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Three Polymethoxyflavones Purified from Ougan ( Citrus reticulata Cv. Suavissima ) Inhibited LPS-Induced NO Elevation in the Neuroglia BV-2 Cell Line via the JAK2/STAT3 Pathway

Yue Wang, Wenjing Zang, Shiyu Ji, Jinping Cao, Chongde Sun
Nutrients 2019 April 5, 11 (4)
In order to establish an efficient method for separation of polymethoxyflavones (PMFs) and explore the anti-inflammatory mechanism of PMF monomers, a citrus variety rich in PMFs, Ougan ( Citrus reticulata cv. Suavissima ), was selected, and three monomers, including nobiletin, tangeretin, and 5-demethylnobiletin, were purified by ultrasonic-assisted extraction, solid phase extraction, and high-speed countercurrent chromatography separation. UPLC-MS was used to identify the three monomers. UPLC determined purities of 99.87% to nobiletin, 99.76% to tangeretin, and 98.75% to 5-demethylnobiletin with the standard curve method. A lipopolysaccharide (LPS)-induced NO releasing model was performed in the mouse microglia BV-2 cell line. Results illustrated that PMF monomers inhibited the NO release and the inflammation-related cytokines, including IL-1β, IL-6, and TNFα elevation. QRT-PCR revealed that PMFs alleviated LPS-induced upregulation of iNOS , IL-6 , JAK2 , TNF α, IL-1β , and NF- κ B and LPS-induced downregulation of I κ B α, while they did not affect TLR1 , TLR2 , TLR4 , and TLR6 . STAT3 expression was repressed by tangeretin and 5-demethylnobiletin, but not by nobiletin. Western blot assay also showed a suppression of expression and phosphorylation of JAK2 by all three PMF monomers, while STAT3 phosphorylation was restrained by tangeretin and 5-demethylnobiletin. The mechanism was primarily verified by the JAK2 inhibitor Ruxolitinib and the STAT3 inhibitor Stattic.


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