Effects Of Salivaricin A On TG Transport And Hydrolysis Pathways In Lipidated L02 Cells

BackgroundHyperlipidemia is a common endocrine systemic disease,which is caused by genetic factors,eating habits,lifestyle and age,menopause,disease and drugs.The main manifestations of hyperlipidemia are excessive levels of cholesterol(TC)or triglyceride(TG)in plasma.It also includes the decrease of high density lipoprotein and the increase of low density lipoprotein.From the point of view of traditional Chinese medicine,postmenopausal women have gradually weakened kidney qi,deficiency of kidney essence,deficiency of kidney yang,loss of department of warming yang,dereliction of duty of transpiration,inability to remove qi and water,loss of body fluid,clear turbidity,and hyperlipidemia.The treatment often adopts the method of warming kidney yang.Shayuanzi is a traditional tonifying medicine,which can warm kidney yang and help gasification to reduce turbidity and fat.Our research shows that Shayuanzi has a significant effect on triglyceride(TG)in castrated high-fat model rats,and has no significant increase in estrogen level in model rats.The total flavonoids of Shayuanzi are the effective components of blood lipid lowering A and the content of Shayuan glycoside is high monomer.TG after liver cell synthesis,the assembly VLDL-TG,transports the liver into the blood,and the TG can also be decomposed by the liver hydrolase,thus forming a dynamic balance.three nuclear receptors involved in TG synthesis,transport and decomposition of the liver are SREBP-lc,PPARs,HNF4?,important transcriptional regulators.The mechanism of TG synthesis pathway has been studied in the early stage of the research group.This experiment mainly explores the mechanism of TG transport pathway in the liver.ObjectiveTo study the effect of A on fatty degeneration of L02 hepatic parenchyma cells and the effect of A on the expression and index of TG transport pathway regulated by nuclear factor 4(HNF4?)in hepatocytes.To explore the possible lipid regulation effect and mechanism of Complanatoside A.MethodsStudy 1:L02 Screening of cell line lipid change model concentration:establishing hepatocyte lipid change model and dividing into 5 groups,Each group had three multiple holes:?blank control group:normal cultured L02 cells,no other treatment;?0.125mM FFA group:L02 cell+0.125mmol/L free fatty acid;?0.25mM FFA group:L02 cell+0.25 mmol/L free fatty acid;?0.5mM FFA group:L02 cell+0.5mmol/L free fatty acid;?1FFA group:L02cell+1mmol/L free fatty acid;?2FFA group:L 02 cells+2 free fatty acids;CCK8 method was used to detect cell viability at various concentrations;Grease red O for lipid droplet staining;TG kit to detect triglyceride content.Study 2:The pharmacological effects of Complanatoside A on the model of fatty degeneration of L02 cells:the safe concentration of A action cells was first screened to determine the low,medium and high dose grouping concentration.then,when the cells adhered to the wall,he density reached about 70%-80%.the cells were pretreated with the A of the 1mM FFA for 24 hours,then the L02 cells were treated with a mold-making fluid containing 1 and different concentrations of the cell.then the index was detected.Cells were divided into 5 groups,each group was divided into three multiple holes,the group and administration were as follows:1 blank control group:complete medium normal culture of L02 cells,no other treatment;2 Model group:L02 cell+FFA(1mm);L02 cell+FFA(1mM)+100?M),L02 cell+FFA(1mM),50?M)and 25?M(1mM),Annexin V-PE label,flow cytometry to detect the apoptotic rate of each group of cells;flow cytometry to detect the cell fat level,Dyeing of oil red O to observe lipid droplets;determination of TG?TC?AST?ALT MDA,SOD,IL-6,TNF-? content in hepatocytes in each group.Study 3:Regulation of Astragaloside A on Nuclear Factor 4?(HNF4?)TG Transport and Hydrolysis Pathway in Lipid L02 CellsComplanatoside A pretreatment cells for 24 hours,Then the L 02 cells were treated with a mold-making fluid containing 1mM FFA and different concentrations A sainagin for 24 hours,Then the index is detected.The groups were as follows:?blank control group;?Model group(1mmol/L FFA);?Sargassin A low dose group:25?M;?A low dose group:50?M;?A low dose group:100?M.The levels and P65 mRNA of TG transport pathway related proteins and gene HNF4?,MTP,ATGL?PPAR??LXR?,IL-6,TNF-?proteins regulated by nuclear factor 4(HNF4?)were detected by WB and RT-PCR.ResultsResults of study 1:the effect of different concentrations of FFA on cell activity was determined by CCK8 method.At the same time,the formation of intracellular lipid droplets and the quantitative detection of intracellular TG levels under oil red O staining light microscope were determined.Finally,1mM FFA induced 24h can successfully cause cell lipid change model,laying the foundation for subsequent experiments.Results of study 2:the results showed that A could improve the accumulation of lipid in FFA induced L02 cells,such as the decrease of TG,TC level,the decrease of liver enzyme AST,ALT,IL-6,TNF-?,MDA and the effect of lipid lowering and protecting hepatocytes.Results of study 3:The experimental study of the pharmacodynamics and mechanism of apropionoside A on lipogenic L02 cells showed that aprotonin A has ameliorating effect on the degree of lipolysis in L02 cells induced by free fatty acids.The mechanism may be reduced by up-regulating the level of HNF4?-MTP protein.Apob100 content acts on the TG transport pathway regulated by HNF4?,up-regulates the expression of PPARa and PPARy,increases ATGL expression,promotes the hydrolysis of liver TG,reduces liver fat deposition,and exerts a lipid-lowering effect,while inhibiting TNFa and IL-6.The expression of LXRamRNA reduces the expression of P65 protein and plays a role in lowering lipids,protecting liver,and alleviating oxidative stress.ConclusionsApropionoside A has a certain effect on lowering liver fat and protecting liver in an in vitro hepatocyte lipidosis model.The mechanism of apropionoside A in regulati ng lipids may increase the level of HNF4?-MTP protein,reducing Apob100 content,a nd acting on the regulation of HNF4?.The lipid-lowering effect exerted by the TG tr ansport pathway increases the expression of PPARa and PPARy,increases the expressi on of ATGL,promotes the hydrolysis of liver TG,and reduces liver fat deposition.T he liver protection mechanism may be the regulation of inflammatory factors and adip okines.And inhibit the expression of TNF?,IL-6,LXRamRNA,reduce the expression of P65 protein,and play a role in lowering lipids,protecting liver,and alleviating ox idative stress.