Polymethoxyflavones in Citrus Regulate Lipopolysaccharide‐Induced Oscillating Decay of Circadian Rhythm Genes by Inhibiting Nlrp3 Expression

Abstract

The aim of this study is to compare the regulatory abilities of citrus flavonoids on the oscillating expression of circadian genes. Seven varieties of citrus fruits and twenty-five citrus flavonoids were selected and evaluated. Per2 luciferase bioluminescence report system and serum shock were used to induce circadian gene expression in mouse microglia BV-2 cells. In vivo experiments were carried out using C57BL6/J mice to evaluate the regulation of flavonoids on the oscillatory expression of liver biorhythm genes. Lipopolysaccharide was used to interfere the gene oscillating expression. QRT-PCR was performed to detect the expression of circadian rhythm-related genes, including Clock, Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-erbα, Rev-erbβ, Rorα, Dbp, and Npas2. The results show that the polymethoxyflavones (PMFs) exerted stronger circadian gene regulatory capability, while the flavonoids containing glycosides showed no biological activity. Also, all tested flavonoids decreased LPS-induced nitric oxide release, but only polymethoxyflavones inhibited circadian rhythm disorder. PMFs inhibited Nlrp3 inflammasome-related genes and proteins, including Nlrp3, IL-1β, ASC, and Caspase1, while other flavonoids only affected IL-1β and Caspase1 expression. This mechanism was preliminarily verified using the Nlrp3 inhibitor INF39.

Effects of (Poly)phenols on Circadian Clock Gene–Mediated Metabolic Homeostasis in Cultured Mammalian Cells: A Scoping Review

“…The studies included in this review were published from 2008 to 2024, with the majority published after 2016, reflecting the growing scientific interest in (poly)phenols and circadian biology. Table 2 [ [67] , [68] , [69] , [70] , [71] , [72] , [73] , [74] , [75] , [76] , [77] , [78] , [79] , [80] , [81] , [82] , [83] , [84] , [85] , [86] , [87] , [88] , [89] , [90] , [91] , [92] , [93] , [94] , [95] , [96] , [97] , [98] , [99] , [100] , [101] , [102] , [103] , [104] , [105] , [106] , [107] , [108] , [109] ] summarizes the main characteristics and outcomes of the included studies.…”

“… Wang et al. [ 87 ], 2021 7 Citrus fruit extracts 6 N/A Mouse BV-2 2-h Serum shock (50% horse serum) Pretreatment citrus flavonoid extracts or NOB/12 h and then 0.1 μg/mL LPS/12 h. Control (0.05% DMSO) and LPS Per2 luciferase reporter system knock-in (PER2::Luc) Almost all flavonoid extracts ↑ PER2 mRNA Nobiletin (NOB) 10 ↑ CLOCK, BMAL1, PER1/2/3, CRY1/2, Rev-Erbα/β, RORα, DBP, and NPAS2 mRNAs Okada and Okada [ 95 ], 2020 Resveratrol (Res) 100 Human lung fibroblast cells (TIG-1-20, young cells; and TIG-1-60, old cells) None Treatment with Res/4 h. Control (0.1% DMSO). ↑ BMAL1 mRNAs in both types of fibroblast cells.…”

“…Six studies recorded the rhythms of PERs for 4–7 d, of which 5 focused on PER2 and only 1 on PER1, using luciferase-expressing reporter cells. Exposure of mouse microglia BV-2 cells to 7 citrus flavonoid extracts stimulated PER2 expression and the findings suggested that an enhanced oscillation of PER2 and inhibitory effect on LPS-induced circadian disruption of PER2 expression were specifically correlated to the presence of polymethoxyflavones with 4 or more methoxy groups, namely, nobiletin and tangeretin [ 87 ]. Consistent with this, tangeretin (3 μM) increased the rhythmic activity of PER2 in PER2 ::Luc Clock Δ19/+ fibroblast cells; however, the analogs naringin and naringenin showed no effect on the circadian rhythm of PER2 in either PER2 ::LucSV or mutant reporter cells [ 82 ].…”

“…In primary mouse hepatocytes, the protein concentrations of CLOCK and SIRT1 were also elevated [ 74 ]. As such, polymethoxyflavones, specifically containing 4 or more methoxy groups, enhanced the expression of CLOCK , BMAL1 , PER1/2/3 , CRY1/2 , Rev-Erbα/β , RORα , and NPAS2 circadian genes in synchronized mouse microglia BV-2 cells pretreated with either citrus flavonoid extracts or nobiletin [ 87 ]. In the same way, pretreatment with nobiletin at very high concentration (200 μM) amplified the shallow mRNA fluctuations of clock oscillators CLOCK , BMAL1 , PER2 , CRY2 , and Rev-Erbα in synchronized primary mouse hepatocytes, while also enhancing the protein expression of CLOCK and BMAL1 in nonsynchronized HepG2 [ 73 ].…”

Advances in extraction and purification of citrus flavonoids

“…Our study also found that citrus flavonoids could induce cellular antioxidant activity by inhibiting the ubiquitination of Nrf2 (Wang et al., 2021b). PMFs in citrus could reverse LPS‐induced circadian rhythm disturbance in mouse liver by inhibiting Nlrp3 inflammasome formation (Wang et al., 2021c). The biological activities of flavonoids are closely related to their molecular structures.…”