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  • In this study SPR AIRS was

    2019-04-25

    In this study, SPR-AIRS was applied to screen p38 ligands from herbal extracts. p38 is an important drug target, which plays a key role in cell proliferation, apoptosis and tumorigenesis [22]. Firstly, recombinant p38 protein was immobilized on SPR chip, the activity and feasibility of the chip was validated by positive inhibitor SB203580. Then thirty-four p38-related medicinal herbs were selected and pre-screened. Two medicinal herbs having high response signal with p38-immobilized chip, Folium Ginkgo and Herba Artemisiae Scopariae, were injected into SPR system for ligand fishing. Subsequently, the recovered compounds were analyzed by UPLC-Q-TOF-MS to confirm their structures. Candidates were identified through the comparison between herbal extracts and recovered samples. Totally, two active compounds, eupatilin (EPT) and ginkgolide B (GKB), were identified by SPR-AIRS. Affinity constants of these compounds were 21.68 ± 2.21 and 44.71 ± 1.80 μM, respectively, determined by SPR assays. Enzyme linked immunosorbent assay (ELISA) revealed that EPT and GKB could inhibit the cellular activities of p38 significantly. Molecular docking showed that EPT and GKB could bind to the ATP binding site of p38. Furthermore, EPT and GKB could significantly inhibit proliferation, induce apoptosis and G2/M cholecystokinin receptor arrest against K562 cell line. This is the first time that EPT and GKB are reported as effective p38 binding ligands. These results prove that SPR-AIRS could be an effective method to screen active compounds acting on a specific protein from complex systems.
    Methods
    Results and discussion
    Conclusion
    Introduction Atherosclerosis leads to narrowing of the vessel and many cardiovascular complications. Vascular calcification is a manifestation of the atherosclerotic lesions, which reduces elasticity and compliance of the vessel wall [1], and is an independent risk factor for subsequent cardiovascular mortality [2], [3]. Previously considered a passive calcium deposition, vascular calcification has now been determined as an active cell-regulated process resembling bone modeling [4]. We and others have demonstrated that osteogenic differentiation of vascular smooth muscle cells (VSMC) is important for the development of vascular calcification [5], [6], [7]. Increased oxidative stress plays a critical role in promoting atherosclerosis [8] and vascular calcification [5]. In the atherosclerotic lesions of the high-fat-fed ApoE deficient mice (ApoE-/-), we have shown that increased oxidative stress is associated with increased vascular calcification [7]. Using a primary VSMC culture system, we have determined that oxidative stress induces vascular calcification through activation of AKT signaling pathways that upregulate the key osteogenic transcription factor, Runx2 [5]. Furthermore, activation of cholecystokinin receptor AKT in diabetic mice by O-GlcNAcylation also promotes VSMC calcification [9]. Consistently, constitutively activated AKT was sufficient to promote VSMC calcification in vitro and vascular calcification in animals in vivo [10]. These studies have supported the concept that activation of AKT signaling mediates oxidative stress and hyperglycemia-induced vascular calcification. Sodium dichloroacetate (DCA) is a small molecule that has been shown to inhibit AKT activation while increasing oxidative stress in cancer cells [11], [12]. Early studies have found that DCA reduce blood glucose in diabetic dogs [13] and fasting hyperglycemia in diabetic patients [14]. It has also been recently been tested in phase I clinical trials to treat patients with advanced solid tumors [15], [16]. DCA exhibits potent anti-cancer activity in cancer cells [17], [18] and animal models [12], [19], [20], via inducing oxidative stress that causes apoptosis in cancer cells [21], [22]. Similarly, DCA exhibited protective effects on hypoxia-induced pulmonary hypertension via inducing apoptosis of the pulmonary smooth muscle cells [23], [24]. Most recently, DCA was found to prevent injury-induced restenosis in animal models by inducing apoptosis of smooth muscle cells [25]. Although the effects of DCA on vascular calcification have not been investigated, the regulation of DCA on the pro-calcification signals, oxidative stress, AKT and cell death, suggests that it may play a role in the development of vascular calcification.