Study On The Effect And Mechanism Of Hesperidin On Anti-atherosclerosis

Objective: Atherosclerosis(AS)was considered to be a complex,multi-factor progressive,chronic inflammatory cardiovascular disease,which was the major reason of morbidity and mortality in many countries.Hesperidin(HES)was a flavanone glycoside,which can be predominantly and abundantly found in citrus fruits.Studies showed that hesperidin can reduce cholesterol levels and play a role on permeability and brittleness of vascular,trauma and stress protection.However,the effect of hesperidin on atherosclerosis has not been investigated.This study was aimed to investigate the effect of hesperidin on atherosclerosis.The effects of hesperidin on atherosclerotic drug target——lectin oxidized low density lipoprotein receptor 1(LOX-1)and its mechanism were studied.It was aimed to elucidate whether hesperidin inhibited macrophage foam to exert anti-atherosclerotic effects by LOX-1/NF-?B signaling pathway.Method: 10 weeks old male Apo E-/-mice were randomly separated into 4 groups: Apo E-/-model group,hesperidin group(HES-100 mg / kg / d),and atorvastatin group(10 mg / kg / d),with 10 animals in each group and fed a high-fat diet.After 14 weeks,organs were obtained for future analysis.Ten C57BL/6J male mice was fed a chow diet and given equal volume of distilled water as control group.Serum levels of TC,TG and LDL-C were determined using kits;Oil Red O staining was used to observe the atherosclerotic plaque and plaque distribution in arterial tree and aortic root.H&E was used to observe the change of macrophage infiltration.The effect of hesperidin on RAW264.7 foam cell formation was observed by Oil Red O staining.The effect of RAW264.7 cells on the binding and phagocytosis of ox LDL was observed by confocal laser confocal microscopy.The effects of hesperidin on LOX-1,p-p65 and p65 protein expression were detected by Western Blot.Effect of hesperidin on LOX-1 expression in plaque were using IHC.Effect of hesperidin on NF-?B p65 subunit enucleation by cell immunofluorescence.The m RNA expression of LOX-1 and inflammatory factors were detected by reverse transcription polymerase chain reaction(RT-PCR).Results: 1.After Apo E-/-mice fed with high fat diet for 14 weeks,there were no change on body weigh in each group.But the levels of TC,TG and LDL-C in Apo E-/-model mice were significantly increased while hesperidin significantly decreased the levels of TC,TG and LDL-C in serum of Apo E-/-mice.2.After Apo E-/-mice fed with high fat diet for 14 weeks,the levels of plaque area significantly increased while hesperidin inhibited the plaque area in aorta root and arterial tree.3.After Apo E-/-mice fed with high fat diet for 14 weeks,Apo E-/-model mice had a large number of foam cells and inflammatory cells,and followed by cholesterol crystallization,liposis and fibrous lesions.However hesperidin can reduce the inflammatory cells and fiber components in plaque.There were no cholesterol crystals in lesions and mostly are the lipid phase lesions.4.Hesperidin can dose-dependently inhibit RAW264.7 foam cell formation and Dil-ox LDL uptake.5.The expression of LOX-1 protein and m RNA was increased in ox LDL-induced RAW264.7 cells,while hesperidin and PDTC can decrease the expression of LOX-1 protein and m RNA.6.Hesperidin can inhibit the expression of LOX-1 protein in lesions of Apo E-/-mice fed with high fat diet.7.Hesperidin can reduce the activation of NF-?B in ox LDL-induced RAW264.7 cells.8.Hesperidin can reduce the m RNA expression of TNF-?,ICAM-1,IL-1,IL-6 and MCP-1.Conclusion: 1.Hesperidin can significantly reduce TC,TG and LDL-C levels in high-fat-induced Apo E-/-mice.2.Hesperidin can significantly reduce the area of plaque in aorta root and arterial tree in high-fat-induced Apo E-/-mice.3.Hesperidin can significantly reduce the formation of inflammatory cells and fibrous components to decrease the development of AS lesions 4.Hesperidin can dose-dependently inhibit ox LDL-mediated RAW264.7 foam cell formation and uptake of Dil-ox LDL.5.Hesperidin can inhibit the expression of LOX-1 in ox LDL-mediated foam cells and high-fat diet-fed Apo E-/-mice plaques.In addition,hesperidin can inhibit the expression of NF-?B activation in foam cells,suggesting that hesperidin may play its anti-atherosclerosis effect through the LOX-1 / NF-?B signaling pathway.6.Hesperidin can inhibit inflammatory factors induced by ox LDL in RAW264.7 cells.