Alleviation Effects And Mechanisms Of Citrus Flavanones On Atherosclerosis

Cardiovascular diseases seriously threaten human health,and atherosclerosis is one of the primary underlying pathological processes.There are multiple anti-atherosclerotic drugs,which always result in side effects for long-term administration.Safe and efficient strategies to alleviate atherosclerosis have drawn special attention.Flavanones are the main flavonoids in citrus,which exhibit the special potential to alleviate atherosclerosis with high safety.However,there are still several issues that need to be resolved for the development and utilization of citrus flavanones.Firstly,although many studies have reported the atherosclerosis alleviation effect of citrus flavanone,while the effects and differences of different flavanones for atherosclerosis alleviation are still unclear.Secondly,citrus flavanones,especially the glycosides,are difficult to be absorbed intestines,which still have good health effects.It is speculated that gut microbiota may play an important role in alleviating atherosclerosis,while the mechanism is not clear.In addition,flavanones often undergo complex biotransformation after oral intake into the body,which is of great significance to the health of humans.However,the effect of citrus flavanone metabolism on atherosclerosis alleviation has not been reported.Therefore,this paper innovatively investigated the mechanisms of citrus flavanones in atherosclerosis alleviation from function confirmation,gut microbiota and metabolisms.The main results are as follows:(1)Confirmation of atherosclerosis alleviation effects of citrus flavanones:The effects of hesperidin and naringin on lipids and gut microbiota in C57BL/6J mice were first investigated,and then their atherosclerosis alleviation effects were confirmed via Apo E^-/-mice.As result,a high-dose(100mg/kg,body weight)of citrus flavanones exhibited significant lipid-lowering effects,and the former was more potent than the latter,while the low dose of citrus flavanones had no obvious effect.Moreover,different kinds and doses of flavanones had different effects on gut microbiota.High-dose of naringin could significantly increase the relative abundance of secondary cholic acid-producing bacteria(g?Eubacterium),and decreased bile acid hydrolase-producing bacteria(g?Bacteroides and g?Lactococcus)in both mice fed with a normal and high-fat diet,enhancing the bile acid emission in feces,which indicated that the alternation in the abundance of the three bacteria may contribute to the reduction of cholesterol and bile acid emission.High-dose naringin and hesperidin were further found that they could significantly reduce arterial plaque in Apo E^-/-mice and naringin was more efficient than hesperidin.In conclusion,citrus flavanones could significantly alleviate atherosclerosis.The results would provide a theoretical basis for the development of citrus flavanones-based diet prevention strategy for atherosclerosis,and also provide the theoretical basis for the following researches.(2)Mechanism of naringin alleviating atherosclerosis mediated by gut microbiota:Based on the above results,the effects of naringin with more significant atherosclerosis alleviation on lipid metabolism pathways of Apo E^-/-mice,and the role of gut microbiota were further investigated.The results showed that naringin significantly down-regulated 40 metabolites in the liver of Apo E^-/-mice,15 of which were involved in lipids,especially cholesterol ester(CE)related to cholesterol metabolism was down-regulated 8 folds.In addition,naringin significantly increased the contents of total lipid and total bile acid(TBA)in feces.At the same time,naringin could reduce the relative abundance of bile acid hydrolase-producing bacteria(e.g.,g?Bacteroides,g?Clostridium and g?Lactococcus),and increased the relative abundance of secondary cholic acid-producing bacteria(e.g.,g?Eubacterium).Further study demonstrated that naringin significantly down-regulated FGF15 in the ileum,and up-regulated CYP7A1 and down-regulated PCSK9 both in gene and protein levels in the liver.Therefore,the mechanism of naringin alleviating atherosclerosis was revealed:it remolded gut microbiota,which could inhibit FXR/FGF15,and then consequently up-regulated CYP7A1 via negative feedback pathway,increasing the conversion of cholesterol to bile acid.In addition,naringin can also inhibit the expression of PCSK9,which promoted the efflux of cholesterol from peripheral tissues,and facilitated the alleviation of atherosclerosis.The results would provide theoretical guidance for revealing the atherosclerosis alleviation mechanism of citrus flavanone,and also provide new in vivo targets for citrus flavanone atherosclerosis alleviation.(3)Different effects and mechanisms of citrus flavanones on atherosclerosis:The alleviating atherosclerosis efficacy of different citrus flavanones(naringin,naringenin,hesperidin and hesperetin)in Apo E^-/-mice were explored,and the corresponding mechanisms were further investigated via flavanone metabolisms in vivo,gut microbiota and signal pathways.The results showed that all the four flavanones could reduce the atherosclerotic plaque of the whole aorta in mice in different degrees,and naringin was the most potent,followed by hesperidin,naringenin and hesperetin.The results indicated that C7-O-neohesperidin was more conducive to atherosclerosis alleviation than rutin glycosides,and substitution of hydroxyl at C4'is better than methoxy.In vivo metabolism studies showed that naringin could hardly be absorbed by the intestines,while a large amount of it remained in the intestines with only a small part absorbed into the liver through the large intestine.Hesperidin could be quickly absorbed into the liver through the small intestine and large intestine,and then metabolized and excreted.Naringenin and hesperetin could be partially absorbed into the liver by the small intestine.Notably,the absorption of hesperetin was less than naringenin.16S RNA analysis demonstrated that the regulation of the other three flavanones on bile acid hydrolase-producing bacteria was not uniform compared with naringin.Further study showed that hesperidin significantly increased the expression of ABCA1,a transport protein in the cholesterol reversal pathway,and naringenin and hesperidin decreased the expression of HMGCR.In conclusion,the different mechanisms of the four flavanones in alleviating atherosclerosis were revealed as follows:naringin mainly inhibited FXR/FGF15 pathway by regulating gut microbiota.Hesperidin could increase the reverse transport of cholesterol by up-regulating the expression of ABCA1.Naringenin and hesperidin alleviated atherosclerosis by inhibiting HMGCR,a key enzyme in the cholesterol synthesis pathway.The results of this study could provide new scientific cognition and reference for the atherosclerosis alleviation mechanism of citrus flavonoids,as well as theoretical guidance for the target design of related functional foods.