Circadian Rhythm Disorders Regulating Ability Of PMFs From Citrus And Related Mechanism

Circadian rhythm is a time-keeping system ubiquitous in organisms,which is closely related to the metabolism and nutrition,and fluctuate periodically with the change of day and night.Circadian rhythm disorder is a common metabolic-related disease.Previous studies have shown that some natural active substances have significant regulatory effects on circadian rhythm.Citrus(Citrus spp.)is widely cultivated in China,and its flavedo is rich in flavonoids,which deserves further research and wilder application.In this study,the main flavonoids identification and quantification in 6 types of citrus fruits were evaluated,and their regulatory effects on circadian rhythm disorders and related mechanisms were studied.The main results were as follows:1.Citrus flavonoids extract can regulate the circadian rhythm of BV-2 cells.4 flavanones and 5 polymethoxyflavones(PMFs)were identified and quantitatively analyzed from flavedo extracts of 7 citrus varieties of 6 types.By Per2::Luc fluorescence screening system,we evaluated the circadian rhythm regulation ability of extract from flavedo of 7 citrus varieties and it was found that ’Fuju’ and ’Zijin Ougan’ showed significant circadian rhythm regulation effect,and inhibited the circadian rhythm disorders induced by lipopolysaccharides(LPS).The content of PMFs in citrus flavonoids extract was positively correlated with the circadian rhythm regulation ability.2.PMFs is the main effective substance of citrus flavonoids in regulating circadian oscillation.In this study,25 citrus flavonoids were selected for the experiment,and the circadian rhythm genes of BV-2 cells were detected.It was found that 8 flavonoids,nobiletin,sinensetin,isosinensetin,tangeretin,gardenin B,5-demethylnobiletin,hesperetin and diosmetin,showed regulatory effects on the genes of Clock,Bmal1,Per1,Per2,Per3,Cry1,Cry2,Rev-erbα,Rev-erbβ,Rorα,Dbp and Npas2.However,flavones and flavanones such as vitexin,isovitexin,vicenin-2,hesperidin,neohesperidin,diosmin,neodiosmin,didymin,naringin,narirutin,rhoifolin,isorhoifolin,eriocitrin,neoeriocitrin,poncirin,apigenin and naringenin did not have this effect.It was also found that nobiletin,sinensetin,isosinensetin,tangeretin,gardenin B and5-demethylnobiletin can effectively inhibit LPS-induced circadian gene oscillation suppression,suggesting that the structure of PMFs containing four or more methoxy substituents can have this effect.3.Nobiletin(NOB)can regulate LPS-induced circadian rhythm disorders in mouse liver cells.We tested the circadian rhythm disorders regulation function of NOB in vivo.C57BL/6J mice were continuously given NOB by gavage and intraperitoneal injection of LPS-induced circadian rhythm disorders in the liver.The expression of circadian rhythm genes in mouse liver under the action of LPS was detected by qRT-PCR.It was found that NOB effectively inhibited the LPS-induced rhythmic disturbance,and maintained the normal expression of Clock,Bmal1,Per1,Per2,Per3,Cry1,Cry2,Rev-erbα,Rev-erbβ,Rorα,Dbp and Npas2,and the effect was sustained for 24 h.4.Inhibition of citrus PMFs on LPS-induced circadian rhythm disorders is associated with NLRP3 inflammasome.We verified the correlation between the inhibition of LPS-induced rhythm disorders by citrus flavonoids and inflammation through NO release analysis,and the correlation was further verified by activation of the expression of inflammatory factors by NLRP3 activator nigericin.We analyzed the expression of circadian rhythm genes,Nlrp3,IL-1β,ASC and Caspase1 genes and their proteins by qRT-PCR and ELISA,and preliminarily determined that the regulation of LPS-induced circadian rhythm disorders by PMFs might be related to Nlrp3 and ASC genes in NLRP3 inflammasome.This study proved that PMFs has its special biological activity in the regulation of circadian rhythm,and preliminarily verified the relevant mechanism of citrus PMFs in regulating LPS-induced circadian rhythm disorders.We have also examined the circadian rhythm disorders regulation effect of NOB in vivo.This study pointed out a new direction for the further development and application of citrus natural products.