[PubMed] [Google Scholar] 35. decreased oleic acid\induced lipid accumulation, enhanced the protein levels of low\density lipoprotein receptor (LDLR), ABCG5, ABCG8 and PPAR, and reduced the expression of sterol regulatory element\binding protein 2 (~32%). PPAR antagonists, GW6471 and MK886, could significantly inhibit the furanone\induced lipid\lowering effect. Furthermore, the furanone showed a significantly lower activity on the activation of the expression of lipogenic genes compared to T0901317. Taken together, the furanone exhibited a weak cytotoxicity but had powerful TC\ and TG\lowering effects most likely through targeting LXR and PPAR, respectively. These findings indicate that the furanone has a potential application for the treatment of dyslipidaemia. sp SCSIO41009.21 Here, we reported for the first time that the furanone named as 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one had an effective lipid\lowering activity via influencing multiple processes of lipid metabolism. 2.?MATERIALS AND METHODS 2.1. Materials Mouse\derived macrophage cell line RAW 264.7 and the human hepatoma cell line HepG2 were purchased from the Cell Bank of Chinese Academy of Sciences. (Shanghai, China). 3\(4,5\dimethyl\2\thiazolyl)\2,5\diphenyl\2H\tetrazolium bromide (MTT, 413Y0511), oleic acid (01008) and Oil Red O (00625) were Sigma\Aldrich products (St. Louis, MO, USA). Liver X receptor (LXR) agonist T0901317 (293754\55\9), fenofibrate (S1794) and the peroxisome proliferator\activated receptor (PPAR) antagonist MK886 were the products of Selleck (Shanghai, China). LXR antagonist, GSK2033 and SR9243, and PPAR antagonist GW6471 were the products of MedChemExpress (Shanghai, China). Dimethyl sulphoxide (DMSO, 821D035) and the goat serum (SL038) were purchased from Solarbio (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) and foetal bovine serum (FBS) were from Gibco (BRL, Gaithersburg, MD, USA). RIPA lysis buffer was a product of Merck (3108491; Darmstadt, Germany). Rabbit polyclonal antibody against LXR (ab3585, 1:200; ab176323, 1:5000) and LXR (ab28479, 1:500); rabbit monoclonal antibody against scavenger receptor B type 1 (SR\B1, ab217318, 1:2000), ATP\binding cassette (ABC) G1 (ab52617, 1:1000) and low\density lipoprotein receptor (ab52818, LDLR 1:1000); and mouse monoclonal antibody against ABCA1 (ab18180, 1:200 or 1:1000) were from Abcam (Cambridge, MA, USA). Mouse monoclonal antibody against sterol regulatory element\binding protein (SREBP)\1c (sc\13551, 1:100), SREBP\2 (sc\271616, 1:200) and PPAR (sc\398394, 1:100) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody against cholesterol 7 alpha\hydroxylase A1 (CYP7A1, TA351400, 1:1000) was the product of OriGene (Shanghai, China). Mouse monoclonal antibody against \actin (66009\1\Ig, 1:5000), rabbit polyclonal antibody against ABCG5 (27722\1\AP, 1:1000) and rabbit monoclonal antibody against proprotein convertase subtilisin/kexin type 9 (PCSK9, 55206\1\AP, 1:500) were the products of Proteintech (Chicago, IL, USA). Complete protease inhibitor and the secondary antibodies, including the goat antimouse IgG (FITC conjugated), were from CWBIO (Beijing, China). Mouse monoclonal antibody against ABCG8 (1B10A5, 1:1000) and enhanced chemiluminescence (ECL) kits were purchased from Thermo Scientific Pierce (Rockford, IL, USA). Total cholesterol (TC) and triglyceride (TG) assay kits were the products of Biosino Bio\technology and Science Inc (Beijing, China). Double\deionized water was produced using a Milli\Q Gradient System from Millipore. All reagents used in this study were of analytical grade. 2.2. Purity determination of the furanone, 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one The furanone, 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one, was isolated from the fungus sp SCSIO41009, as previously reported.21 Its purity was determined by multiple analytical methods. Ultra\performance liquid chromatography (UPLC) spectrum was performed on an Acquity UPLC BEH C18 column (2.1??50?mm i.d., 1.7?m) connected to a Waters Acquity H Class UPLC System (Waters) with a PDA detector (wavelength of 212?nm). High\resolution electrospray ionization mass spectrometry (HRESIMS) spectrum was recorded on a Bruker maXis Q\TOF mass spectrometer in positive ion mode. 1D and 2D NMR spectra were measured on a Bruker AV 500? MHz or AVANCE HD 700?MHz NMR spectrometer with tetramethylsilane as an internal standard.21 2.3. Preparation of lipoproteins Plasma was from healthy volunteers in the Affiliated Hospital of Weifang Medical University or college. To obtain LDL fraction, plasma was subjected to sequential ultracentrifugation as previously explained.22, 23 In brief, the plasma denseness was adjusted to 1 1.006?g/mL for ultracentrifugation at 10C (400?000??for 24?hours). The top layer containing very low\denseness lipoproteins was eliminated, and the denseness was re\modified to.Lee\Rueckert M, Escola\Gil JC, Kovanen PT. collectively, the furanone exhibited a fragile cytotoxicity but experienced powerful TC\ and TG\decreasing effects most likely through focusing on LXR and PPAR, respectively. These findings indicate the furanone has a potential software for the treatment of dyslipidaemia. sp SCSIO41009.21 Here, we reported for the first time the furanone named as 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one experienced an effective lipid\lowering activity via influencing multiple processes of lipid metabolism. 2.?MATERIALS AND METHODS 2.1. Materials Mouse\derived macrophage cell collection Natural 264.7 and the human being hepatoma cell collection HepG2 were purchased from your Cell Standard bank of Chinese Academy of Sciences. (Shanghai, China). 3\(4,5\dimethyl\2\thiazolyl)\2,5\diphenyl\2H\tetrazolium bromide (MTT, 413Y0511), oleic acid (01008) and Oil Red O (00625) were Sigma\Aldrich products (St. Louis, MO, USA). Liver X receptor (LXR) agonist T0901317 (293754\55\9), fenofibrate (S1794) and the peroxisome proliferator\triggered receptor (PPAR) antagonist MK886 were the products of Selleck (Shanghai, China). LXR antagonist, GSK2033 and SR9243, and PPAR antagonist GW6471 were the products of MedChemExpress (Shanghai, China). Dimethyl sulphoxide (DMSO, 821D035) and the goat serum (SL038) were purchased from Solarbio (Beijing, China). Dulbecco’s revised Eagle’s medium (DMEM) and foetal bovine serum (FBS) were from Gibco (BRL, Gaithersburg, MD, USA). RIPA lysis buffer was a product of Merck (3108491; Darmstadt, Germany). Rabbit polyclonal antibody against LXR (ab3585, 1:200; ab176323, 1:5000) and LXR (ab28479, 1:500); rabbit monoclonal antibody against scavenger receptor B type 1 (SR\B1, ab217318, 1:2000), ATP\binding cassette (ABC) G1 (ab52617, 1:1000) and low\denseness lipoprotein receptor (ab52818, LDLR 1:1000); and mouse monoclonal antibody against ABCA1 (abdominal18180, 1:200 or 1:1000) were from Abcam (Cambridge, MA, USA). Mouse monoclonal antibody against sterol regulatory element\binding protein (SREBP)\1c (sc\13551, 1:100), SREBP\2 (sc\271616, 1:200) and PPAR (sc\398394, 1:100) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody against cholesterol 7 alpha\hydroxylase A1 (CYP7A1, TA351400, 1:1000) was the product of OriGene (Shanghai, China). Mouse monoclonal antibody against \actin (66009\1\Ig, 1:5000), rabbit polyclonal antibody against ABCG5 (27722\1\AP, 1:1000) and rabbit monoclonal antibody against proprotein convertase subtilisin/kexin type 9 (PCSK9, 55206\1\AP, 1:500) were the products of Proteintech (Chicago, IL, USA). Total protease inhibitor and the secondary antibodies, including the goat antimouse IgG (FITC conjugated), were from CWBIO (Beijing, China). Mouse monoclonal antibody against ABCG8 (1B10A5, 1:1000) and enhanced chemiluminescence (ECL) packages were purchased from Thermo Scientific Pierce (Rockford, IL, USA). Total cholesterol (TC) and triglyceride (TG) assay packages were the products of Biosino Bio\technology and Technology Inc (Beijing, China). Double\deionized water was produced using a Milli\Q Gradient System from Millipore. All reagents used in this study were of analytical grade. 2.2. Purity dedication of the furanone, 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one The furanone, 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one, was isolated from your fungi sp SCSIO41009, as previously reported.21 Its purity was determined by multiple analytical methods. Ultra\overall performance liquid chromatography (UPLC) spectrum was performed on an Acquity UPLC BEH C18 column (2.1??50?mm i.d., 1.7?m) connected to a Waters Acquity H Class UPLC System (Waters) having a PDA detector (wavelength of 212?nm). Large\resolution electrospray ionization mass spectrometry (HRESIMS) spectrum was recorded on a Bruker maXis Q\TOF mass spectrometer in positive ion mode. 1D and 2D NMR spectra were measured on a Bruker AV 500?MHz or AVANCE HD 700?MHz NMR spectrometer with tetramethylsilane as an internal standard.21 2.3. Preparation of lipoproteins Plasma was from healthy volunteers in the Affiliated Hospital of Weifang Medical University or college. To obtain LDL portion, plasma was subjected to sequential ultracentrifugation as previously explained.22, 23 In brief, the plasma denseness was adjusted to 1 1.006?g/mL for ultracentrifugation at 10C (400?000??for 24?hours). The top layer containing very low\denseness lipoproteins was eliminated, and the denseness was re\modified to 1 1.063?g/mL for ultracentrifugation at 400,000??g for an additional 24?hours to obtain the upper coating containing low\denseness lipoproteins (LDL). EDTA\2Na (0.1%, w/v) was added to chelate the metal ions, thereby reducing oxidation during ultracentrifugation. The protein content material from the fractions was dependant on the Bradford technique. The LDL small percentage was.Lee\Rueckert M, Escola\Gil JC, Kovanen PT. In HepG2 F2RL3 cells, it reduced oleic acidity\induced lipid deposition considerably, enhanced the proteins degrees of low\thickness lipoprotein receptor (LDLR), ABCG5, ABCG8 and PPAR, and decreased the appearance of sterol regulatory component\binding proteins 2 (~32%). PPAR antagonists, GW6471 and MK886, could considerably inhibit the furanone\induced lipid\reducing impact. Furthermore, the furanone demonstrated a considerably lower activity over the activation from the appearance of lipogenic genes in comparison to T0901317. Used jointly, the furanone exhibited a vulnerable cytotoxicity but acquired effective TC\ and TG\reducing effects probably through concentrating on LXR and PPAR, respectively. These results indicate which the furanone includes a potential program for the treating dyslipidaemia. sp SCSIO41009.21 Here, we reported for the very first time which the furanone named as 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one acquired a highly effective lipid\decreasing activity via influencing multiple procedures of lipid metabolism. 2.?Components AND Strategies 2.1. Components Mouse\produced macrophage cell series Organic 264.7 as well as the individual hepatoma cell series HepG2 were purchased in the Cell Loan provider of Chinese language Academy of Sciences. (Shanghai, China). 3\(4,5\dimethyl\2\thiazolyl)\2,5\diphenyl\2H\tetrazolium bromide (MTT, 413Y0511), oleic acidity (01008) and Essential oil Crimson O (00625) had been Sigma\Aldrich items (St. Louis, MO, USA). Liver organ X receptor (LXR) agonist T0901317 (293754\55\9), fenofibrate (S1794) as well as the peroxisome proliferator\turned on receptor (PPAR) antagonist MK886 had been the merchandise of Selleck (Shanghai, China). LXR antagonist, GSK2033 and SR9243, and PPAR antagonist GW6471 had been the merchandise of MedChemExpress (Shanghai, China). Dimethyl sulphoxide (DMSO, 821D035) as well as the goat serum (SL038) had been bought from Solarbio (Beijing, China). Dulbecco’s improved Eagle’s moderate (DMEM) and foetal bovine serum (FBS) had been from Gibco (BRL, Gaithersburg, MD, USA). RIPA lysis buffer was something of Merck (3108491; Darmstadt, Germany). Rabbit polyclonal antibody against LXR (ab3585, 1:200; ab176323, 1:5000) and LXR (ab28479, 1:500); rabbit monoclonal antibody against scavenger receptor B type 1 (SR\B1, ab217318, 1:2000), ATP\binding cassette (ABC) G1 (ab52617, 1:1000) and low\thickness lipoprotein receptor (ab52818, Lynestrenol LDLR 1:1000); and mouse monoclonal antibody against ABCA1 (stomach18180, 1:200 or 1:1000) had been from Abcam (Cambridge, MA, USA). Mouse monoclonal antibody against sterol regulatory component\binding proteins (SREBP)\1c (sc\13551, 1:100), SREBP\2 (sc\271616, 1:200) and PPAR (sc\398394, 1:100) had been bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody against cholesterol 7 alpha\hydroxylase A1 (CYP7A1, TA351400, 1:1000) was the merchandise of OriGene (Shanghai, China). Mouse monoclonal antibody against \actin (66009\1\Ig, 1:5000), rabbit polyclonal antibody against ABCG5 (27722\1\AP, 1:1000) and rabbit monoclonal antibody against proprotein convertase subtilisin/kexin type 9 (PCSK9, 55206\1\AP, 1:500) had been the merchandise of Proteintech (Chicago, IL, USA). Comprehensive protease inhibitor as well as the supplementary antibodies, like the goat antimouse IgG (FITC conjugated), had been from CWBIO (Beijing, China). Mouse monoclonal antibody against ABCG8 (1B10A5, 1:1000) and improved chemiluminescence (ECL) sets had been bought from Thermo Scientific Pierce (Rockford, IL, USA). Total cholesterol (TC) and triglyceride (TG) assay sets had been the merchandise of Biosino Bio\technology and Research Inc (Beijing, China). Dual\deionized drinking water was produced utilizing a Milli\Q Gradient Program from Millipore. All reagents found in this research had been of analytical quality. 2.2. Purity perseverance from the furanone, 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one The furanone, 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one, was isolated in the fungus infection sp SCSIO41009, as previously reported.21 Its purity was dependant on multiple analytical strategies. Ultra\functionality liquid chromatography (UPLC) range was performed with an Acquity UPLC BEH C18 column (2.1??50?mm we.d., 1.7?m) linked to a Waters Acquity H Course UPLC Program (Waters) using a PDA detector (wavelength of 212?nm). Great\quality electrospray ionization mass spectrometry (HRESIMS) range was recorded on the Bruker maXis Q\TOF mass spectrometer in positive ion setting. 1D and 2D NMR spectra had been measured on the Bruker AV 500?MHz or AVANCE HD 700?MHz NMR spectrometer with tetramethylsilane as an interior regular.21 2.3. Planning of lipoproteins Plasma was extracted from healthful volunteers on the Associated Medical center of Weifang Medical School. To acquire LDL small percentage, plasma was put through sequential ultracentrifugation as previously defined.22, 23 In short, the plasma thickness was adjusted to at least one 1.006?g/mL for ultracentrifugation in 10C (400?000??for 24?hours). Top of the layer containing extremely low\thickness lipoproteins was taken out, and the thickness was re\altered to at least one 1.063?g/mL for ultracentrifugation in 400,000??g for yet another 24?hours to get the upper level containing low\thickness lipoproteins (LDL). EDTA\2Na (0.1%, w/v) was put into chelate the metal ions, reducing thereby.Br J Pharmacol. the proteins degrees of peroxisome proliferator\turned on receptors (PPAR) and ATP\binding cassette (ABC) transporters, that could end up being inhibited by LXR antagonists partly, GSK2033 and SR9243. In HepG2 cells, it considerably decreased oleic acidity\induced lipid deposition, enhanced the proteins degrees of low\thickness lipoprotein receptor (LDLR), ABCG5, ABCG8 and PPAR, and decreased the appearance of sterol regulatory component\binding proteins 2 (~32%). PPAR antagonists, GW6471 and MK886, could considerably inhibit the furanone\induced lipid\reducing impact. Furthermore, the furanone demonstrated a considerably lower activity in the activation from the appearance of lipogenic genes in comparison to T0901317. Used jointly, the furanone exhibited a weakened cytotoxicity but got effective TC\ and TG\reducing effects probably through concentrating on LXR and PPAR, respectively. These results indicate the fact that furanone includes a potential program for the treating dyslipidaemia. sp SCSIO41009.21 Here, we reported for the very first time the fact that furanone named as 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one got a highly effective lipid\decreasing activity via influencing multiple procedures of lipid metabolism. 2.?Components AND Strategies 2.1. Components Mouse\produced macrophage cell range Organic 264.7 as well as the individual hepatoma cell range HepG2 were purchased through the Cell Loan company of Chinese language Academy of Sciences. (Shanghai, China). 3\(4,5\dimethyl\2\thiazolyl)\2,5\diphenyl\2H\tetrazolium bromide (MTT, 413Y0511), oleic acidity (01008) and Essential oil Crimson O (00625) had been Sigma\Aldrich items (St. Louis, MO, USA). Liver organ X receptor (LXR) agonist T0901317 (293754\55\9), fenofibrate (S1794) as well as the peroxisome proliferator\turned on receptor (PPAR) antagonist MK886 had been the merchandise of Selleck (Shanghai, China). LXR antagonist, GSK2033 and SR9243, and PPAR antagonist GW6471 had been the merchandise of MedChemExpress (Shanghai, China). Dimethyl sulphoxide (DMSO, 821D035) as well as the goat serum (SL038) had been bought from Solarbio (Beijing, China). Dulbecco’s customized Eagle’s moderate (DMEM) and foetal bovine serum (FBS) had been from Gibco (BRL, Gaithersburg, MD, USA). RIPA lysis buffer was something of Merck (3108491; Darmstadt, Germany). Rabbit polyclonal antibody against LXR (ab3585, 1:200; ab176323, 1:5000) and LXR (ab28479, 1:500); rabbit monoclonal antibody against scavenger receptor B type 1 (SR\B1, ab217318, 1:2000), ATP\binding cassette (ABC) G1 (ab52617, 1:1000) and low\thickness lipoprotein receptor (ab52818, LDLR 1:1000); and mouse monoclonal antibody against ABCA1 (stomach18180, 1:200 or 1:1000) had been from Abcam (Cambridge, MA, USA). Mouse monoclonal antibody against sterol regulatory component\binding proteins (SREBP)\1c (sc\13551, 1:100), SREBP\2 (sc\271616, 1:200) and PPAR (sc\398394, 1:100) had been bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody against cholesterol 7 alpha\hydroxylase A1 (CYP7A1, TA351400, 1:1000) was the merchandise of OriGene (Shanghai, China). Mouse monoclonal antibody against \actin (66009\1\Ig, 1:5000), rabbit polyclonal antibody against ABCG5 (27722\1\AP, 1:1000) and rabbit monoclonal antibody against proprotein convertase subtilisin/kexin type 9 (PCSK9, 55206\1\AP, 1:500) had been the merchandise of Proteintech (Chicago, IL, USA). Full protease inhibitor as well as the supplementary antibodies, like the goat antimouse IgG (FITC conjugated), had been from CWBIO (Beijing, China). Mouse monoclonal antibody against ABCG8 (1B10A5, 1:1000) and improved chemiluminescence (ECL) products had been bought from Thermo Scientific Pierce (Rockford, IL, USA). Total cholesterol (TC) and triglyceride (TG) assay products had been the merchandise of Biosino Bio\technology and Research Inc (Beijing, China). Dual\deionized drinking water was produced utilizing a Milli\Q Gradient Program from Millipore. All reagents found in this research had been of analytical quality. 2.2. Purity perseverance from the furanone, 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one The furanone, 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one, was isolated through the fungus infection sp SCSIO41009, as previously reported.21 Its purity was dependant on multiple analytical strategies. Ultra\efficiency liquid chromatography (UPLC) range was performed with an Acquity UPLC BEH C18 column (2.1??50?mm we.d., 1.7?m) linked to a Waters Acquity H Course UPLC Program (Waters) using a PDA detector (wavelength of 212?nm). Great\quality electrospray ionization mass spectrometry (HRESIMS) range was recorded on the Bruker maXis Q\TOF mass spectrometer in positive ion setting. 1D and 2D NMR spectra had been measured on the Bruker AV 500?MHz or AVANCE HD 700?MHz NMR spectrometer with tetramethylsilane as an interior regular.21 2.3. Planning of lipoproteins Plasma was extracted from healthful volunteers on the Associated Medical center of Weifang Medical College or university. To acquire LDL small fraction, plasma was put through sequential ultracentrifugation as previously referred to.22, 23 In short, the plasma thickness was adjusted to at least one 1.006?g/mL for ultracentrifugation in 10C (400?000??for 24?hours). Top of the layer containing extremely low\thickness lipoproteins was taken out, and the thickness was re\altered to at least one 1.063?g/mL for ultracentrifugation in 400,000??g for yet another 24?hours to get the upper level containing low\density lipoproteins (LDL). EDTA\2Na (0.1%, w/v) was added to chelate the metal ions, thereby reducing oxidation during ultracentrifugation. The.Ward NC, Page MM, Watts GF. antagonists, GW6471 and MK886, could significantly Lynestrenol inhibit the furanone\induced lipid\lowering effect. Furthermore, the furanone showed a significantly lower activity on the activation of the expression of lipogenic genes compared to T0901317. Taken together, the furanone exhibited a weak cytotoxicity but had powerful TC\ and TG\lowering effects most likely through targeting LXR and PPAR, respectively. These findings indicate that the furanone has a potential application for the treatment of dyslipidaemia. sp SCSIO41009.21 Here, we reported for the first time that the furanone named as 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one had an effective lipid\lowering activity via influencing multiple processes of lipid metabolism. 2.?MATERIALS AND METHODS 2.1. Materials Mouse\derived macrophage cell line RAW 264.7 and the human hepatoma cell line HepG2 were purchased from the Cell Bank of Chinese Academy of Sciences. (Shanghai, China). 3\(4,5\dimethyl\2\thiazolyl)\2,5\diphenyl\2H\tetrazolium bromide (MTT, 413Y0511), oleic acid (01008) and Oil Red O (00625) were Sigma\Aldrich products (St. Louis, MO, USA). Liver X receptor (LXR) agonist T0901317 (293754\55\9), fenofibrate (S1794) and the peroxisome proliferator\activated receptor (PPAR) antagonist MK886 were the products of Selleck (Shanghai, China). LXR antagonist, GSK2033 and SR9243, and PPAR antagonist GW6471 were the products of MedChemExpress (Shanghai, China). Dimethyl sulphoxide (DMSO, 821D035) and the goat serum (SL038) were purchased from Solarbio (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) and foetal bovine serum (FBS) were from Gibco (BRL, Gaithersburg, MD, USA). RIPA lysis buffer was a product of Merck (3108491; Darmstadt, Germany). Rabbit polyclonal antibody against LXR (ab3585, 1:200; ab176323, 1:5000) and LXR (ab28479, 1:500); rabbit monoclonal antibody against scavenger receptor B type 1 (SR\B1, ab217318, 1:2000), ATP\binding cassette (ABC) G1 (ab52617, 1:1000) and low\density lipoprotein receptor (ab52818, LDLR 1:1000); and mouse monoclonal antibody against ABCA1 (ab18180, 1:200 or 1:1000) were from Abcam (Cambridge, MA, USA). Mouse monoclonal antibody against sterol regulatory element\binding protein (SREBP)\1c (sc\13551, 1:100), SREBP\2 (sc\271616, 1:200) and PPAR (sc\398394, 1:100) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody against cholesterol 7 alpha\hydroxylase A1 (CYP7A1, TA351400, 1:1000) was the product of OriGene (Shanghai, China). Mouse monoclonal antibody against \actin (66009\1\Ig, 1:5000), rabbit polyclonal antibody against ABCG5 (27722\1\AP, 1:1000) and rabbit monoclonal antibody against proprotein convertase subtilisin/kexin type 9 (PCSK9, 55206\1\AP, 1:500) were the products of Proteintech (Chicago, IL, USA). Complete protease inhibitor and the secondary antibodies, including the goat antimouse IgG (FITC conjugated), were from CWBIO (Beijing, China). Mouse monoclonal antibody against ABCG8 (1B10A5, 1:1000) and enhanced chemiluminescence (ECL) kits were purchased from Thermo Scientific Pierce (Rockford, IL, USA). Total cholesterol (TC) and triglyceride (TG) assay kits were the products of Biosino Bio\technology and Science Inc (Beijing, China). Double\deionized water was produced using a Milli\Q Gradient System from Millipore. All reagents used in this study were of analytical grade. 2.2. Purity determination of the Lynestrenol furanone, 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one The furanone, 5\hydroxy\3\methoxy\5\methyl\4\butylfuran\2(5H)\one, was isolated from the fungus sp SCSIO41009, as previously reported.21 Its purity was determined by multiple analytical methods. Ultra\performance liquid chromatography (UPLC) spectrum was performed on an Acquity UPLC BEH C18 column (2.1??50?mm i.d., 1.7?m) connected to a Waters Acquity H Class UPLC System (Waters) with a PDA detector (wavelength of 212?nm). High\resolution electrospray ionization mass spectrometry (HRESIMS) spectrum was recorded on a Bruker maXis Q\TOF mass spectrometer in positive ion mode. 1D and 2D NMR spectra were measured on a Bruker AV 500?MHz or AVANCE HD 700?MHz NMR spectrometer with tetramethylsilane as an internal standard.21 2.3. Preparation of lipoproteins Plasma was obtained from healthy volunteers at the Affiliated Hospital of Weifang Medical University. To obtain LDL fraction, plasma was subjected to sequential ultracentrifugation as previously described.22, 23 In brief, the plasma density was adjusted to 1 1.006?g/mL for ultracentrifugation at 10C (400?000??for 24?hours). The upper layer containing very low\density lipoproteins was removed, and the density was re\adjusted to 1 1.063?g/mL for ultracentrifugation at 400,000??g for an additional 24?hours to obtain the upper layer containing low\density lipoproteins (LDL). EDTA\2Na (0.1%, w/v) was added to chelate the metal ions, thereby reducing oxidation during ultracentrifugation. The protein content of the fractions was determined by the Bradford method. The LDL fraction was stored at 4C until use. Oxidized LDL (Ox\LDL) was prepared by the method described previously.23 In brief, LDL (~10?mg/mL) was incubated with CuSO4 (10?mol/L) at 37C for 24?hours; then, the reaction was stopped by addition of 500?mol/L EDTA\2Na. The.