从AMPK介导的自噬研究衰老大鼠小肠粘膜屏障功能障碍的分子机制及淫羊藿总黄酮的调节作用

Intestine plays a prominent role in organismal aging and intestinal mucosal barrier dysfunction is an important characteristic in intestinal aging. Recent studies have shown that the decrease of autophagy induced by AMPK in the intestine plays a major role in the intestinal barrier dysfunction in aging Drosophila, which is unclear in aging mammals. Our previous studies showed that autophagy was decreased, and the expression of activated AMPK was reduced in aging rats, suggesting that the decrease of autophagy induced by AMPK may be one of the mechanisms in intestinal aging in mammals. Many Studies have reported that Epimedium Flavoniods, one of the main active components of extracted from Herba Epimedii, have good anti-aging effects. Our preliminary experiments discovered that Epimedium Flavoniods could improve intestinal mucosal barrier dysfunction. However, whether Epimedium Flavoniods improve intestinal mucosal barrier dysfunction of aging rats via AMPK-induced autophagy in intestinal epithelia remains unclear. This project will study the levels of serum D-lactic acid, DAO, inflammatory factors, LC3, and Beclin1 in aging rats via ELISA, Immunohistochemistry and Western Blot to explore the mechanisms of intestinal aging in rats and the effects of Epimedium Flavoniods, providing a new strategy for the treatment of intestinal aging.

肠道衰老是机体衰老的重要表现，粘膜屏障功能障碍是导致肠道衰老的基础，但其机制仍不明确，亦缺乏有效防治措施。最近果蝇的研究显示，AMPK介导的自噬与其肠道衰老相关，但哺乳动物衰老肠道的屏障功能障碍是否与AMPK介导的自噬减少有关仍有待研究；我们前期研究发现，衰老大鼠肠道细胞自噬与活化的AMPK减少，提示AMPK介导的自噬减少可能是哺乳动物肠道衰老的机制之一。淫羊藿总黄酮为中药淫羊藿的主要活性部位，具有抗衰老等药理作用。我们预实验结果显示，淫羊藿总黄酮可改善衰老大鼠肠道粘膜屏障功能，但其能否调控AMPK介导的自噬未有研究。基于此，本项目拟采用ELISA、免疫组化、Western Blot等方法，检测衰老大鼠血清D-乳酸、DAO等的水平、炎症因子、LC3、Beclin1等表达情况，从自噬的角度探讨衰老大鼠肠道屏障功能障碍的分子机制、以及淫羊藿总黄酮的干预作用，为肠道衰老的防治提供新策略。

肠道衰老是机体衰老的重要表现，粘膜屏障功能障碍是导致肠道衰老的基础，本项目从自噬的角度探讨衰老大鼠肠道屏障功能障碍的分子机制、以及淫羊藿总黄酮的干预作用，为肠道衰老的防治提供新策略。结果显示：衰老模型组大鼠血清D-乳酸含量与成年对照组相比显著增加，用药之后有所降低。与对照组相比，衰老模型组大鼠小肠claudin-1，occludin，p-AMPK，Lc3Ⅱ，Beclin1表达量降低；给予淫羊藿总黄酮保护后，小肠紧密连接蛋白与自噬相关蛋白的表达量增加。以上结果提示淫羊藿总黄酮可能通过上调AMPK介导的自噬，增加肠上皮细胞间紧密连接蛋白的表达，从而改善衰老大鼠肠道粘膜屏障功能障碍。进一步研究显示：与成年组大鼠相比，24月龄衰老大鼠肠道上皮细胞排列疏松紊乱，Bax/Bcl-2比值显著增加，Nrf2、HO-1、Foxo3a、Sirt1 表达显著降低；而淫羊藿总黄酮可改善衰老大鼠肠道上皮细胞排布、增加肠粘膜厚度，显著降低Bax/Bcl-2 比值，并显著增加Nrf2、HO-1、Foxo3a、Sirt1 表达。以上结果提示淫羊藿总黄酮可能通过抗氧化应激改善衰老大鼠肠道粘膜屏障功能障碍。此外，我们还进一步研究了淫羊藿总黄酮的主要活性成分淫羊藿苷对衰老大鼠肠道屏障功能的调节作用，研究显示：给予淫羊藿苷处理的大鼠，与衰老组大鼠相比，肠道组织的SOD、CAT、GSH-PX值升高，紧密连接蛋白claudin-1和claudin-5的表达增加，提示淫羊藿苷也可通过抗氧化应激改善肠道屏障功能。