Study On The Effect Of Bitter Chinese Medicine On Lipid Metabolism Mediated By Autophagy-lysosome Pathwa

Background and aimDysfunction of lipid metabolism is considered to be caused by the disorder of qi activity in traditional Chinese medicine(TCM).Stagnation of qi and body fluid would induce phlegmdampness,and stagnation of qi and blood lead to blood stasis.Herbs with pungent in flavor could move qi to relieve distension and herbs with bitter in flavor could eliminate dampness.Thus herbs with bitter and pungent in flavor hold potential to disperse stagnation and purging heat and improve the blance of human body.While herbal medicines generally contain diverse chemicals and make it difficult to fully understand their chemical basis and molecular mechanism that be responsible for their functions.To identify the potential bio-active ingredients for uncovering the implications of TCMs on regulating lipid metabolism,a TCM compound library with 376 compounds was collected and their effects were assessed from the aspects of both phenotype and pathway,the action rule of pungent and bitter medicines was to be revealed through this application study.Methods1.Screening of active ingredients based on three lipid deposition phenotypes(1)A phenotypic high content screening assay for administrating lipid droplet accumulation in 3T3-L1 adipocytes was developed and applied for the TCM compound library.Nile red and Bodipy 493/503 were used for staining lipid droplets.The primary screening concentration was 10 μM and the cells were continuously incubated with test compounds for 6 days.Subsequently,immunofluorescent labeling was used for detection of the expression of lipid droplet biomarkers to validate the anti-adipogenic effects of identified hit compounds and analyze their molecular mechism.The examed biomarkers included silent mating type information regulation 2 homolog 1(SIRT1),peroxisome proliferator-activated receptor gamma(PPARy),CCAAT/enhancer-binding protein alpha(C/EBPα),fatty acid synthesis(FAS),apolipoprotein E(APOE),and uncoupling protein-1(UCP-1).Thereafter,to explore the potential role of bitter taste receptors(T2Rs)on lipid droplet metabolism,expression of T2R10 and T2R14 were detected by immunofluorescent labeling.Meanwhile,molecular docking and calcium mobilization assay were adopted for investigating the effects of cardamonin on TRPA1.Label free real-time cell analysis(RTCA)was applied for evaluating the effect of cardamonin on neonatal rat cardiomyocyte.A protein-protein interaction network was constructed incombination of its inhibitory effects on TRPA1 and lipid droplet biomarkers.Finally,study of drug combination was performed for potential synergistic effects on lipid droplet formation,cell index(CI)was calculated according to Tou-Talalay Method.Synergistic effects were assessed for two-compound combination of berberine hydrochloride,quercetin,lovastatin,aspirin,and metformin.(2)GM03123 cells were adopted as the cell model of cholesterol deposition and applied to the TCM compound library,for the aim of identifying potential ingredients that attenuated cholesterol deposition.The primary screening was conducted at 10 μM for continuous treatment of 72 h.Cholesterol was stained with filipin and the cell body was stained with CMRA.High content screening assay was used for analyzing the cholesterol in each cell.The identified hits from primary screening were further validated by dose responses within 0-30 μM for 48 h and 72 h treatment.(3)The cationic amphiphilic drug(CAD)induced lysosomal phospholipidosis increased cardiotoxicity and hepatotoxicity.An image-based high content assay was optimized by propranolol(30 μM)induced phospholipidosis in HepG2 cells,which facilitated to identify potential anti-phospholipidosis ingredients from 165 compounds.Phospholipid was stained with LipidToxTM phospholipidosis during the compound treatment for 48 h.The average phospholipid per cell was analyzed by spot detector protocol.2.Regulation of autophagic-lysosomal pathway(4)Transcriptional factor EB(TFEB)was the key factor of autophagic-lysosomal pathway,the TFEB stable overexpression cell line was adopted for high content nuclear translocation analysis.Primary screening was performed at the concentration of 10 μM for 4 h treatment.Percentage of Positive TFEB and Mean Nuclear TFEB Intensity were analyzed for assessing the activity on inducing nuclear translocation.Dose-response effects were confirmed for identified hit compounds by 1.5 folds dilution within 30 μM.Further,BAPTA-AM(10 μM)was pretreated with the cells for 30 min to chelate the Ca2+,for exploring the role of Ca2+ on the hit compounds induced TFEB nuclear translocation.(5)Further,microtubule-associated protein light chain 3(LC3)was another critical biomarker involved in autophagyic-lysosomal pathway,effects of 26 representative compounds on inducing LC3 formation were assessed by spot detection.GFP-and mCherry-LC3 spot intensity per cell were analyzed by high content imaging assay.Lysosomal and mitochondrial activity were monitored for the representative compound C10 treatment in 3T3-L1 preadipocytes.Results(1)A high content screening assay was developed and 16 active compounds were identified from the TCM compound library with 50%inhibitory concentration(IC50)values determined.The positive correlationship of lipid droplet with PPARy,C/EBPa,FAS,APOE,and UCP1 were determined from single cell level(P<0.05).Agrimol B improved SIRT1 expression and further down-regulated PPARγ,C/EBPα,FAS,APOE,and UCP-1.Cardamonin and parthenolide exhibited similar inhibitory effects on PPARy signal pathway.Meanwhile,the effects of ten anti-adipogenic candidates were validated by decreased PPARγ-C/EBPαexpression.Immunofluoresent staining analysis showed that two bitter taste receptors T2R10 and T2R14 were detected in 3T3-L1 adipocytes,their expression increased during the adipocyte maturation.Cardamonin selectively blocked TRPA1 ion channel,the IC50 value was 454 nM,and it did not obviously influence cardiomyocyte activity.The protein-protein interaction study suggested that addictive effects could be achieved of cardamonin by blocking TRPA1 and lipid droplet deposition.The combination study revealed that synergistic anti-adipogenic effects were observed and validated by combining quercetin and metformin(CI<0.7),while antagonistic effects were observed by combining aspirin with berberine hydrochloride or quercetin(CI>1).(2)Twelve compounds were confirmed to markedly decrease cholesterol deposition.Four ingredients derived from Salvia miltiorrhiza exhibited the strongest efficacy and the IC50 values were within 5 μM.Origins of the other active ingredients identified included Agrimonia pilosa,Coptis chinensis,Radix Ophiopogonis,Hippophae rhamnoides,Lonicera japonica,and Humulus japonicas,etc.Among the hit compounds,three polyphenols exhibited "U" shape dose responses.This study identified 11 novel ingredients for the treatment of NPC1 mutation induced cholesterol deposition with IC50 values determined,which provided a reliable basis for the further in vivo and clinical studies.(3)The mTORC1 inhibitor torin 1(250 nM)and rapamycin(1 μM)mildly attenuated phospholipidosis.A natural compound isolated from Agrimonia pilosa Ledeb.,Y15,markedly blocked phospholipidosis and exhibited dose-dependent responses.These results implied that Agrimonia pilosa Ledeb may hold potential efficacy for inhibiting drug-induced phospholipidosis.In contrast,the ingrients derived from Salvia miltiorrhiza failed to reverse phospholipidosis.This study implied that activating autophagic lysosomal pathway possessed mild effects on reversing phospholipidosis.(4)TFEB nuclear translocation assay identified 23 hit compounds,four compounds were derived from Salvia miltiorrhiza and the EC50 values were less than 6 μM.Its ethanol extract induced TFEB nuclear translocation with EC50 value of 8.8 μg·mL-1,while the boiled water extract exhibited slight effect with EC50 value up to 33.4 μg·mL-1.The other hit compounds were mainly derived from bitter taste herbals,Agrimonia pilosa,Stephania tetrandra S.Moore,Lotus plumule,Rabdosia rubescens,and Chelidonium majus;pungent herbs ginger,feverfew,and Alpinia katsumadai.Further,the percentages of positive TFEB were obviously decreased in C10,F07,F47,B17,D27,and D06 treated cells with cellular Ca2+ chelated(P<0.05),which suggested that Ca2+played a role on these compounds induced nuclear translocation.On the contrary,the efficacy of Y15 and X39 were markedly increased(P<0.05).(5)Thirteen compounds were observed to induce autophagosome accumulation from 26 tested compounds.A representative ingredient,Y15,significantly induced LC3 puncta formation in time-and dose-dependent manners.Ingredients derived from Salvia miltiorrhiza mildly decreased cellular GFP-and mCherry-LC3 puncta accumulation.Meanwhile,effects were obsent among the other eight compounds(isorham,luteolin,reserpine,orindonin,pyrogallic acid,berberine hydrochloride,quercetin,and C10).C10 significantly improved lysosomal and mitochondrial activity.Additionally,rapamycin and torin 1 significantly inhibited lipid droplet accumulation,3-methyladenine blocked autophagic influx induced by rapamycin and torin 1,while it failed to abrogate their anti-adipogenic effects.ConclusionIn summary,a high content screening platform was developed by ingeniously combination of five phenotypic assays.This study identified 16 compounds that inhibited lipid droplet accumulation,12 compounds reduced cholesterol deposition,one compound blocked phospholipidosis,23 compounds induced TFEB nuclear translocation,and 13 compounds induced LC3 puncta formation.A series of candidates that targeted at ALP and regulated lipid metabolism were identified.The major findings were as follows:(1)Four candidate compounds(Y15,D27,C10,and D06)activated autophagic-lysosomalpathway and significantly reduced lipid droplet accumulation in 3T3-L1 adipocytes.(2)Seven candidate compounds(T05,X02,F07,X29,X39,Y15,and F46)activated autophagic-lysosomal pathway and attenuated cholesterol deposition in GM03123 cells.(3)Five bitter candidate compounds(berberine hydrochloride,quercetin,andrographolide,aristolochic acid A,and R-ginsenoside Rh2)inhibited lipid droplet accumulation without inducing TFEB nuclear translocation or LC3 puncta formation.(4)One compound derived from Agrimonia pilosa decreased propranolol and amiodarone induced phospholipidosis.