床治疗及研发糖肾产品提供借鉴。