Anti-thrombotic Study Of Berberine Via Gut Microbiota Metabolic Pathway Of Choline/TMA/TMAO

Thrombosis is the leading cause of cardiovascular events,and developing effective drugs to decrease risk of thrombosis is urgent in prevention and treatment of cardiovascular diseases.Berberine(BBR)is the main active compound of rhizoma coptidis.Previous studies showed that BBR could decrease the risk of thrombosis via reducing platelet aggregation and activation.However,some data demonstrated that the absorption of BBR was very limited.After a single oral administration of 400mg BBR,the peak plasma concentration was only 0.4ng/ml and the rest of BBR was excreted through feces.The antithrombotic effect of BBR could not be explained directly by its low bioavailability,and the mechanism of BBR on anti-thrombosis is necessary to be studied.Recent study showed that gut microbiota played an important role in diet-dependent susceptibility of cardiovascular events.Gut microbiota produce metabolic dependent and no-metabolic dependent substances,which could influence cardiovascular action directly or indirectly.And gut microbiota has become the new target of anti-thrombosis.Previous studies have confirmed that the Cut C enzyme in gut microbiota could metabolize choline in the diet into trimethylamine(TMA),and then TMA is catalyzed into trimethylamine n-oxide(TMAO)by flavin monooxygenase-3(FMO3).Clinical cohort study and meta-analysis results showed that circulating TMAO was closely related to the incidence of cardiovascular events and was recognized as a new independent risk factor for cardiovascular events.The study showed that TMAO could lead to platelet hyperreactivity via increasing release of calcium in platelet.At present,microbiota-driven therapy(probiotic,prebiotic and synbiotic)was the main intervention targeting Choline/TMA/TMAO metabolic pathway,however,the effects of microbiota-driven therapy were still controversial.In addition,previous studies showed that a variety of traditional Chinese medicines,including BBR,play an important role in regulating gut microbiota.Therefore,we propose that BBR reduces the risk of thrombosis by regulating the Choline/TMA/TMAO metabolic pathway.To validate the hypothesis,C57BL/6J mice,anaerobic bacteria and Cut C enzyme were used and the methods of Western-Blot,Q-PCR and LC-MS/MS were applied in the present study.This study includes four partsPart 1 Effects of microbiota-driven therapy on circulating TMAO and its related metabolites:a meta-analysis and systematic reviewObjective:This study was designed to systematically evaluate the effects of microbiota-driven therapy on decreasing TMAO and its related metabolites.Methods:PubMed,EMBASE,Cochrane Library,CNKI and other Chinese databases were searched to identify randomized controlled trials investigating microbiota-driven therapy on decreasing TMAO and its related metabolites.The searching terms include "trimethylamine or trimethylamine N-oxide or TMA or TMAO or Betaine or L-carnitine" and "prebiotic or prebiotic or synbiotic or dried yeast or lactobacillus".Two researchers extracted the data independently and the disagreement will be solved by a third researcher.The quality of included study was evaluated using Cochrane tool.And meta-analysis,meta-regression analysis and publication bias analysis were performed by RevMan 5.3 or Stata 12.0 software.Results:A total of 8 English studies with 258 patients(126 patients in the microbiota-driven therapy group and 132 patients in the control group)were included.Compared with the control group,microbiota-driven therapy did not reduce circulating TMAO[SMD=-0.12,95%CI(-0.31,0.06),p=0.189],choline[SMD=-0.342,95%CI(-1.093,0.410),p=0.373],betaine aldehyde[SMD=-0.704,95%CI(-1.789,0.382),p=0.204],and L-carnatine[SMD=-0.350,95%CI(-1.906,1.206),p=0.659].The meta-regression showed that the intervention time of microbiota-driven therapy was not associated with the effect size(p>0.05).Conclusion:Microbiota-driven therapy did not reduce circulating TMAO,choline,betaine aldehyde and L-carnatine levels(p>0.05),and the effects sizes were not associated with the intervention time(p>0.05).Therefore,it is necessary to explore new interventions to reduce circulating TMAO level.Part 2 Effects of BBR on the risk of thrombosis induced by high choline dietObjective:The study was designed to observe the effects of BBR on risk of thrombosis induced by high choline dietMethods:C57BL/6J mice were randomly divided into Chow group,Choline group,BBR group and DMB group.After 6 weeks of intervention,a carotid injury model was established by applying 10%FeCl3 solution externally to the exposed left common carotid artery model for 1 min.The time of carotid artery thrombosis in mice was recorded by ultrasound and fluorescence microscope.Platelet aggregation rate and P selection was observed by platelet aggregator and flow cytometry.The bleeding time after cut-off tail was also observed.Results:Compared with Chow group,high-choline diet feeding for 6 weeks could shorten the time of thrombosis,increase the platelet aggregation rate and expression of P selectin(p<0.05).And compared with Choline group,BBR intervention decresed platelet aggregation rate and expression of P selectin of platelet,prolonged the time of thrombosis(p<0.05),without increasing the time of bleeding after cutting off tail(p>0.05).After TMAO injection,the effects of BBR were offset(p<0.05).Conclusion:BBR reduced the risk of thrombosis induced by high choline diet,and TMAO might be the important targetPart 3 Effects of BBR on regulating Choline/TMA/TMAO metabolic pathwayObjective:The study was designed to elucidate molecular mechanism of BBR in regulating circulating TMAO.Methods:C57BL/6J mice were randomly divided into Chow group,Choline group,BBR group and DMB group.Plasma TMA and TMAO levels were observed after 6 weeks of intervention.The mechanism of BBR regulating Choline/TMA/TMAO metabolic pathway was explored by LC-MS/MS,Elisa,Q-PCR and Western-Blot,with the methods of anaerobic culture of gut microbiota and purification of Cut C enzyme.Results:Plasma TMA and TMAO levels in C57BL/6J mice were significantly increased after 6 weeks high choline diet(p<0.001),while BBR intervention significantly decreased plasma TMA and TMAO levels(p<0.05).BBR inhibited the process of choline transforming to TMA via directly inhibiting the activity of C.ut C enzyme(IC50=31 μM),however,there were no effects of BBR on decreasing FMO3 enzyme level or inhibiting FMO3 activity in liver(p>0.05).Conclusion:BBR reduced the transformation of Choline into TMA by inhibiting the activity of Cut C enzyme in gut microbiota.Part 4 The molecular docking and molecular dynamics simulation of BBR and Cut C enzymeObjective:The study was designed to elucidate the interaction between BBR and Cut C enzyme based on molecular docking and molecular dynamics simulation.Methods:(1)The method of molecular docking:Download 3D structure of Cut C enzyme from RCSB database and 3D structure of BBR in the PubChem Compound database.BBR structure was modified by ChemBio 3D and stored in mol format.The structure of Cut C enzyme was modified by PyMOL and stored in PDB format for molecular docking.The"Hydrogen" in Autodock Tools(ADT)was used to add hydrogen of Cut C enzyme and"Compute Gasteiger" was used to calculate local charge of Cut C enzyme.The active pocket of Cut C enzyme for docking was defined as 24cm×22cm×30cm(center:X=-51.504,Y=13.565,and Z=-14.899)according to previous reference,and the distance between the grid points was defined as 1A.Autogrid was used to calculate the grid point energy with Lamarckian genetic algorithm.Default parameters were used for initial population number,mutation probability,crossover probability and maximum optimization number in the genetic algorithm,and each pair was docked for 10 times.(2)The method of molecular dynamics simulation:Amber18 was used for molecular dynamics simulation,amber 99sb-ildn was used for protein force field,and gaff general force field was used for small molecules.First,the optimal BBR-Cut C enzyme complex from molecular docking was placed in a cube water box.The water molecule model was TIP3P,and the closest distance from the edge of the box to the Cut C enzyme was lnm.The energy of the reaction system was minimized by the 10,000-step maximum descent method,and the beam solute molecules were prebalanced by 100ps for the system.The pressure bath used Berendsen,and the temperature bath used v-rescale.The PME algorithm was adopted for electrostatic action,the van der Waals truncation value was 1nm,and the hydrogen atom restriction algorithm was Shake.After the pre-balancing,a dynamic simulation of 50ns was observed,the electrostatic action was PME algorithm,the vandewaal truncation value was 1nm,the pressure bath algorithm was parrinello-rahman,the temperature bath algorithm was v-rescale,and the hydrogen atom restriction algorithm used h-bonds.Track and energy data were stored every 20ps.Results:The molecular docking between BBR and Cut C enzyme showed that BBR was complementary in shape with active pockets of Cut C enzyme and could be tightly bound in the active pockets.The active pocket of Cut C enzyme includes hydrophobic residues such as Tyr490,Tyr670,Phe671,Phe677 and Tyr788,and BBR also formed hydrophobic aromatic rings;BBR could be stabilized in the active pocket of Cut C enzyme by hydrophobic accumulation and van der Waals action with these hydrophobic residues.In addition,there were also two hydrogen bonds complex between BBR and Cut C enzyme,and the bond length was short and the strength was large.Moreover,the benzene ring of BBR and the benzene ring of Tyr 670 in Cut C enzyme formed a Pi-Pi stacking.Further molecular dynamics simulation showed that RMSD and ROG of the complex fluctuated slightly,and their binding energy was as low as-40 kcal/molConclusion:BBR occupied the active pocket of the Cut C enzyme and bound tightly to the Cut C enzyme,the whole system was stable after the combination of BBR and Cut C enzyme.