Treatment Effect Of Serum Containing Quzhi Ruangan Formula On PPARα Of Nonalcoholic Fatty Liver Cell Model Induced By High Oleic Acid And Downstream Lipid Metabolism Genes

Objective1.To verify the therapeutic effect of serum containing Quzhi Ruangan Formula in NAFLD cell model which induced by high oleic acid.2.To elucidate the effects of Quzhi Ruangan Formula and its spleen-invigorating drug group,blood-activating drug group and adjuvant drug group on the genes related to lipid metabolism mediated by PPARαin NAFLD cell model.Methods1.Preparation of drug-containing serum:56 six-week-old SD rats of SPF grade were adaptively fed for 3 days,and randomly divided into:normal group,Quzhi Ruangan Formula whole prescription group,Quzhi Ruangan Formula invigorating spleen group,Quzhi Ruangan Formula activating blood medicine group,Quzhi Ruangan Formula adjuvant drug group,10 rats in each group.The normal group was gavaged with normal saline,and the other experimental groups were gavaged with high doses of the corresponding drug groups for three days.Blood was collected from the abdominal aorta 1hour after the last administration,and the upper serum was separated by centrifugation.The serum of the same drug group was mixed and then inactivated.Store at-80°C after aliquoting.2.Analysis of drug-containing serum components:By UHPLC-QE-MS ultra-high performance liquid chromatogram-tandem four-pole mass spectrometry for liquid chromatography-tandem mass spectrometry to analyze the active components of drug-containing serum in four groups,and the differences of drug-containing serum components in the whole prescription and disassembled prescription groups were compared.3.NAFLD cell model preparation:human normal liver cell line（L-02）in the logarithmic growth phase was seeded in a 6-well plate for 24 hours,and then used complete medium（containing 10%fetal bovine serum+1%double antibody+RPMI-1640）Add oleic acid working solution（0.5mmol/L）to culture,use oil red O staining to observe the accumulation of cell lipids,CCK-8 method to determine the survival rate of modeled cells,and determine the optimal concentration of oleic acid modeled by the above method.4.Intervention concentration of drug-containing serum and determination of therapeutic effect:The cell survival rate of each concentration of drug-containing serum was observed by CCK-8 experiment,and the normal human liver cell line（L-02）in the logarithmic growth phase was inoculated in 96 wells,and after 24 hours add each dose of drug-containing serum in each group and incubate for 24 hours,add CCK-8 reagent for 2hours,and then test the absorbance on the machine.The cells were cultured on seed plates,and after 24 hours of transfection with the constructed PPER plasmid,each dose of drug-containing serum in each group was added for culture,and the expression activity of luciferase was detected by lysis the next day.The low,middle and high dose groups of drug-containing serum in each group were determined by the above two methods.Oil red O staining was performed to observe the red-orange lipid area after adding medicated serum to the oleic acid model cells overnight,and it was confirmed that the medicated serum of Quzhi Ruangan Formula had a therapeutic effect.5.Establishment of PPARαgene knockdown cell model and drug efficacy test:Human normal liver cell line（L-02）in the logarithmic growth phase was seeded in a6-well plate for 24 hours,and fluorescently labeled si RNA-PPARα^kdwas transfected into the cells for 6 hours.After modeling,high-dose drug-containing serum was added to each experimental group,and cells were collected 24 hours later.6.Detection of expression levels of PPARαand downstream lipid metabolism genes:Total RNA and total protein were extracted from the collected cells,and PPARαand its downstream lipid breakdown-related genes（CPT-1,ACOX-1）m RNA and protein expression of lipid synthesis-related genes（SREBP-1c,FAS）.7.Statistical analysis:Statistical software SPSS was used for analysis and processing.One-way analysis of variance was used among different groups,and the statistical results were presented as mean standard deviation（（?）±S）.LSD test was used for homogeneous variance,and Dunnett’s T3 test was used for heterogeneous variance.P<0.05 means statistical significance.Statistical graphs were drawn using Graph Pad Prism.version8.3.1.Results1.Detection of drug-containing serum components:The components of the drug-containing serum of rats in the Quzhi Ruangan Formula,spleen-invigorating drug group,blood-activating drug group,and adjuvant drug groups were analyzed respectively.302 substances were detected,including 113 terpenoids,77 flavonoids,43 alkaloids,15fatty acids,30 phenols,and 24 other substances.A total of 110 substances were detected in the medicated serum of the invigorating spleen group,of which 54 were the same or similar to the whole prescription of Quzhiruangan Formula.A total of 106 substances were detected in the drug-containing serum of the blood-activating group,and 63 of them were the same or similar to the whole prescription of Quzhi Ruangan Formula.A total of 128substances were detected in the drug-containing serum of the adjuvant drug group,and 79of them were the same or similar to the whole prescription of Quzhi Ruangan Formula.2.Evaluation of NAFLD cell model:after oil red O staining,a large amount of orange-red lipid accumulation was observed in the cells under the microscope.CCK-8results showed that the cell survival rate was 96%,and the NAFLD cell model was successfully constructed.3.Dosing concentration of drug-containing serum:through luciferase reporter gene detection,determine the half effective concentration of drug-containing serum in each group,as follows:the half effective concentration of drug-containing serum of Quzhi Ruangan Formula is EC50=3.249±0.015%;The half effective concentration value of the serum containing Quzhi Ruangan formula for invigorating the spleen is EC50=2.543±0.008%;The half effective concentration value of the drug-containing serum is EC50=3.249±0.031%.The CCK-8 experiment showed that in the four groups of administration,the 7.5%concentration of drug-containing serum began to decrease the cell survival rate,so 2.5%,5.0%,and 7.5%were selected as the low,middle and high-dose groups of drug-containing serum.4.The drug-containing serum has a therapeutic effect:the model cells were given Quzhiruangan Formula,the spleen-invigorating,blood-activating,and adjuvant group with drug-containing serum intervention,and the red-orange lipid area in the cells was observed to decrease under the microscope.5.The expression of PPARαm RNA and protein in the NAFLD cell model in each group after the drug-containing serum intervened:Compared with the normal group,the expression of PPARαm RNA and protein in the model group decreased,and the difference was statistically significant（P<0.01 or P<0.05）.Compared with the model group,the expression of PPARαm RNA and protein in the whole prescription of Quzhi Ruangan Formula and its disassembled prescriptions in low,middle and high dose groups was increased,and the difference was statistically significant（P<0.01 or P<0.05）.Compared with the normal group,the expression of PPARαm RNA in the PPARα^kdgroup and the PPARα^kdmedication group decreased significantly,and the difference was statistically significant（P<0.01 or P<0.05）.6.The expression of CPT-1,ACOX1 m RNA and protein expression of lipolysis-related genes downstream of PPARαpathway after intervention of NAFLD cell model with drug-containing serum in each group:Compared with the normal group,the expression of CPT-1 and ACOX1 m RNA and protein decreased in the model group,the expression of SREBP-1c m RNA,FAS m RNA and protein increased,and the difference was statistically significant（P<0.01 or P<0.05）.Compared with the model group,the expression of CPT-1,ACOX1 m RNA and protein increased in the whole prescription of Quzhi Ruangan Formula and its disassembled prescriptions in the low,middle and high dose groups.Compared with the normal group and model group,the PPARα^kdgroup and the PPARα^kdmedication group,the m RNA and protein expressions of CPT-1 and ACOX1decreased significantly,and the difference was statistically significant（P<0.01 or P<0.05）.7.The expression of lipid synthesis-related genes SREBP-1c,FAS m RNA and protein in the downstream of the PPARαpathway after the drug-containing serum intervened in the NAFLD cell model in each group:Compared with the normal group,the expression of SREBP-1c,FAS m RNA and protein in the model group increased,the difference was statistically significant（P<0.01 or P<0.05）.Compared with the model group,the expression levels of SREBP-1c and FAS m RNA in the Quzhi Ruangan Formula and its disassembled prescription groups were decreased,and the difference was statistically significant（P<0.01）.Compared with the normal group and the model group,the expression of SREBP-1c m RNA and FAS m RNA increased in the PPARα^kdgroup and the PPARα^kdtreatment group,and the difference was statistically significant（P<0.01 or P<0.05）.Conclusion1.In NAFLD model cells,Quzhi Ruangan Formula medicated serum can reduce lipid accumulation,and has a therapeutic effect on nonalcoholic fatty liver cell models;2.Quzhi Ruangan Formula,spleen-invigorating medicine group,blood-activating medicine group and adjuvant medicine group could up-regulate the expressions of PPARα,CPT-1 and ACOX-1,and down-regulate the expressions of SREBP-1c and FAS in the NAFLD cell model.The regulation effect of the drug group on PPARαand downstream lipid metabolism genes is the most obvious,and the regulation effect of the whole prescription,spleen-invigorating and blood-activating drug group is weaker than that of the adjuvant drug group,indicating that Quzhi Ruangan Formula has the most significant effect on PPARαpathway and its downstream lipid metabolism.Metabolic genes are regulated.