Study The Mechanism Of Qige Decoction Alleviating NAFLD Based On ACSL1 Regulating Arachidonic Acid Metabolism

BackgroundNon-alcoholic fatty liver disease(NAFLD)is the most common chronic liver disease,and it is also one of the main causes of liver cirrhosis and liver cancer-related deaths.NAFLD can be divided into simple steatosis(SS)and non-alcoholic steatohepatitis(NASH),NASH-related liver cirrhosis according to pathology.The pathogenesis of NAFLD has not yet been fully elucidated.Disorders of lipid metabolism run through the pathogenesis of NAFLD.Long-chain acyl-Co A synthase 1(ACSL1)can catalyze the synthesis of acyl-Co A from long-chain fatty acids between C:12 and C:22.Both lipid synthesis and catabolism are related to ACSL1.In the previous experiments,the expression of ACSL1 was downregulated in the liver tissue of NAFLD rats based on transcriptomics(N=3,log2 Fold Change=-0.415,Pvalue=0.00063586,the experimental data is not yet available Published).In the preliminary experiment,the expression of rat liver ACSL1 protein was down-regulated by WB verification.Further based on bioinformatics,we performed single gene set enrichment analysis(GSEA)on ACSL1 and found that the low expression of ACSL1 is related to the metabolism of arachidonic acid.ACSL1 may be an effective intervention target for the treatment of NAFLD.However,the pathogenesis of ACSL1 in NAFLD still needs further study.Although diet control and exercise can alleviate NAFLD,for severe NAFLD patients,effective lipid-lowering treatment is needed to regain healthy blood lipid levels.The commonly used drugs for lowering blood lipids mainly include statins and fibrates,but these drugs are expensive,and long-term use may cause gastrointestinal reactions and adverse reactions such as myalgia and rhabdomyolysis.Therefore,choosing an effective,safe and cheap drug is of great significance for patients with hyperlipidemia,coronary heart disease,and NAFLD.The previous experiments of our research group found that Astragaloside IV-pμerarin has the effect of protecting gastric mucosa damage,and the compatibility of tangerine peel has a synergistic effect.Therefore,our research group has been devoted to the pharmacological research of Qige Decoction(Astragaloside IV:pμerarin: nobiletin =30:10:5)for many years.In the early stage,we found that preadministration can prevent liver damage in alcoholic rats,and can also improve lipid metabolites such as glycerophospholipid metabolism,tryptophan metabolism,and arachidonic acid in alcoholic liver damage.Qige Decoction may have the effect of lowering lipids.Furthermore,our research group found that Qige Decoction can reduce cholesterol(TC)and triglycerides(TG)in rats induced by high-fat diet + carbon tetrachloride(molecular formula: CCL4).Rat liver and urine metabolomics found that Qige Decoction can reduce lipids such as oleic acid,linoleic acid,docosapentaenoic acid,and glycerophospholipids(relevant data has not been officially published).Linoleic acid is an essential amino acid in the body and can be metabolized into arachidonic acid.Therefore,Qige Decoction may improve the lipid disorder of NAFLD by regulating arachidonic acid.Therefore,this experiment will further study the mechanism of Qigegen Decoction to improve NAFLD and provide a theoretical basis for the clinical application of Qigegen Decoction.Objective1.To study the pathogenesis of ACSL1 in regulating arachidonic acid metabolism in NAFLD;2.To study the mechanism of Qige Decoction and its active ingredients in alleviating NAFLD in vivo and in vitro.MethodsPart 1: The pathogenesis of ACSL1 in NAFLD.(1)study the differential expression of ACSL1 in NAFLD based on bioinformatics:NAFLD gene chip data and high-throughput data are downloaded from GEO database.The limma package and edge R package in R language are used to analyze gene data sets.The differential change of ACSL1 in NAFLD was found,and single gene set enrichment analysis(GSEA)analysis of ACSL1 was used to analyze the data set(2)in vitro: The SS model is constructed by high-fat and high-cholesterol feed,The NASH model is constructed by high-fat and high-cholesterol feed + CCL4.record the weight of the rat,HE and oil red staining to detect the liver lipid accumulation of the rat.Automatic biochemical analyzer is used to detect rat serum ALT,AST,TC,TG,HDL,LDL.WB,immunohistochemistry test ACSL1,FADS2,CYB5R3,COX1,SCD,ELOVL6,CYB5 protein expression.(3)In vivo :First,300 μM oleic acid + 150 μM palmitic acid + 100 μM linoleic acid(OPL)was used to co-culture with rat BRL cells to construct NAFLD models in vitro.Cell lipids were extracted with methanol and chloroform,the content of TG in cells was detected by an automatic biochemical analyzer,combined with oil red staining to evaluate the construction of the model,and the expression of ACSL1 in NAFLD was detected by WB.Next,use lentivirus to construct an in vitro model of ACSL1 overexpression and downregulation.WB and immunofluorescence are used to evaluate the construction of the model.Furthermore,LC-MS/MS was used to analyze the lipidomic changes of control,sh ACSL1,Over ACSL1 with and without fatty acid intervention,and finally WB and CO-IP were used to verify the expression of related genes and the effect of protein interaction.The second part: Qige Decoction and its active ingredients alleviate NAFLD in vivo and in vitro.(1)In vivo : high-fat and high-cholesterol feed is used to build SS model,and high-fat and high-cholesterol feed+CCL4 is used to build NASH model.SS model rats were given low(5g/kg),medium(10g/kg),and high(20g/kg)Qige Decoction.In the SS model,5g/kg Qige Decoction has been screened as an effective dose for intervention in NAFLD rats,so NASH rats were given 5g/kg Qige Decoction.Record the weight of the rat.HE and oil red staining clearly showed the accumulation of liver lipids in the liver of rats.Automatic biochemical analyzer was used to detect rat serum liver function ALT,AST,TC,TG,HDL,LDL,WB,immunohistochemistry were used to verify the related proteins ACSL1,FADS2,CYB5R3,COX1,SCD,ELOVL6,CYB5 expression.(2)In vitro: Screening of 7 active ingredients of Qige Decoction,including:astragalosi de IV,pμerarin,nobiletin,3’-hydroxy pμerarin,calycosin,naringenin,qμercetin for lipid-loweri ng experiments.First,the MTT experiment was used to clarify the non-toxic concentration o f the 7 components in vitro,and then the active components were co-cultured with OPL in r at BRL cells.The lipids were extracted with methanol,chloroform,and isopropanol,and TG was detected by an automatic biochemical analyzer.Oil red staining evaluates the effect of active ingredients on the lipid accumulation of BRL cells.ResultsPart 1: The pathogenesis of ACSL1 in NAFLD.(1)study the differential expression of ACSL1 in NAFLD based on bioinformatics: a total of 4 sets of NAFLD data sets were download from GEO database.Among them,ACSL1 was down-regulated in GSE33814 and GSE39140,and there was no significant difference in GSE89632 and GSE126848.GSEA analysis showed that the high expression of ACSL1 is related to the peroxisome and adipokine signaling pathway,while the low expression of ACSL1 is related to the metabolism of arachidonic acid.(2)In vivo: SS and NASH models were successfully constructed in vivo.For SS rats,compared with control group,the weight of rats in the model group increased slowly on the15 th day,and decreased significantly on the 20 th day.HE staining showed that the cytoplasm of liver in the normal group was relatively tight,the liver lobules of SS rats were normal,the hepatocytes were vacuolated,and there was no obvious inflammatory cell infiltration.Oil red staining showed that there were a lot of neutral lipid droplets in SS.Compared with control group,ALT and AST in SS group had no significant change,TC,TG and LDL increased significantly,while HDL decreased.WB showed that compared with control group,ACSL1 in model group was significantly decreased,while FADS2,cyb5r3 and cox1 were significantly increased.There was no statistical significance between SCD,ELOVL6 and CYB5 groups.The results of immunohistochemistry showed that the expression of ACSL1 was down regulated,while the expression of FADS2,ELOVL6,COX1 and LOX5 was increased in the model group compared with the normal control group.On the 30 th day,compared with the control group,the weight of NASH rats was lower than that of the normal control group.Alt,AST,TC,TG and LDL were significantly increased,while HDL was decreased.HE staining showed hepatic bullous steatosis,lobular inflammatory infiltration and ballooning degeneration in NASH rats.Oil red staining showed that there was a large amount of neutral fat in NASH.WB showed that: compared with normal control group,ACSL1 in NASH group was significantly decreased,while SCD,ELOVL6,CYB5,COX1 and LOX5 were significantly increased,but there was no significant difference between FADS2 and CYB5R3 groups(3)In vitro: 300μM oleic acid + 150μM palmitic acid + 100μM linoleic acid(OPL)co-cultured with rat BRL for 1 day,the intracellular TG was significantly increased,and oil red staining indicated a large amount of neutral fat accumulated in the cells.WB indicated that the expression of ACSL1 was reduced.WB and immunofluorescence indicated that ACSL1 overexpression and downregulation model was successfully constructed in vitro.Compared with the model group,154 down-regulated metabolites and 519 up-regulated metabolites were obtained.OPL-BRL and OPL-sh ACSL1 groups had 195 down-regulated metabolites and 357 up-regulated metabolites,PL-BRL and OPL-Over ACSL1 A total of158 down-regulations and 322 up-regulation differential metabolisms were obtained between the two groups.Differential metabolite enrichment analysis suggests that the down-regulation of ACSL1 is closely related to the metabolism of arachidonic acid.The verification experiment found that: compared with OPL-BRL and OPL-sh ACSL1 groups,FADS2,SCD were down-regulated,ELOVL6,COX1,and LOX5 were up.ACSL1 has no interaction relationship with COX1,CYB5 and CYB5R3.The second part: Qige Decoction and its active ingredients alleviate NAFLD in vivo and in vitro.(1)In vivo: For SS rats,compared with the control group,the weight of the model group increased significantly in the first 2 weeks,and the growth rate of the later rats was slow.On the 20 th day,the weight of the model group was significantly reduced compared with that of the control group.Compared with the model group,the weight of rats with5g/kg Qige Decoction decreased.HE staining suggests that the liver plate structure around the central vein of the liver lobules is neatly arranged.the liver cells are swollen,and vacuoles of varying sizes are seen,with nuclear displacement in the model group in the control group.The liver cells are swollen,and there are small vacuoles of different sizes in the liver cells,and the nuclear shift is not obvious in the 5g/kg Qige Decoction group.Serum biochemical results showed that compared with the model group,5g/kg Qige Decoction could significantly reduce TC,TG and LDL in rats.There was no significant improvement in pathology and biochemistry in the 10g/kg Qige Decoction and 20g/kg Qige Decoction groups.WB results showed that: compared with the control group,the expression of ACSL1 in the model group was down-regulated,and compared with the model group,the expression of ACSL1 was up-regulated in the 5g/kg Qige Decoction and positive drugs.Compared with the normal group,the expression of FADS2,CYB5R3 and COX1 was upregulated in the model group.Compared with the model group,the expression of FADS2,CYB5R3,and COX1 was reduced in the 5g/kg Qige Decoction group,which was confirmed by immunohistochemistry to further corroborate the results of WB.For NASH rats,HE staining suggests that the liver of NASH rats has bullous steatosis,lobular inflammatory infiltration,and balloon-like degeneration of hepatocytes.the inflammation infiltration was reduced,and the liver cells still had vacuolar degeneration in the 5g/kg Qige Decoction group.Oil red staining indicated that a large amount of neutral fat accumulated in NASH.The lipid accumulation of 5g/kg Qige Decoction was less than that of the model group.Serum biochemical prompts: Compared with the normal group,the model group ALT,AST,TC,TG,LDL were significantly increased,and HDL decreased,while the 5g/kg Qige Decoction group could significantly decrease the ALT,AST,TC,and LDL levels of NASH rats.LDL,but no significant changes to TG and HDL.WB prompt:5g/kg Qige Decoction can increase the expression of ACSL1 in NASH rats and decrease the expression of SCD,CYB5R3,CYB5,and LOX5(2)In vitro: chloroform and methanol extraction of lipids found that 100μM astragaloside IV,1m M puerarin,40μM nobiletin,100μM naringenin can reduce the TG content of liver cells,while isopropanol extraction found 100μM astragaloside IV,40μM nobiletin,100μM naringenin,25μM quercetin can reduce the TG content of hepatocytes.Oil red staining indicates that the active ingredients interfere with the cells.After fixation,oil red staining indicates that the model group cells have a large amount of lipid accumulation,while Astragalus A The lipid accumulation of glycosides,Citrus citrifolia,and naringenin decreased slightly,but the other groups did not improve significantly.Conclusion(1)Down-regulation of ACSL1 can promote the production of arachidonic acid,leading to disorders of liver lipid metabolism and inducing liver inflammation.The possible mechanism is through up-regulating the expression of FADS2,SCD,LOX5,COX1 and other proteins related to arachidonic acid metabolism.(2)Qige Decoction can alleviate lipid accumulation and inflammation in SS and NASH rats in vivo.The possible mechanism is to improve the metabolism of arachidonic acid in liver tissue by up-regulating the expression of ACSL1.(3)The effective ingredients of Qige Decoction,astragaloside IV,nobiletin,and naringenin can reduce the lipid accumulation of BRL rat liver cells.