背景:2型糖尿病(T2DM)是一种以血糖水平升高为特征的慢性代谢紊乱,由于其患病率的增加,构成了重大的全球健康问题。胰岛素抵抗(IR)在T2DM的发生发展中起着重要作用,并且通常与肥胖等因素有关。缺乏身体活动,和久坐不动的生活方式。最近,人们对探索天然产物改善胰岛素敏感性和葡萄糖代谢的潜力越来越感兴趣。其中,锁阳。,一种可食用的寄生植物,已显示出有希望的抗糖尿病作用。然而,关于其对IR有益作用的研究仍处于起步阶段。因此,本研究旨在探讨锁阳类黄酮富集部位(CSF)在T2DMIR治疗中的应用,以及阐明所涉及的化学和生化机制。
方法:首先,利用UHPLC/ESI-LTQ-Orbitrap-MS进行CSF的化学分析。随后,糖原合成,使用高葡萄糖高胰岛素诱导的IR模型对HepG2细胞进行糖异生和葡萄糖消耗测定,以说明CSF对IR的有利影响。然后,我们进行了一项创新的网络药理学分析,以预测对CSF抗IR的保护作用有贡献的潜在化学成分和中枢基因.为了进一步阐明分子间的相互作用,进行了分子对接研究,重点关注CSF活性成分与关键靶标之间的结合相互作用。此外,RNA测序分析用于揭示导致CSF有益作用的潜在生化信号通路。为了验证这些发现,Westernblot和qPCR分析用于验证与IR相关的途径以及导致IR改善的潜在信号级联。
结果:UHPLC/ESI-LTQ-Orbitrap-MS分析成功鉴定出总共36种源自CSF的类黄酮。此外,CSF显示显着改善IR的HepG2细胞的糖原合成和葡萄糖消耗以及抑制糖异生。一项创新的网络药理学分析揭示了代谢综合征相关信号通路的关键枢纽基因AKT1和PI3K,这有助于CSF对IR的有利影响。值得注意的活性成分,包括槲皮素,鞣花酸和柚皮素被确定为这些影响的潜在贡献者。蛋白质印迹和qPCR测定的结果提供了令人信服的证据,即CSF通过调节PI3K-Akt信号通路来改善胰岛素敏感性。随后的RNA测序分析,与蛋白质印迹分析相结合,深入研究了CSF对IR的有利作用的潜在机制,可能与内质网(ER)蛋白抑制的增强有关。
结论:CSF在HepG2细胞的IR模型中表现出显著的增强胰岛素敏感性的能力。这通过糖原合成和葡萄糖消耗的增强来证明,以及它对糖异生的抑制作用。此外,CSF显示胰岛素介导的PI3K-Akt信号传导途径的改善。潜在的活性成分被鉴定为槲皮素,鞣花酸和柚皮素.负责CSF对IR的有益作用的潜在生化机制与其减轻ER应激的能力密切相关。从而提供对其保护作用的全面了解。
BACKGROUND: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by elevated blood glucose levels, posing a significant global health concern due to its increasing prevalence. Insulin resistance (IR) plays a major role in the development of T2DM and is often linked to factors such as obesity, physical inactivity, and a sedentary lifestyle. Recently, there has been growing interest in exploring the potential of natural products for improving insulin sensitivity and glucose metabolism. Among these, Cynomorium songaricum Rupr., an edible parasitic plant, has shown promising antidiabetic effects. However, research on its beneficial effects on IR is still nascent. Therefore, this study aims to investigate the application of a Cynomorium songaricum flavonoid-enriched fraction (CSF) in the treatment of IR in T2DM, along with elucidating the chemical and biochemical mechanisms involved.
METHODS: First, UHPLC/ESI-LTQ-Orbitrap-MS was utilized to perform a chemical profiling of CSF. Subsequently, glycogen synthesis,
gluconeogenesis and glucose consumption assays were conducted on HepG2 cells with a high glucose high insulin-induced IR model to illustrate the favorable impacts of CSF on IR. Then, an innovative network pharmacology analysis was executed to predict the potential chemical components and hub genes contributing to CSF\'s protective effect against IR. To further elucidate molecular interactions, molecular docking studies were performed, focusing on the binding interactions between active constituents of CSF and crucial targets. Additionally, an RNA-sequencing assay was employed to uncover the underlying biochemical signaling pathway responsible for CSF\'s beneficial effects. To validate these findings, western blot and qPCR assays were employed to verify the pathways related to IR and the potential signaling cascades leading to the amelioration of IR.
RESULTS: The UHPLC/ESI-LTQ-Orbitrap-MS analysis successfully identified a total of thirty-six flavonoids derived from CSF. Moreover, CSF was shown to significantly improve glycogen synthesis and glucose consumption as well as inhibit
gluconeogenesis in HepG2 cells of IR. An innovative network pharmacology analysis unveiled key hub genes-AKT1 and PI3K-integral to metabolic syndrome-related signaling pathways, which contributed to the favorable impact of CSF against IR. Noteworthy active ingredients including quercetin, ellagic acid and naringenin were identified as potential contributors to these effects. The results of western blot and qPCR assays provided compelling evidence that CSF improved insulin sensitivity by modulating the PI3K-Akt signaling pathway. Subsequent RNA-sequencing analysis, in tandem with western blot assays, delved deeper into the potential mechanisms underlying CSF\'s advantageous effects against IR, potentially associated with the enhancement of endoplasmic reticulum (ER) proteostasis.
CONCLUSIONS: CSF exhibited a remarkable ability to enhance insulin sensitivity in the IR model of HepG2 cells. This was evident through enhancements in glycogen synthesis and glucose consumption, along with its inhibitory impact on
gluconeogenesis. Furthermore, CSF demonstrated an improvement in the insulin-mediated PI3K-Akt signaling pathway. The potential active constituents were identified as quercetin, ellagic acid and naringenin. The underlying biochemical mechanisms responsible for CSF\'s beneficial effects against IR were closely linked to its capacity to mitigate ER stress, thereby offering a comprehensive understanding of its protective action.