High Content Screening Of Traditional Chinese Medicine Active Ingredients Inhibiting 3T3-L1 Cell Lipid Formation

Objective:Large number of traditional Chinese medicines(TCM)has been claimed to be able to anti-hyperlipidemia,however,the main active ingredients of the TCMs of lipid-lowering effects have not been fully clarified.Further,the conventional methods are far more than enough to meet the demands of efficiency to evaluate the tested samples in large scale in pararel.Therefore,the current research aimed to identify bioactive ingredients of TCMs though establishing a phenotype-based,stable and robust high-content screening(HCS)assay.In addition,compound induced liver injury of the candidates was investigated to assure their safety.Methods:3T3-L1 preadipocytes were differentiated with hormone coaktail to induce lipid droplet accumulation.The lipid droplet was stained with Nile red and the fluorescence was captured using Cellomics ArrayScan(?)VT1 HCS Reader,consistent analyzing template was employed to calculate the fluorescence intensity of lipid droplet,which was represented by RFU(relative fluorescence unit)per μm2.The Z factor was determined between the differentiated mature 3T3-L1 adipocytes and 10 μmol·L-1 Lovastatin treated cells to evaluate the performance of the HCS assay.135 compounds derived from multiple TCMs and 24 fractions extracted from the plant of Polygonum cuspidalum were evaluated for their effects on inhibiting lipid droplet accumulation in this HCS assay,the primary screening concentration was 10 μmol·L-1 for single compound and 500 μg·mL-1 for fraction.Nuclear labeled by Hoechst 33342 representing 3T3-L1 adipocyte cell viability was used to exclude false positive effects.For the tested samples with inhibition up to 50%,dose response curves were further identified,the IC50 value of anti-adipogenic effect was determined according the fitted dose response curve using GraphPad Prism 5.0.Further,for those candidates reduced adipocyte cell number,compound-induced liver injury were tested by a multi-parameter ToxInsight assay,cell loss,reduced glutathione level(GSH),reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were used as toxicity markers.Results:The Z factor value of the HCS assay was determined as 0.62(n=27),indicating that the assay was excellent in quality and stability.11 potential triglyceride inhibitors were identified by primary screening from a collection of 135 compounds with structure diversity,and 1 fraction was discovered from the 24 tested factions.The hits were further validated by rescreening and the IC50 values of candidates were acquired.The IC50 value of cycloastragenol(CAG)isolated from Radix astragali was 15.7 μmol·L-1.The IC50 value of oleanolic acid(OA)was determined as 14.6 μmol·L-1.2 active ingredients were discovered from Salvia miltiorrhiza Bge.,1 was a phenolic acid with IC50 value of 3.3 μmol·L-1,the other was a quinine with IC50 value of 2.2 pmol·L-1.1 polyphenol was identified with IC50 value of 9.5 μmol·L-1.2 alkaloids were identified from Berberidaceae plants,with IC50 values of 2.43 and 2.44 μmol·L-1,respectively;1 lignin was identified from Schisandra chinensis with IC50 value of 1.4 μmol·L-1;2 triterpenes were identified from ginsengs with IC50 values of 2.43 and 2.44 μmol·L-1,,respectively.The IC50 value of a flavonoid from the plant of Flos sophorae was determined as 2.5μmol·L-1.The results of liver toxicity evaluation showed that merely an alkaloid B-51 and a polyphenol B-22 induced slight injury of HepG2 cells at 40 μmol·L-1,the other candidates were free of hepatocyte injury within tested concentrations.Calcium mobilization assay exhibited that CAG stimulated intracellular calcium influx in 3T3-L1 preadipocyte with dose dependent trend,the EC50 value was determined as 21.9 μmol·L-1,Since there were proofs that elevated calcium level played a vital role in suppressing adipocyte differentiation,the induced cytoplasmic calcium by CAG might be implicated in anti-adipogenic process.Conclusion:A robust phenotype-based HCS imaging assay was developled to identify anti-adipogenic ingredients.11 candidate compounds were figured out from a collection of 135 compounds and 1 fraction was identified from Polygonum cuspidatum extracts.The current study partially explained the material basis of anti-hyperlipidemia effects for more than 10 TCMs.