The Molecular Mechanisms Of Persimmon Tannin Structural Units Inhibiting Preadipocytes 3T3-L1 Differentiation Based On Lipid Rafts Receptor

Our previous study showed A-ECG dimer and A-EGCG dimer were the characteristic structural units of persimmon tannin in inhibiting 3T3-L3 preadipocytes differentiation,but the underlying mechanisms are unknown.The aim of this study was to investigate the molecular mechanisms of persimmon tannin characteristic structural units underlying their inhibiting effects on the differentiation of 3T3-L1 preadipocytes based on the cell membrane lipid raft receptors.The interactions between four persimmon tannin characteristic structural units and cell membranes were compared in synthetic fluorescent liposome system.The mechanisms of the interactions between four persimmon tannin characteristic structural units and cell membranes as well as the structure-activity relationship were determined by molecular dynamics(MD)technique.Experimental analysis techniques including super high-resolution scanning electron microscopy(SEM),atomic force microscopy(AFM),laser confocal microscopy(LSCM),combined with flow cytometry(FCM),real-time fluorescent quantitative PCR(RT-PCR),and Western blot were used to investigate the damage of cell membrane lipid rafts caused by the persimmon tannin characteristic structural units A-ECG dimer and A-EGCG dimer.Whether cholesterol in the lipid raft and the receptor 67 LR were the targets of A-ECG dimer and A-EGCG dimer in inhibiting the proliferation and differentiation of 3T3-L1 preadipocytes were further explored.The main findings were as follows:1.The relationship of the inhibitory effects of persimmon tannin characteristic structural units on the differentiation of 3T3-L1 preadipocytes with their interactions with cell membrane.Four persimmon tannin characteristic structural units A-ECG dimer,A-EGCG dimer,A-EC dimer,and B-EC dimer were measured for their inhibitory effect on differentiation of 3T3-L1 preadipocytes.Among them,A-ECG dimer and A-EGCG dimer had the predominant capacity to inhibit cell differentiation.A-EC dimer could also inhibit cell differentiation to a certain extent,but the effect was far less than those of A-ECG dimer and A-EGCG dimer,while B-EC dimer had little inhibitory effect on cell differentiation.At the same time,we compared the interaction between the four dimers and the cell membrane and found that A-ECG dimer and A-EGCG dimer could destroy the morphology of the cell membrane surface,inducing cavities on the membrane surface and causing roughness of the membrane.Meanwhile,A-ECG dimer and A-EGCG dimer could significantly reduce the fluidity of cell membranes and increase the hydrability and permeability of cell membranes.Little effects of A-EC dimer and B-EC dimer on cell membrane were observed.We found that the inhibiting effects of the four persimmon tannin characteristic structural units on 3T3-L1 preadipocytes differentiation was highly positively correlated with its capability to interfere with cell membranes.The stoichiometry software ChemPro was applied to analyze the molecular chemical properties of the four dimers and to compare their hydrophobicity,the surface polarity topological region and the hydrogen bond forming ability.It was found that A-ECG dimer and A-EGCG dimer had higher hydrophobic parameter(LogP),indicating that indicated they were more likely to interact with the cell membrane.Meanwhile,their abilities to form hydrogen bonds was much greater than those of A-EC dimer and B-EC dimer.These chemical differences between the four dimeric molecules might lead to the differences in their interactions with the cell membrane.2.The relationship of the interaction between persimmon tannin characteristic structural units and synthetic DPPC liposome in vitro with their inhibitory effect on differentiation of 3T3-L1 preadipocytes.In order to further verify the difference between the interaction of the four dimers and the cell membrane,we synthesized DPPC liposomes in vitro to mimic the cell membrane,and explored the accumulation,distribution,localization and affinity with cell membranes of the four dimers with liposome by fluorescence quenching,fluorescence polarization,ultrafiltration,and DSC methods.The results showed that A-ECG dimer and A-EGCG dimer could not only accumulate on the surface of the liposome,but also enter into the liposome with some groups inserting into the interior of the liposome,thereby quenching the fluorescence inside the liposome.A-EC dimer and B-EC dimer could only act on the surface of the liposome and quenching the fluorescence of fluorescent probelocalized on the surface of the liposome.The DSC method also demonstrated that A-ECG dimer and A-EGCG dimer could not only change the molecular properties of the liposome surface but also change the molecular properties inside the liposome,while A-EC dimer and B-EC dimer only changed the properties of liposome surface.Both static quenching and ultrafiltration showed that the affinity of A-ECG dimer and A-EGCG dimer to liposomes was much higher than that of A-EC dimer and B-EC dimer.The binding constant of A-ECG dimer was 160 times higher than that of the binding constant of B-EC dimer to liposomes.The results of fluorescence polarization also indicated that A-ECG dimer and A-EGCG dimer significantly reduced cell membrane fluidity,while A-EC dimer and B-EC dimer had no significant effect on cell membrane fluidity.The trend of the action of the four dimers with the liposomes was A-ECG dimer>A-EGCG dimer>A-EC dimer>B-EC dimer.The interaction between the characteristic structural unit of persimmon tannin and synthesized DPPC liposome in vitro was highly positively correlated with the inhibition of adipogenic differentiation of3T3-L1 preadipocytes.3.Molecular dynamics simulation verify the interaction between the persimmon tannin characteristic structural units and cell membranes and analyze the structure-activity relationship.In order to further explore the difference between the four dimers and cell membranes and the molecular mechanisms,we used molecular dynamics simulation were used to analyze and explain the difference of the membrane-perturbing effect from the atomic view.It was found that the four dimers bound to the phospholipid bilayer through hydrogen bonds,while A-ECG dimer and A-EGCG dimer could form more hydrogen bonds with the phospholipid membrane than A-EC dimer and B-EC dimer.Moreover,A-EC dimer and B-EC dimer could only form hydrogen bonds with oxygen atoms O7,O9,O10,O11 on the surface of phospholipid membrane,while A-ECG dimer and A-EGCG dimer could not only form hydrogen bonding with oxygen atoms on the surface of phospholipid membrane,and their galloyl groups in the molecule could also insert into the interior of the phospholipid membrane to form hydrogen bonds with the interioroxygen atoms O14,O16,O33,and O35.A-ECG dimer and A-EGCG dimer had lower binding energy to phospholipid bilayer,indicating that A-ECG dimer and A-EGCG dimer had higher affinity with phospholipid membrane.Furthermore,energy decomposition showed that two galloyl groups in the molecule contributed over 50% of the total binding energy,suggesting galloyl groups in the molecules were important to the capacity of binding to membrane.Moreover,A-ECG dimer and A-EGCG dimer significantly reduced the lateral diffusion of the phospholipid membrane and the ordered arrangement of lipids.The results of the theoretical simulations fully validated our previous experimental results.The trend of the action of the four dimers with the phospholipid membrane was A-ECG dimer>A-EGCG dimer>A-EC dimer>B-EC dimer.which suggested that the galloyl groups in A-ECG dimer and A-EGCG dimer played a crucial role in their interaction with the phospholipid membrane.4.Persimmon tannin characteristic structural units A-ECG dimer and A-EGCG dimer binds to lipid raft cholesterol,disrupts the lipid rafts structure and inhibit activation of lipid rafts-associated receptor IR/IGF-1R to inhibit the differentiation of 3T3-L1 preadipocytes.Considering that lipid rafts acted as an important functional microdomain for the recruitment and transduction of various signals on the cell membrane,we continued to explore the effect of A-ECG dimer and A-EGCG dimer on the lipid rafts and the target of A-ECG dimer and A-EGCG dimer in lipid rafts.Results from the in vitro synthesized "lipid rafts-like" liposome system indicated that A-ECG dimer and A-EGCG dimer could act on lipid rafts,significantly disturbing the structure of lipid rafts.In the 3T3-L1 cell system,by fluorescent probes labeling and microscope observation,it was found that A-ECG dimer and A-EGCG dimer could reduce the cholesterol content in lipid rafts and destroyed lipid rafts integrity.By comparing the mechanism of A-ECG dimer and A-EGCG dimer and lipid rafts cholesterol binding reagents(β-MCD and Filipin)underlying the inhibitory effect on 3T3-L1 preadipocytes differentiation,we found that the inhibiting mechanisms of A-ECG dimer and A-EGCG dimer on cell differentiation were completely consistent with β-MCD and Filipin,which disturbed the cell cycle,hindered cell mitosis,inhibited the expression of downstream target genes PPARγ,SREBP1 C,C/EBPα,down-regulated FAS,SCD1 and other liposynthetic genes,then reduced intracellular lipid accumulation,and ultimately blocked the differentiation of preadipocytes.A-ECG dimer and A-EGCG dimer and β-MCD could synergistically inhibit cell differentiation,further indicating that A-ECG dimer and A-EGCG dimer acted on lipid rafts cholesterol,destroying lipid rafts structure,thereby inhibiting cell differentiation.Molecular dynamics simulation of the constructed POPC/POPE/CHOL phospholipid bilayer revealed that the interaction between A-ECG dimer or A-EGCG dimer and POPC/POPE/CHOL phospholipid bilayer was stronger than that of POPC/POPE phospholipid bilayer.A-ECG dimer or A-EGCG dimer was capable of simultaneously forming multiple hydrogen bonds with cholesterol in the phospholipid membrane.The theoretical results further confirmed the interactive effects of A-ECG dimer and A-EGCG dimer with lipid rafts cholesterol.Additionally,A-ECG dimer and A-EGCG dimer disrupted lipid raft structure by binding lipid rafts cholesterol and hindering the migration of insulin-induced insulin receptors(IR)and insulin-like growth factor-1 receptors(IGF-1R)from non-lipid rafts fraction to lipid rafts fraction and auto-phosphorylation of the receptors in the lipid rafts fraction.The inhibition of the receptors’ activation led to the breakdown of the downstream proliferation and differentiation signaling pathways.5.The association of persimmon tannin characteristic structural unit inhibiting the differentiation of 3T3-L1 preadipocytes with the lipid rafts receptor 67 LR.67LR is known as a cell surface molecular receptor of EGCG.In present study,with or without pretreatment of anti-67 LR antibody,the effects of A-ECG dimer and A-EGCG dimer on cell cycle,cell proliferation,insulin signaling pathway and cell terminal differentiation were compared.The results demonstrated that A-EGCG dimer repressed cell cycle,prevented cell mitosis,inhibited activation of the insulin signaling pathway(IRS1/PI3K-AKT/MEK-ERK)and cell terminal differentiation through the 67 LR pathway.A-ECG dimer could also interfere with cell cycle,inhibit cell mitosis,activationof the insulin signaling pathway(IRS1/PI3K-AKT/MEK-ERK),and terminal differentiation of cells,but this process was not dependent on the 67 LR pathway.Molecular docking revealed that the binding of A-EGCG dimer to 67 LR was similar to that of EGCG w.One of the galloyl group inserted into the hydrophobic pocket of the protein,forming three hydrogen bonds with Trp176,Arg180,and Arg184,while for A-ECG dimer,it was a pyrocatechol ring inserted into the hydrophobic pocket to form a hydrogen bond with His169.In the entire amino acid sequence,residues 169-180 of the palindromic sequence were reported to be involved in the interaction of the small molecule inhibitor with 67 LR.Our docking results indicated that the galloyl group of EGCG and A-EGCG dimer could interact with this binding site and the pyrocatechol ring of A-ECG dimer has a limited binding effect.The molecular docking results gave further support to our experimental results.