In Vitro Catabolism Of Litchi Pericarp Polyphenol By Intestinal Microbiota And The Mechanism Of Its Inhibitory Effects On The Formation Of Macrophages-Derived Foam Cells

Litchi Pericarp has significant anti-atherosclerotic activity for richly containing procyanidin,but procyanidin have a very low small intestinal absorption rate.Studies have also shown that the metabolism of intestinal flora may play an import role in the bioactivity of procyanidins.In this study,the human intestinal flora was used to metabolize Litchi pericarp polyphenols in vitro,UPLC-Q-TOF-MS/MS was used to analyze the composition of Litchi pericarp polyphenols(LP)and its metabolites(LM).Simultaneously,high-throughput sequencing method was used to analyze the changes of intestinal microflora during metabolism.Finally,transcriptome sequencing was used to analyze the influence of LP and LM on the expression of macrophage-related genes,to explore the effect and mechanism of LM on inhibiting macrophage foaming.The main findings are as follows:1.Changes of total phenol content(TPC)and antioxidant activity during the metabolism of LPThe changes of TPC and antioxidant activity of LM were analyzed at different time points.Results showed that TPC was reduced to 95.03%,92.74%,89.95%,84.21%and 79.06%of 0 hour,respectively,at 4,8,12,24 and 48 hours of metabolism.The comparison between groups at different time points showed that FRAP and ABTS +.scavenging capacities were 1.2,0.89,0.95,0.80,and 0.80 times and 1.08,1.14,0.91,0.87 and 0.89 times for the LP group at 4-48 hours,respectively.2.Analysis of individual phenols in LP and LMThe analysis of the results of UPLC-Q-TOF-MS/MS shows that the main phenols in LP are 2,4,6-Trihydroxybenzoic acid,Protocatechuic acid,Protocatechuic aldehyde,Gallocatechin,Procyanidin B,Epicatechin-(4?->6)-epicatechin-(2?->7,4?->8)-epicatechin,A-type Propelargonidin Trimer,Kaempferol-3-O-glucoside,Kaempferol-3-O-rutinoside,A-type Proanthocyanidin dimer,A-type procyanidin trimer,4-Hydroxyacetophenone.The main metabolic products of intestinal microflora are benzoic acid,Phenylacetic acid,2,4,6-Trihydroxybenzoic acid,Vanillin,3-Methylcatechol,catechin dimer,3,5,7,3'-tetrahydroxy-4'-methoxyisoflavone,Kaempferide,C15H1405(reduced metabolites of polyphenols),(±)8-Gingerol?schizandriside?9-hydroxy-6-gingerol?Triptophenolide and so on.Among them,the newly detected compounds in the metabolites of bacteria are mainly 3,5,7,3'-tetrahydroxy-4'-methoxyisoflavone,Kaempferide,C15H14O5,(±)8-Gingerol?schizandriside?9-hydroxy-6-gingerol?Triptophenolide.3.Changes of dominant genus during the fermentation of LPThe results of high throughput sequencing show that the top 10 dominant genus are Bacteroides,Clostridium,Enterococcus,Streptococcus,Phascolarctobacterium,Peptostreptococcus,Oscillospira,Pseudomonas,Citrobacter,and Desulfovibrio in the process of metabolism.Among them,Clostridium,Bacteroides,Peptostreptococcus and Citrobacter showed a downward trend during metabolism,decreasing by 50.87%,57.16%,28.15%and 7.37%,respectively;while the other genus showed an upward trend,which was 1.47,1.09,1.85,1.63,1.74,10.95 times of 0 h,at the end of metabolism.Eight of them with larger changes were Bacteroides,Desulfovibrio,Bilophila,Fusobacterium,Ochrobactrum,Eggerthella,Oscillospira and Sutterella in metabolic processes.4.The effect of LP and LM inhibiting on macrophage foamingLP and LM can significantly reduce the lipid content in macrophages,intracellular cholesterol ester,total cholesterol and the ratio of cholesterol ester and total cholesterol reduced to 69.78%,64.87%,85.71%,81.75%and 81.22,79.40%after 15?g/mL LP and LM interfered with ox-LDL-induced macrophage;25 p,g/mL decreased to 50.08%,43.73%,73.33%,68.13%,and 68.31%,64.21%,respectively.In addition,the content of ROS and MDA in the cells decreased to 86.72%,76.97%and 74.99%,75.24%after LP and LM interfered with ox-LDL-induced macrophage;25 ?g/mL decreased to 64.56%and 62.50%of the MDA content in the model group,respectively.15 ?g/mL LP and LM can significantly reduce the content of TNF-a and IL-6 in macrophages,which were reduced to 68.65%,68.31%and 88.73%,89.02%,and 25 ?g/mL decreased to 58.75%,58.23%,84.31%and 84.80%,respectively.5.The mechanism of LP and LM inhibiting on macrophage foamingThe results of transcriptome sequencing showed that LP mainly regulatesthe Cytokine-cytokine receptor interaction,TNF and NF-kappa B signaling pathways to significantly up-regulate the expression of Tnfaip3 and ncehl genes,down regulation of the expression of Cxcll 1,Pdgfa and Cxc110 genes to inhibit the formation of inflammatory factors and cholesteryl esters,promote the negative regulation of inflammatory reactions,and then inhibit the foam of macrophages.LM mainly regulates the Toll-like receptor signaling pathway to mediate cholesterol exodus,significantly up-regulates the expression of Notch3,Tnfaip3 and Egr2 genes,downregulates the expression of Slprl,Chstl and Adgre5 genes,inhibiting the expression of inflammatory factors,promoting cholesterol efflux,and then inhibiting macrophage cell foams.The results of transcriptome sequencing analysis showed that there are differences in the main mechanisms of LP and LM inhibition of macrophage foaming.