Effects of active ingredients from Limonium Sinense (Girard) Kuntze extracts: a potential link with hypoxia-inducible factor (HIF) activation

Abstract

Limonium Sinense (Girard) Kuntze is a traditional Chinese medicinal herb with a wide range of therapeutic uses, including fever, hepatitis, haemostasis, anaemia, menorrhagia, irregular menstruation, and other disorders. Recent studies revealed the antioxidant, anti-tumour, anti-hepatitis, anti-viral, and immunomodulatory activities of Limonium Sinense amongst others. However, the precise mechanisms underlying its pharmacological activities remain largely unknown. This study aimed to investigate the effects of bioactive ingredients from Limonium Sinense and elucidate their underlying mechanisms. The therapeutic effects of Limonium Sinense were predicted using a network pharmacology approach. Integrated analysis including bioinformatic analysis and in vitro experiments was employed to explore the effects of bioactive ingredients from Limonium Sinense. A structure-based virtual screening approach was conducted to identify potential inhibitors of prolyl hydroxylase domain protein 2 (PHD2) from Hydroxybenzoic acids and their derives. The network pharmacology analysis revealed the involvement of the breast cancer pathway and several cancer-related pathways in the biological effects of Limonium Sinense. Water extracts from Limonium Sinense (LSW) showed a strong growth inhibitory effect on multiple cells in both 2D and 3D cultures. Global transcriptomic profiling and connectivity map (CMap) analysis identified several similarly acting therapeutic candidates, including Tubulin inhibitors and hypoxia-inducible factor (HIF) modulators. The effect of LSW on the cell cycle was verified with flow cytometry showing a G2/M phase arrest. Integrated analysis suggested a role for gallic acid in mediating HIF activation. Additionally, three potent PHD2 inhibitors were identified, which can potentially bind to the 2OG binding pocket of PHD2, leading to the inactivation of the enzyme. Taken together, this study provides novel insights into the bioactive ingredients in Limonium Sinense, highlighting the rich natural resource and therapeutic values of herbal plants. The whole plants of Limonium Sinense were collected from the coastal region in Jiangsu, eastern China (33°09'33.0" N, 120°46'40.4" E). The water extracts from Limonium Sinense (LSW) exhibit a significant capability to induce cell cycle arrest at G2/M checkpoint, leading to the inhibition of cell growth. On the other hand, LSW prominently stimulates the upregulation of HIF-1α at the protein level, leading to its accumulation. The HIF-1α then translocates into the nucleus, where it binds with co-factors HIF-1β, p300/CBP and HRE, ultimately activating the transcription of its target genes. CBP, CREB-binding protein; HRE, hypoxia response element; p300, E1A binding protein p300; HIF-1α, Hypoxia-inducible factor 1 alpha; HIF-1β, Hypoxia-inducible factor 1 beta; LSW: Limonium Sinense water extract.

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Identifiers

ORCID for Hualong Zhao: orcid.org/0000-0002-5009-1444

ORCID for Yihua Wang: orcid.org/0000-0001-5561-0648

ORCID for Phil Williamson: orcid.org/0000-0002-0231-8640

ORCID for Rob Ewing: orcid.org/0000-0001-6510-4001

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