| Cardiovascular and cerebrovascular disease is the main reason for the death of the population in developed countries,and the mortality rate in our country has increased year by year,accounting for the first cause of the death.Atherosclerosis is an important pathological basis of cardiovascular and cerebrovascular diseases,and to prevent and treat atherosclerosis is the fundamental measure for preventing cardiovascular and cerebrovascular diseases.The formation of foam cells is a typical pathological feature in the early stage of atherosclerosis,therefore,it is great significant to prevent and reverse the formation of foam cells in the prevention and treatment of atherosclerosis.It has been found that cholesterol influx,esterification and efflux are the key links in regulating the dynamic balance of cholesterol in macrophages,and it is important,as a new target to prevent,slow down and treat AS,for reducing the intracellular cholesterol accumulation,preventing the formation of foam cells and the AS occurrence.Polyphenols are very rich in Pomegranate,with the most abundant polyphenol content in pomegranate peel.Pomegranate peel,a traditional Chinese medicine,has often been used as anthelmintic anti diarrheal,etc.At present,pomegranate peel extracts has been widely used as an important biomedical material.Many studies have shown that polyphenol phytochemical,the main active substances of pomegranate extracts,have many effects,such as anti-bacteria,anti-cancer,anti-inflammatory,lipid-lowering,antioxidant,anti-aging,prevention and treatment of diabetes and cardiovascular disease,et al.However,whether the cholesterol homeostasis can be regulated by pomegranate peel polyphenols,has not been reported.In view of this,the experimental AS rats were intervented by pomegranate juice as test materials firstly.On the basis of the above,Pomegranate Peel was as raw material for its most abundant polyphenols,and its polyphenols extract and main components were used as test object for the experiment,and the foam cell model was established from the mouse macrophage RAW264.7 cells,and the present study was designed to examine the effects of Pomegranate Peel Polyphenols on lipid accumulation,cholesterol infulx and cholesterol efflux in foam cells,and then to analyze their effects on the formation of foam cell formation,and to further reveal its mechanism and target from the signal transduction.The main investigation contents and results are as follows:(1)The effects of pomegranate juice on total cholesterol,low density lipoprotein cholesterol,C reactive protein,total bile acid,alanine aminotransferase,MDA and other indicator were detected in AS rats established by high fat diet.The results showed that pomegranate juice reduced significantly serum TC,LDL,LDL/HDL,ALT,MDA levels,and showed a good scavenging effect on DPPH free radical,which indicates pomegranate juice has a certain anti-lipid peroxidation and has a certain preventive effect on hyperlipidemia.Meanwhile,pomegranate juice reduced serum CRP and arteriosclerosis index in AS rats,indicating that pomegranate juice can reduce the risk of AS.In addition,pomegranate juice reduced ALT and serum total bile acid content in AS rats,indicating that pomegranate juice has protective effect on the liver.(2)The main components of pomegranate peel polyphenols were tested by HPLC method.The results showed that the main active ingredient were ellagic acid,gallic acid,punicalagin,catechins,epicatechin,chlorogenic acid and so on.Among them,the highest content of three components:ellagic acid,gallic acid and punicalagin,their content was 206.42,112.46 and 98.12 mg/g,respectively.(3)The effect of pomegranate polyphenols on the accumulation of cholesterol in raw264.7 macrophages was determined by oil red O staining and detection of changes in intracellular cholesterol levels,and the key genes related with cholesterol intake were detected by real-time fluorescence quantitative PCR and western blot.The results showed that the increasing of cholesterol content induced by ox-LDL was reduced significantly,and the intracellular lipid droplets was also reduced significantly with a good dose-dependence,after pretreated 1 h by pomegranate peel polyphenol.The expression of CD36 mRNA and protein was significantly down-regulated,and no significant effect on SR-A protein expression was observed.These results suggested that pomegranate peel polyphenol pretreatment reduced cholesterol accumulation in macrophages which may be achieved by down-regulating the CD36 receptor.(4)To study the effect of Pomegranate Peel Polyphenols on cholesterol efflux in raw264.7 macrophage-derived foam cells and its molecular mechanism.The results showed that pomegranate peel polyphenols promoted Apo-A1 and HDL-mediated cholesterol efflux with concentration-dependent,Pomegranate Peel Polyphenols significantly increased ABCA1,LXR alpha and SR-B1 gene protein expression with no obvious influence in ABCG1.This suggests that pomegranate peel polyphenols can promote cholesterol efflux through LXR?-ABCA1/SR-B1 pathway.In addition,Pomegranate Peel Polyphenols not significantly affected nCEH and ACAT1 protein expression,but significantly decreased the activity of ACAT1,increased nCEH activity,which was beneficial to the transformation of cholesterol ester to free cholesterol,thereby promoting cholesterol efflux.(5)In order to explore the main constituentsof pomegranate polyphenos,the effects of punicalagin,ellagic acid and gallic acid on cholesterol intake,efflux and regulation signal pathway in mouse macrophage raw264.7 cells were further studied.The results of oil red O staining and cholesterol determination showed that punicalagin and ellagic acid significantly reduced the intracellular cholesterol accumulation and cholesterol content in raw264.7 cells,down-regulated the receptor CD36 protein expression,in a dose-dependent manner.But,gallic acid at low concentration reduced the accumulation of cholesterol,and not at the higher concentration(50 ?mol/L).In addition,punicalagin,ellagic acid and gallic acid promoted cholesterol outflow,and increased ABCA1,ABCG1 and LXRa protein expression,and gallic acid also increased PPARy.This indicates punicalagin and ellagic acid are the major contributors to inhibit macrophage cholesterol influx,and the inhibition is achieved by down regulating the expression of scavenger receptor CD36.In promoting cholesterol efflux from foam cells,punicalagin,ellagic acid and gallic acid showed a certain activity.But the regulation pathways were different,punicalagin and ellagic acid may promote cholesterol efflux by LXRa-ABCAl/ABCG1 pathway,independent of PPAR?,and the regulation target may be LXR?.Gallic acid may promote cholesterol efflux by PPAR?-LXRa-ABCA1/ABCG1 pathway,and the regulation targets may be PPARy and LXRa. |
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