Study On The Effect Of Mongolian Medicine Wuwei Qingzhuo San Against Oleic Acid-induced Fat Accumulation In HepG2 Cells Based On The Method Of In-vitro Pharmacology

Objective:To establish a model of valgus intestinal sac method for studying the components of Wei Wei Qing Zhuo San(WWQZS)absorbed by intestinal sac.To investigate the transitional components in blood of WWQZS by using Serum Pharmacochemistry Method.To study the effect of intestinal absorbed solution and serum containing WWQZS on the lipid accumulation in HepG2 cells induced by oleic acid in vitro.Methods:The intestinal absorbed solution containing WWQZS was prepared by using valgus intestinal sac method and the components in which were analyzed by UPLC-MS technology.A lipid accumulation model in HepG2 cells induced by oleic acid was established in vitro.The intervention effect of different concentrations of intestinal absorption solution containing WWQZS on lipid accumulation in HepG2 cells induced by oleic acid was evaluated based on the index of thiazolium blue experiment(MTT value),oil red O staining and changes of TG levels.The transitional components in blood were detected by UPLC-MS analysis.The cell viability of HepG2 cells mediated by different concentration of oleic acid or the serum containing WWQZS was detected by the thiazole blue(MTT value)experiment,also,the effect of the serum containing WWQZS on the accumulation of lipids in HepG2 cells induced by oleic acid in vitro was investigated by oil red O staining and the determination of triglyceride(TG value).Results:Compound Discoverer 3.0 was used after UPLC-MS detection to infer the structures of compound comparing with multi-stage fragment ion information in known literature.Six ingredients were identified by comparing the samples of blank intestinal absorption solution under the condition of positive and negative ions information,they were piperine,piperlonguminine,gallic acid,pomegranate skin tannin,hydroxyl safflower yellow pigment A,safflower 5-glucoside.The model cells were treated with different concentrations of intestinal absorption solution containing WWQZS and then the oil red O staining was performed;the result shows that a large amount of lipid accumulated around the cell membrane in the model group,and a large area of red lipid droplets wrapped around the cell membrane.The result of TG content measurement was that the intestinal absorption solution at different concentrations decreased the TG content in HepG2 cells in a dose-dependent manner,which was extremely significant compared with the model group.The Xcalibur 2.2 software was used to compare the detected structure data of compound with the known multilevel fragment information in the literature,and compared with the blank serum sample,a compound,piperine,was finally identified in the positive ion mode.The results of oil red O staining after the intervention in model cells with different concentrations of serum containing drugs showed that the cells grew ovally with clear edges and abundant cytoplasm,the red lipid droplets were not observed at the cell edges.The cells in the model group showed obvious red lipid droplets surrounding the nucleus,indicating this is a successful hyperlipidemic cell model.Further observation by optical microscope showed that with the increase of the concentration of serum containing WWQSZ,the red lipid droplets in cells gradually decreased and the color became lighter.After the administration of the drug-containing serum in HepG2 cell induced with oleic acid,Compared with the control group,the content of triglyceride(TG)in the model group increased significantly(P<0.01),indicating that the HepG2 cells lipid accumulation model induced by 1mmol/l oleic acid was successfully established;Compared with the model group,serum containing WWQZS in the range of 2.5%to 40%in reducing TG content was significantly(P<0.01).Conclusions:After the analysis by UPLC-MS,the components that were absorbed by the intestinal in the intestinal absorption solution containing WWQZS were piperine,piperlonguminine,gallic acid,pomegranate skin tannin,hydroxyl safflower yellow pigment A,safflower 5-glucoside.Piperine is the only transitional component in the drug-containing serum;These ingredients are may be the pharmacodyamic material basis for WWQZS.In the pharmacology study,taking TG content and the result of oil red O staining as indicators,the contents of lipid droplets and TG in HepG2 cells were effectively reduced by serum and intestinal absorption solution containing WWQZS at different concentrations,which has a certain efficacy in lipid lowering and provides some important experimental data for the further development and utilization of WWQZS.