Study On The Effects Of Proanthocyanidin A2 Attenuating Atherosclerosis Based On Intestinal Flora

Numerous studies show that proanthocyanidin A2(PCA2)has obvious anti--atherosclerosis effects.However,the absorption and utilization of proanthocyanidins are very low.The contradiction between low bioavailability and significant bioactivity suggests that proanthocyanidins may have other bioavailability pathways in vivo.It has been found that the interaction between proanthocyanidins and intestinal flora may play an important role in the biological activity of proanthocyanidins.Therefore,this study aims to analyze the effect of interaction between intestinal flora and PCA2 on the anti-atherosclerosis activity of PCA2.The atherosclerosis mice model by Apo E^-/-mice fed with high-fat diet and the pseudo germ-free mice model by antibiotic cocktails were established.The main results are as follows:(1)PCA2 could significantly inhibit the formation of atherosclerosis in Apo E^-/-mice fed with high-fat diet,in which intestinal flora played an important role.12-week high-fat diet significantly promoted the formation of AS in Apo E^-/-mice.Compared with HFD group,TG levels significantly decreased(P<0.05),while the level of HDL-C significantly increased in HFD+A2 group(P<0.05).PCA2 significantly increased the activity of SOD in plasma and liver and significantly decreased MDA content of Apo E^-/-mice fed with high-fat diet(P<0.05).PCA2 significantly decreased the levels of inflammatory factors VCAM-1 and ICAM-1 in the aorta of Apo E^-/-mice fed with high-fat diet(P<0.05).Compared to control mice,the atherosclerotic lesion area and macrophages in the whole aorta were much higher in mice fed with high-fat diet,which were markedly reversed by PCA2(P<0.05).The disordered arrangement of hepatocytes and the enlargement of adipocytes were ameliorated by PCA2.Moreover,when the intestinal flora were suppressed by antibiotics,the effect of PCA2 on atherosclerosis induced by high-fat diet was significantly abolished.The contents of PCA2 metabolites in plasma and urine of Apo E^-/-mice were significantly higher than that of pseudo germ-free mice.The content of p-hydroxyphenylacetic acid and 3-(4-hydroxyphenyl)propionic acid in plasma of normal mice was 165.92 and 2327.21 times of that of pseudo germ-free mice.It suggested that the metabolites of PCA2 might be the material basis of its anti-atherosclerosis activity.(2)Effects of PCA2 on the structure of intestinal flora in Apo E^-/-mice fed with high-fat dietThe composition and structure of intestinal flora in mice feces were determined by 16S r RNA analysis.The results showed that the proportion of Firmicutes to Bacteroidetes in intestinal flora of Apo E^-/-mice fed with high fat diet was significantly higher than that of control group(P<0.05).The proportion of Firmicutes to Bacteroidetes significantly decreased in intestinal flora of Apo E^-/-mice fed with high fat diet after intervention with PCA2(P<0.05),and the abundance of Akkermansia increased significantly,which suggested that PCA2 could adjust the composition and structure of intestinal flora.However,the diversity of intestinal flora in pseudo germ-free mice treated with antibiotics was significantly reduced,and only Protebacteria exist at the phylum level.There was a significant positive correlation between the abundance of Akkermansia,norank?f?Lachnospiraceae,Lactobacillus,norank?f??Bacteroidales?S24-7?group and the content of major metabolites of PCA2(P<0.05).(3)The role of intestinal flora in the regulation of lipid metabolism by PCA2The differential metabolites in plasma of mice were analyzed by UPLC-Q-TOF-MS.Thirty-two different lipid metabolites were obtained.Compared with control group,the content of phosphatidyl ethanolamine,phosphatidylserine and phytosphingosine decreased and the content of phosphatidylinositol and lysophosphatidylethanolamine increased fed with high-fat diet.After the intervention of PCA2,the contents of these metabolites were partly restored.Pathway analysis showed that the PCA2 regulated lipid metabolism disorders induced by high-fat diet mainly involving four related metabolic pathways:Glycosylphosphatidylinositol anchor biosynthesis,Sphingolipid metabolism,Pentose phosphate pathway,and Glycerophospholipid metabolism.Among them,the impact value of Glycerophospholipid metabolism was the highest.RT-PCR results showed that the PCA2 treatment significantly increased the expression of PPAR?,CYP7A1 and ABCA1 m RNA(P<0.05),and the effect of PCA2 on the expression of related lipid metabolism genes was significantly weakened after inhibiting the intestinal flora of mice by antibiotics(P<0.05).These results indicate that intestinal flora can convert PCA2 into p-hydroxyphenylacetic acid and 3-(4-hydroxyphenyl)propionic acid,which may be the material basis of PCA2anti-atherosclerosis.PCA2 can also improve the intestinal flora disorder induced by high-fat diet.The interaction between intestinal flora and PCA2 plays a key role in the regulation of lipid metabolism and atherosclerosis-attenuated by PCA2.