Pharmacological Effects And Molecular Mechanism Of Extracts From Radix Bupleuri On Liver Uptake Of HDL

ObjectiveIn this study,we evaluated the aqueous extract and ethanol extract quality of Bupleurum chinense by establishment of Bupleurum characteristic map,hepatocyte uptake of HDL cell model in vitro,cellular selection and transcriptional sequencing.From the standpoint of "Reverse cholesterol transport" we evaluate the pharmacodynamics of the extracts systematically.According to"Gui Jing" in traditional Chinese medicine theory,the active constituents acting on the liver were screened out from Bupleurum extract.After Bupleurum administration,transcriptional level of liver cells was further analyzed at the level of gene transcription,which provided the scientific basis for the mechanism of Bupleurum chinense on the regulation of hepatic fatty degeneration.Methods1.The aqueous extract and ethanol extract of Bupleurum were used as the research object.After the purification,the corresponding characteristic maps were established.The stability,reproducibility and precision of the purification process were carried out to determine the attribution of the main chromatographic peaks in the extract.As a reference standard for Bupleurum extract,it provides complete information on the screening of active ingredients by hepatocyte solid phase chromatography.2.The main components of the aqueous and alcohol extract of Bupleurum were different.The toxic effects of the two extracts on the hepatocytes were investigated by CCK-8 kit.The growth curve was plotted and the safe dose was determined for the efficacy experiment.Based on reverse cholesterol transport regulation of lipid metabolism,we investigate Bupleurum extracts using HepG2 cells in vitro uptake of Dil-HDL models and compared the efficacy effects on lipid metabolism.3.According to target theory,the pharmacophore effect of Bupleurum extract was verified by cell membrane solid phase chromatography,and the active components of the extract were compared with the hepatocytes.4.The high fat model of C57BL/6 mice was established by feeding high fat diet.The pathological sections of mice liver were measured and the data were analyzed by transcriptome sequencing.And the liver gene expression profile in mice was analyzed by using the Poisson distribution.At the meanwhile the differentially expressed genes in liver expressionafter the Bupleurum administration were screened.The gene expression was correlated with lipid metabolism genes for further investigation.5.The FGF21 protein expression was tested by western blot.Results1.The results showed that the main saponins of Bupleurum root extract were saikosaponin a and d,indicating that the composition of Bupleurum was related to extraction methods.The reproducibility of the purification method,good precision and stability,can guarantee the extract quality stable and controllable.2.After Bupleurum administration in Hep G2 cells for 24h,the median lethal dose of Bupleurum aqueous extract(IC50=48.46±3.15 mg/mL,α=0.05)was greater than alcohol extract(IC50=31.17±0.81 mg/mL,a=0.05),indicating that Bupleurum alcohol extract cytotoxicity was stronger.In the safe range of administration,Bupleurum aqueous extract could significantly promote hepatocyte uptake of HDL(p<0.05),while its alcohol extract showed inhibitory effect(p<0.05).3.Saikosaponin b1(SSb1)and b2(SSb2)could bind with the specific sites on the liver cells,and saikosaponin a(SSa)and saikosaponin d(SSd)were not stored in the liver cell membrane adsorption.4.Fat mice after administration of aqueous extract of Bupleurum,fatty liver disease has become to improve,13 DEGs were detected(p<0.0000),including Gm3776,Cyp2b9,FGF21 and so on.Gm3776,Cyp2b9,Lgalsl and other 8 genes were significantly down-regulated after administration of Bupleurum,and FGF21,Moxdl and G0s2 were significantly up-regulated.According to differential expression gene information,found that FGF21 related to lipid regulation in vivo.5.At the level of protein expression,Bupleurum aqueous extract could promote FGF21 secretion,which may be related to the mechanism of promoting hepatic uptake of HDL.ConclusionIn this study,the quality of aqueous and ethanol extract of Bupleurum chinense were evaluated comprehensively and effectively by the characteristic map establishment.The toxicity of extracts and extracts of Bupleurum chinense was evaluated by CCK-8 kit.The toxicity of Bupleurum extract to hepatocytes was stronger than that of aqueous extract by comparing IC50 and growth curve.In vitro cell experiments confirmed that aqueous extract can improve HDL level in hepatocyte and its alcoholic acid showed a retroaction.It suggested that Bupleurum decoction can be used to enhance prevention and treatment of Bupleurum on lipid disorders related to disease.The results showed that the secondary saponins(SSb1 and SSb2)could be bound by hepatocytes,which were potential to promote the uptake of HDL in hepatocytes.Bupleuri extract can increase the FGF21 transcription level in mice liver and improve the pharmacological effect of improving fatty liver disease caused by high fat diet.