Molecular Regulation Mechanism Of Persimmon Tannin On Cholesterol Efflux

The purpose of this study is to investigate the molecular regulation mechanisms of persimmon tannin on the cholesterol homeostasis in vitro and in vivo.Firstly,high-cholesterol HepG2 and Caco-2 cell models were established to investigate the effects of persimmon tannin on cholesterol efflux and absorption combining with real-time RT-PCR and western-blot analysis.Secondly,we explored the interaction between persimmon tannin and Caco-2 cell membrane and the influence of persimmon tannin on the cell membrane receptor EGFR.Otherwise,in order to study the regulatory effect of persimmon tannin on reverse cholesterol transport?RCT?and transintestinal cholesterol efflux?TICE?pathway in vivo,the high-cholesterol diets were used to induce cholesterol accumulation in C57BL/6J male mice.Finally,the transport mechanism of persimmon tannin characteristic units and their effects on the transportation of fluorescent-labeled cholesterol were evaluated on the Caco-2 monolayers.The main study results are as follows:1.Effects of persimmon tannin on cholesterol accumulation in high-cholesterol cell modelsHigh-cholesterol HepG2 and Caco-2 cell models were induced by 20?g/m L cholesterol and 2?g/mL 25-hydroxycholestrrol for 24 h,and they were used to investigate the molecular regulation mechanism of persimmon tannin on cholesterol absorption and cholesterol efflux.Our results indicated that the cellular TC and extracellular LDL-C contents in high-cholesterol groups were significantly?p<0.05?higher than that of normal control group.Intervention with persimmon tannin noticeably?p<0.05?relieved the high cholesterol-induced increase in TC,LDL-C and ApoB levels but markedly?p<0.05?elevated the HDL-C concentration.Additionally,persimmon tannin reduced the uptake of 22-NBD-cholesterol and inhibited the accumulation of cholesterol significantly in HepG2 and Caco-2 cell lines.The mRNA and proteins levels were quantified by real-time RT-PCR and western-blot analysis,respectively.The expression of NPC1L1 involved in cholesterol absorption and SREBP-2 gene critical for cholesterol synthesis were significantly inhibited by persimmon tannin in comparison with high-cholesterol group.Moreover,persimmon tannin showed potential in promoting cholesterol efflux through up-regulating mRNA and/or protein levels of ABCA1,ABCG1,SRB1,CYP7A1 and ABCG5/G8.The alterations in protein levels of ABCA1 and CYP7A1 were consistent with those in their mRNA levels.However,transfection with LXR?siRNA did not abrogate the increased effects of persimmon tannin on mRNA and protein expression of CYP7A1 and ABCA1.Therefore,the regulatory effects of persimmon tannin on cholesterol homeostasis in both cell lines were LXR?independent.2.Effects of persimmon tannin on the integrality of cell structure and the phosphorylation of EGFR In Caco-2 cells,treatment with 2.5 mM methyl-?-cyclodextrin?MCD?resulted in a significant depletion of cellular membrane cholesterol?p<0.05?,Filipin III staining analysis was further verified the extraction of MCD on cellular cholesterol.Fluorescent probes?Laurdan/DPH/TMA-DPH?were used to label cell membrane and evaluate the effects of persimmon tannin on the fluidity of cell membrane,which was based on the change of fluorescent polarization.The results indicated the depletion of cellular cholesterol decreased the polarization of fluorescent probes,suggesting the increase in fluidity of cell membrane.Pre-incubation with persimmon tannin could protect the fluidity of cell membrane from the abnormalities caused by MCD.Additionally,persimmon tannin prevented DCA,Triton X-100 and MCD-induced destruction of cell membrane structure.On transcriptional and translational levels,persimmon tannin alleviated the upregulation of MCD on the genes expression and protein phosphorylation of EGFR and ERK1/2.Additionally,the treatment with persimmon tannin reversed the down-regulation of cholesterol,the up-regulation of MCD on the mRNA and protein expression of PPAR?.Persimmon tannin might affect the cell physiological function via regulating the membrane receptor protein EGFR and ERK1/2-PPAR?signaling pathway.3.Effects of persimmon tannin on macrophage-RCT and TICE pathway in C57BL/6J mice fed with high-cholesterol diets In order to investigate whether persimmon tannin could promote cholesterol efflux from macrophage,J774 A.1 macrophages were incubated in RPMI 1640 with1%BSA,25?g/m L of Ac-LDL containing 20?g/m L cholesterol or 4?M22-NBD-cholesterol?fluoresterol?for 24 h,cellular lipid was stained with oil red O,and western-blot was used to analysis the protein expression of ERK1/2 and PPAR?.The results showed that persimmon tannin could inhibit cellular cholesterol accumulation and promote 22-NBD-cholesterol efflux through inhibiting the phosphorylation of ERK1/2 and up-regulating the protein levels of PPAR?.High-cholesterol diets resulted in inflammatory cell infiltration and elevation of TC,TG and LDL-C concentration in both liver and serum,but intervention with persimmon tannin could alleviate these phenomena.Macrophage-RCT in vivo was evaluated by injecting 22-NBD-cholesterol-loaded J774 A.1 macrophages intraperitoneally into C57BL/6J mice.Administration of persimmon tannin significantly?p<0.05?decreased the cholesterol concentration in both serum and liver,and increased faecal cholesterol excretion compared with the high-cholesterol group.In transcriptional levels,persimmon tannin enhanced the expression of cholesterol efflux-related genes?ABCA1,LCAT,ABCG5/G8 and CYP7A1?and their upstream nuclear receptors?PPAR?,PPAR?and LXR??.Moreover,the regulation of persimmon tannin on RCT-related genes might be mediated by its inhibition on ERK1/2 in mice.Therefore,persimmon tannin promoted macrophage-RCT through the regulation on ERK1/2-PPAR?pathway both in vitro and in vivo.However,persimmon tannin has no significant effect on TICE in vivo.Therefore,the hypocholesterolemic effect of persimmon tannin mainly benefited from its regulation on RCT.4.The influence of persimmon tannin characteristic units on cellular22-NBD-cholesterol uptake and transport in Caco-2 cell monolayers Caco-2 monolayers were established and evaluated with transepithelial electrical resistance?TEER?,P_appof Lucifer Yellow CH,alkaline phosphatase and cell morphology.Effects of persimmon tannin characteristic units,A-type EC dimer and their corresponding structure monomers on cellular 22-NBD-cholesterol accumulation and transportation in Caco-2 cell monolayers were investigated and compared.Additionally,the correlation between the transport mechanism of persimmon tannin structure units and their effects on cholesterol transport was also explored.The data showed that all of these six phenols could inhibit fluorescent22-NBD-cholesterol cellular accumulation significantly?p<0.01?through suppression of NPC1L1 gene expression.Further study demonstrated that P-gp,tight junctions,their chemical structures and hydrophobicity were related with the transport mechanism of the six phonel compounds in Caco-2 monolayers,and these factors further affected their bioavailability and the regulation on cholesterol transport.Although both persimmon tannin and its structure uints could reduce cellular cholesterol uptake via inhibiting gene expression of NPC1L1,due to the complexity of persimmon tannin,the bioactivity of persimmon tannin structure units could not represent that of persimmon tannin.