http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–9 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2013.879187
ORIGINAL PAPER
Induction of apolipoprotein A-I gene expression by black seed (Nigella sativa) extracts Michael J. Haas1, Luisa M. Onstead-Haas1, Emad Naem1, Norman C. W. Wong2, and Arshag D. Mooradian1 Pharmaceutical Biology Downloaded from informahealthcare.com by National University of Singapore on 05/30/14 For personal use only.
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Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Florida, Jacksonville, FL, USA and 2Departments of Medicine and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada Abstract
Keywords
Context: Black seed [Nigella sativa L. (Ranunculaceae)] has been shown in animal models to lower serum cholesterol levels. Objectives: In order to determine if extracts from black seed have any effects on high-density lipoprotein (HDL), we characterized the effects of black seed extract on apolipoprotein A-I (apo A-I) gene expression, the primary protein component of HDL. Materials and methods: Hepatocytes (HepG2) and intestinal cells (Caco-2) were treated with black seed extracts, and Apo A-I, peroxisome proliferator-activated receptor a (PPARa), and retinoid-x-receptor a (RXRa) were measured by Western blot analysis. Apo A-I mRNA levels were measured by quantitative real-time polymerase chain reaction and apo A-I gene transcription was measured by transient transfection of apo A-I reporter plasmids. Results: Extracts from black seeds significantly increased hepatic and intestinal apo A-I secretion, as well as apo A-I mRNA and gene promoter activity. This effect required a PPARa binding site in the apo A-I gene promoter. Treatment of the extract with either heat or trypsin had no effect on its ability to induce apo A-I secretion. Treatment with black seed extract induced PPARa expression 9-fold and RXRa expression 2.5-fold. Furthermore, the addition of PPARa siRNA but not a control siRNA prevented some but not all the positive effects of black seed on apo A-I secretion. Discussion: Black seed extract is a potent inducer of apo A-I gene expression, presumably by enhancing PPARa/RXRa expression. Conclusions: We conclude that black seed may have beneficial effects in treating dyslipidemia and coronary heart disease.
Black seed, cardiovascular disease, HDL
Introduction Black seed [Nigella sativa L. (Ranunculaceae)] has been used as an herbal remedy for various ailments for centuries in countries such as India and Pakistan as well as throughout the Middle East and the Mediterranean countries (Sayed, 1980). Recent studies have shown that black seed has antitumorigenic effects, due in part to its thymoquinone content, as well as antioxidative, anti-inflammatory, and glucose and lipid modulating effects (Burtis & Bucar, 2000; Houghton et al., 1995; Worthen et al., 1998). As to the latter, black seed has been shown to lower total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL) in rats fed a diet rich in black seed (Dahri et al., 2005; Meral et al., 2001). Several studies have demonstrated that high-density lipoprotein (HDL) levels are inversely correlated with the risk of developing coronary artery disease (CAD) (Gordon et al., Correspondence: Michael J. Haas, Ph.D., Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Florida Jacksonville, 653-1 West 8th Street L14, Jacksonville, FL 32209, USA. Tel: +1 904 244 8619. Fax: +1 904 244 5650. E-mail: [email protected]
History Received 21 October 2013 Revised 10 December 2013 Accepted 22 December 2013 Published online 17 March 2014
1977; Mooradian, 2003). HDL and its primary protein component apolipoprotein A-I (apo A-I) have several antiatherogenic properties which may influence the development of CAD (Haas & Mooradian, 2010a,b; Hachem & Mooradian, 2006). First, HDL participates in the process of reversecholesterol transport, by which cholesterol is transported from peripheral sources such as lipid-laden macrophage cells to the liver where it is either converted to bile acids or directly excreted in the feces (Mooradian et al., 2008). Second, in healthy individuals, HDL has antioxidative, anti-inflammatory, and antithrombotic properties that are thought to decrease macrophage activation, vascular reactivity and thrombus formation (Haas & Mooradian, 2010a,b). Although exercise, weight loss, and dietary modulation can raise HDL levels, the changes are modest and difficult to maintain. Certain drugs such as fibrates and some statins have been shown to induce apo A-I mRNA levels and gene transcription, but only modestly (Cerda et al., 2012; Hachem & Mooradian, 2006). Niacin and cholesterol-ester transfer protein (CETP) inhibitors have been shown to have robust positive effects on plasma HDL levels but they have not been documented to reduce cardiovascular risk in all studies
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(AIM-HIGH Investigators, 2011; Ghosh & Ghosh, 2012). The recent Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides: Impact on Global Health Outcomes (AIM-HIGH) trial showed no benefit of extended release niacin on cardiovascular outcomes (AIM-HIGH Investigators, 2011). Therefore, there remains a need for drugs or other small molecule compounds that elevate apo A-I and HDL significantly. Since black seed has lipid-modulating effects as well as antioxidant and anti-inflammatory properties, we examined the effects of various extracts prepared from the seed on apo A-I gene expression in hepatocyte and intestinal cell lines.
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Materials and methods Lipofectamine and Trizol were purchased from Life Technologies (Carlsbad, CA). 14C-Chloramphenicol was obtained from PerkinElmer (Waltham, MA). Fenofibrate was purchased from Sigma Chemical Company (St. Louis, MO). Plasmid purification kit was purchased from Qiagen (Valencia, CA). An anti-human apo A-I antibody (178422) and an antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MAB374) were from EMD Millipore (Billerica, MA). Antibodies to retinoid-x-receptor a (RXRa) (D-20) and peroxisome proliferator-activated receptor a (PPARa) (H-98), and control siRNA and human PPARa siRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An antibody to human albumin (109-4133) was purchased from Rockland Chemical (Gilbertsville, PA). Secondary antibodies prepared in goats and conjugated to horseradish peroxidase (HRP) were purchased from Southern Biotech (Birmingham, AL). Tissue culture media, media supplements, trypsin, and fetal bovine serum (FBS) were purchased from BioWittaker (Walkersville, MD). Immobilized TPCK trypsin was purchased from Pierce Biotechnology (Rockford, IL). All other chemicals were purchased from Fisher Scientific (Pittsburg, PA) or Sigma Chemical Company. Cell culture HepG2 and Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA). HepG2 cells were maintained in Dulbecco’s Modified Eagle’s Medium containing 10% FBS and penicillin and streptomycin (100 units/ml and 100 mg/ml, respectively). Caco-2 cells were maintained in Eagle’s Minimal Essential Medium containing 10% FBS, non-essential amino acids, sodium pyruvate, and 100 units/ml penicillin and 100 mg/ml streptomycin. The cells were housed in a humidified incubator at 37 C with an atmosphere of 95% air and 5% CO2. Cell viability was measured via mitochondrial dehyrodrogenase activity using 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (Mossman, 1983). Black seed extract preparation Two methods were used to prepare black seed extracts – ethanol extraction and volatile oil extraction. Ethanol extractions were performed essentially as previously described (Al-Jenoobi et al., 2010) by shaking 5 g of seeds in 25 ml of
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100% molecular biology-grade ethanol for 96 h. The ethanol was then evaporated and the residue was dissolved in 300 ml of dimethylsulfoxide (DMSO). Volatile oil extracts were prepared by grinding 20 g of black seed, which was mixed with 100 ml of distilled water and distilled in a Bialetti espresso maker (Bioletti Industrie Spa, Brescia, Italy) (Fararh et al., 2002). After distillation, the liquid was allowed to cool and the aqueous and lipid phases were allowed to separate. The pH of each phase was 6.8 and the amount of protein in each phase was measured using the bicinchoninic acid (BCA) method (Smith et al., 1985). Both the aqueous and lipid fractions were tested for activity. Black seed extracts contains several fixed oils including arachidic acid and eicosadienoic acid (Houghton et al., 1995). They also contain thymoquinone, dithymoquinone, p-cymene, carvacrol, t-anethole, 4-terpineol, and longifoline (Burtis & Bucar, 2000) and several alkaloids including nigellicine, nigellidine, and the isoquinoline nigellimine (Atta-ur-Rahman et al., 1995). In most experiments, the cells were cultured in serum-free medium (SFM) and treated with either a solvent (dH2O or ethanol, where appropriate) or the indicated dilution of black seed. Western blotting Apo A-I and albumin were measured in conditioned medium by Western blots. Briefly, the protein content of each media sample was determined using the BCA method and 5 mg of protein per lane was fractionated by electrophoresis on a 10% sodium dodecylsulfate (SDS) polyacrylamide gel. After electrophoresis, the protein was transferred to nitrocellulose (GE Healthcare, Pittsburg, PA) and after blocking for 2 h in phosphate buffered saline-Tween 20 (PBST) (10 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) containing 5% non-fat dry milk, the antihuman apo A-I primary antibody (1:1000) or anti-human albumin primary antibody (1:2000) was added to the membrane and incubated overnight at 4 C. After washing in PBST, the membrane was incubated with the goat-anti-rabbit HRP secondary antibody (1:5000). After washing in PBST, the membrane was incubated in enhanced chemiluminescence (ECL) reagent for the detection of the signal by autoradiography. PPARa, RXRa, and GAPDH expression was measured in control and black seed extract-treated cells by Western blot. After 24 h of treatment, the media were removed and the cells were washed three times with ice-cold PBS and lysed in 300 ml of sample buffer [50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-Cl) (pH 7.4), 1% SDS, and 1 mM ethylenediamine tetraacetic acid]. After brief sonication to reduce the viscosity of the samples, particulates were removed by centrifugation at 10 000 g for 10 min at 4 C. Protein content of each sample was measured using the BCA assay and 50 mg of protein was loaded onto a 10% SDS polyacrylamide gel. After electrophoresis, the samples were transferred to nitrocellulose, which was then blocked in Trisbuffered saline [50 mM Tris-Cl (pH 7.4), 150 mM NaCl] containing 0.1% Tween 20 (TBST) and 10% newborn calf serum (NCS). The primary antibodies were diluted in TBST/ 10% NCS (PPARa, 1:200; RXRa, 1:200; GAPDH, 1:1000)
DOI: 10.3109/13880209.2013.879187
and incubated with the membranes overnight at 4 C. After washing in TBST, the secondary antibodies were diluted in TBST/10% NCS (1:5000) and added to the membranes for 1 h at room temperature. After washing, the membranes were incubated with the ECL reagent and images were captured on film.
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Real-time quantitative polymerase chain reaction (PCR) Total RNA was extracted from HepG2 cells treated with black seed extract for 24 h as previously described (Chomczynski & Sacchi, 1987). Reverse transcription was performed with 2 mg of total RNA and random hexamers with AMV reverse transcriptase (Promega, Madison, WI). The cDNA was then used for PCR with primers for apo A-I (forward, 50 -AGC TTG CTG AAG GTG GAG GT-30 ; reverse, 50 -ATC GAG TGA AGG ACC TGG C-30 ), albumin (forward, 50 -TTG CAT GAG AAA ACG CCA GTA-30 ; reverse, 50 -GTC GCC TGT TCA CCA AGG A-30 ), and GAPDH (forward, 50 -CTC TCT GCT CCT CCT GTT CGA C-30 ; reverse, 50 -TGA GCG ATG TGG CTC GGC T-30 ). Amplification was performed with SYBRgreen dye (Bio-Rad, Hercules, CA) at 95 C for 1 min, 55 C for 1 min, and 72 C for 1 min for 40 cycles. Melt curve analysis was performed for each amplification to verify that only one product was formed in each well. Relative changes in RNA levels were calculated using the DDCt method using GAPDH as the control. Transient transfection analysis of apo A-I promoter activity HepG2 cells were transfected with 1 mg of the apo A-I reporter plasmid pAI.474.CAT and 1 mg of the control plasmid pCMV.SPORT.b-gal, to normalize transfection efficiency, using Lipofectamine. After 24 h, the cells were treated with a 105-fold dilution of black seed extract for 24 h, and assayed for chloramphenicol acetyltransferase (CAT) (Gorman et al., 1982) and b-galactosidase activity (Herbomel et al., 1984). To identify the black seed-responsive element in the apo A-I gene promoter, a series of apo A-I deletion constructs were used. The plasmids pAI.474.CAT, pAI.425.CAT, pAI.375.CAT, pAI.325.CAT, pAI.186.CAT, pAI.170.CAT, pAI.144.CAT, and pAI.46.CAT contain 474, 425, 375, 325, 186, 170, 144, and 46 bp of the 50 -flanking region of the apo A-I gene promoter. The plasmid pCMV.SPORT.b-gal was cotransfected with each apo A-I construct in order to normalize the transfection efficiency. Inhibition of PPARa expression using siRNA HepG2 cells were transfected with either control siRNA or siRNA targeting PPARa with lipofectamine, and after 72 h, treated with either a solvent of black seed (105-fold dilution) for 24 h. Apo A-I levels were measured in the conditioned medium by Western blot as described above. Data analysis Values are expressed as the mean ± the standard deviation (SD). Statistical significance was assessed using Student’s
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t-test for independent variables and a one-way analysis of variance. p Values less than 0.05 were considered significant.
Results The effect of black seed extract on apo A-I secretion HepG2 cells were treated with 109, 108, 107, 106, 105, and 104-fold dilutions of ethanol black seed extract (2.0 mg/ ml protein equivalent) for 24 h and apo A-I protein levels were measured in the conditioned medium (Figure 1). Apo A-I protein levels (Figure 1A and B) increased from 541 ± 50 arbitrary units (A.U.) in control cells to 622 ± 61, 1341 ± 156, 1250 ± 101, 1356 ± 133, and 1324 ± 127 A.U. in cells treated with 109, 108, 107, 106, 105, and 104-fold dilutions of ethanol black seed extract (N.S., p50.001, 0.003, 0.0005, 0.003, and 0.004, respectively). In contrast, albumin levels in control cells were 591 ± 50 A.U. and 588 ± 33, 584 ± 31, 583 ± 41, 604 ± 39 589 ± 33 A.U. in cells treated with 109, 108, 107, 106, 105, and 104-fold dilutions of black seed extract [all not significant (N.S.)]. Thymoquinone is a substantial component of black seed extract and has been shown to be responsible for many of the biological activities of the mixture. To determine if thymoquinone induces apo A-I protein synthesis, HepG2 cells were treated with increasing doses of thymoquinone and apo A-I levels were measured in the medium (Figure 1C and D). Apo A-I levels were 618 ± 45 A.U. and 648 ± 32, 611 ± 27, 631 ± 33, 609 ± 43, and 617 ± 55 A.U. in cells treated with 109, 108, 107, 196, and 105 M thymoquinone (D) (all N.S.) These results suggest that thymoquinone is not the component that regulates apo A-I protein secretion. The effect of aqueous and lipid portion of black seed extracts on apo A-I protein secretion In addition to ethanol black seed extracts, volatile oil and aqueous black seed extracts were prepared and tested for their ability to induce apo A-I gene expression. The aqueous fraction had significantly higher protein content per milliliter (1.1 mg/ml) than the volatile oil fraction (0.6 mg/ml) (Figure 2A). In HepG2 cells treated with the aqueous fraction, apo A-I levels in conditioned media were 235 ± 12, 254 ± 28, 249 ± 15, 234 ± 5, 241 ± 13, and 235 ± 8 A.U. in cells treated with 109, 108, 107, 106, 105, and 104-fold dilutions of aqueous black seed extract (all N.S.) (Figure 2B). In contrast, the volatile oil fraction increased apo A-I concentrations from 332 ± 43 A.U. to 391 ± 7, 789 ± 17, 973 ± 66, 903 ± 95, 675 ± 54, 493 ± 36 A.U. in cells treated with 109, 108, 107, 106, 105, and 104-fold dilutions of black seed volatile oil extract (N.S., p50.001, 0.001, 0.007, 0.0001, 0.001, and 0.008, respectively) (Figure 2C). These results suggest that a heat-resistant volatile oil from black seed extract induces apo A-I protein levels. To determine whether or not a protein or proteins in the black seed extract are involved in inducing apo A-I production, we examined the effect of trypsin digestion. Aliquots of black seed volatile oil extract were digested with TPCKtrypsin for 24 h at 37 C, at which time they were used to treat the HepG2 cells (Figure 2D). Undigested extract increased
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Figure 1. The effect of ethanol black seed extract and thymoquinone on apo A-I protein secretion. HepG2 cells were treated with the indicated amounts of black seed extract for 24 h and apo A-I and albumin levels (A) were measured by Western blot. The blots were quantified and are shown in (B). Black seed extract induced apo A-I protein synthesis but had no effect on albumin levels. HepG2 cells were treated with the indicated amounts of thymoquinone and apo A-I and albumin levels were measured in the conditioned medium by Western blot (C). The blots were quantified and are shown in (D). Apo A-I levels were normalized to albumin gene expression. Thymoquinone had no effect on apo A-I and albumin protein secretion. N ¼ 6; *p50.05, treated cells relative to untreated cells.
apo A-I protein levels from 568 ± 44 A.U. to 1565 ± 78 A.U. (p50.001, relative to control cells) and trypsin pre-treated extract increased apo A-I protein levels to 1589 ± 173 A.U. (p50.001, relative to control cells) (Figure 2D and E). These results suggest that the component or components that induced apo A-I protein secretion are in the volatile oil fraction and are resistant to heat and proteolysis. The effect of black seed extract on apo A-I mRNA and gene promoter activity To determine if the changes in apo A-I protein levels are due to changes in apo A-I mRNA, HepG2 cells were treated with a 105-fold dilution of black seed extract for 24 h and apo A-I and albumin mRNA levels were measured by quantitative real-time PCR and normalized to GAPDH mRNA levels (Figure 3). Apo A-I mRNA levels increased 1.8-fold in HepG2 cells relative to control cells (p50.001) (Figure 3A). Albumin mRNA levels were similar in each group (N.S.) (Figure 3B). To determine if black seed induces apo A-I promoter activity, HepG2 cells were transfected with the plasmids pAI.474.CAT and pCMV.SPORT.b-gal (to normalize transfection efficiency) and treated with either the solvent (DMSO) or black seed extract for 24 h (Figure 3C). Black seed extract increased apo A-I reporter gene expression from 21.1 ± 1.2% acetylation to 20.2 ± 2.2, 23.4 ± 0.4, 28.5 ± 1.1, and 31.3 ± 1.9% acetylation in cells treated with 109, 108,
107, 106, and 105-fold dilutions of black seed extract (N.S., p50.04, 0.006, 0.001, and 0.001, respectively) (Figure 3C). These results suggest that black seed induces apo A-I protein secretion by inducing apo A-I gene promoter activity and mRNA expression. Identification of a black seed response element in the apo A-I gene promoter To determine the regions of the apo A-I gene promoter that respond to black seed extract, HepG2 cells were transfected with a series of apo A-I reporter plasmids containing successive deletions of the apo A-I gene promoter. The cells were also transfected with the plasmid pCMV.SPORT.b-gal to normalize transfection efficiency. The cells were then treated with black seed extract (a 105-fold dilution) for 24 h and CAT and b-galactosidase activity were measured (Figure 3D). Black seed extract induced reporter gene expression by 1.44 ± 0.05, 1.44 ± 0.04, 1.39 ± 0.03, and 1.38 ± 0.06-fold in cells transfected with the plasmids pAI.474.CAT, pAI.425.CAT, pAI.375.CAT, and pAI.325.CAT (p50.001, 0.001, 0.003, and 0.001, respectively). However, black seedtreatment had no effect on apo A-I promoter activity in cells transfected with pAI.186.CAT (1.1 ± 0.06-fold), pAI.170.CAT (1.0 ± 0.08-fold), pAI.144.CAT (1.0 ± 0.08fold), and pAI.46.CAT (0.9 ± 0.13-fold) (all N.S.) (Figure 3D). These findings suggest that the region of the apo A-I promoter between nucleotides 325 and 186 is
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DOI: 10.3109/13880209.2013.879187
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Figure 2. The effect of aqueous and volatile oil black seed fractions and trypsin digestion on apo A-I gene expression. HepG2 cells were left untreated or were treated with the indicated dilutions of either the aqueous (A) or volatile oil (B) black seed fraction for 24 h and apo A-I levels were measured in the conditioned medium. The volatile oil fraction of the black seed extract induced apo A-I gene expression; however, the aqueous fraction had no effect. HepG2 cells were either untreated or treated with a 105-fold dilution of black seed or a 105-fold dilution of black seed digested with trypsin for 24 h. Apo A-I and albumin levels were measured by Western blot (C). (D) The blots were quantified and apo A-I and albumin expression are shown. Trypsin digestion had no effect on the ability of black seed to induce apo A-I gene expression. N ¼ 6; p50.05, trypsin-digested versus trypsinundigested extract.
essential for the induction of promoter activity in cells exposed to black seed extract. To determine if black seed extract effects apo A-I gene expression in intestinal cells, Caco-2 cells were treated with black seed extract for 24 h and apo A-I protein was measured in the conditioned medium and apo A-I and GAPDH mRNA levels were measured by real-time quantitative PCR (Figure 3E and F). Black seed induced apo A-I secretion 2-fold (from 465 ± 50 A.U. in control cells to 1048 ± 98 A.U. in cells treated with black seed extract; p50.001) (Figure 3E) and apo A-I mRNA levels 2.2-fold (p50.03) (Figure 3F). These results suggest that black seed induces apo A-I gene expression in hepatocytes as well as intestinal cells.
The effect of black seed extract on PPARa and RXRa expressions The black seed extract-responsive region in the apo A-I gene promoter between 325 and 186 contains a PPARa/RXRa binding site. PPARa and RXRa have been shown to induce apo A-I gene transcription (Hossain et al., 2008). To determine if PPARa and or RXRa expressions are regulated by exposure to black seed extract, HepG2 cells were treated with black seed extract for 24 h and PPARa and RXRa levels were measured by Western blot (Figure 4). Treatment with black seed extract increased PPARa (Figure 4A) and RXRa (Figure 4B) levels 9.0-fold (p50.001) and 2.4-fold (p50.003), respectively. In contrast, GAPDH levels did not
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Figure 3. The effect of black seed extract on apo A-I mRNA expression and apo AI promoter activity in liver and intestinal cells. HepG2 cells were either untreated or treated with a 105-fold dilution of black seed for 24 h and apo A-I, albumin, and GAPDH mRNA levels were measured by quantitative real-time PCR. Apo A-I (A) and albumin (B) mRNA levels were normalized to GAPDH mRNA. Black seed treatment induced apo A-I mRNA levels but had no effect on albumin mRNA expression. (C) HepG2 cells were transfected with the plasmids pAI.474.CAT and pCMV.SPORT.bgal and either left untreated or treated with the indicated dilution of black seed for 24 h. Treatment of black seed extract induced apo A-I promoter activity but had no effect on the CMV promoter. N ¼ 6; *p50.05, treated versus untreated cells. (D) HepG2 cells were transfected with plasmids containing successive deletions of the apo A-I gene promoter and either left untreated or treated with a 105-fold dilution of black seed extract for 24 h. The fold-change in black seed cells relative to control cells is presented for each plasmid. Induction of apo A-I promoter activity by black seed required the region between 326 and 186. N ¼ 3; *p50.05. Caco-2 cells were treated with black seed extract for 24 h and apo A-I expression was measured by Western blot (E) and apo A-I mRNA levels were measured by quantitative real-time PCR (F). Black seed induced apo A-I protein secretion and apo A-I mRNA levels. N ¼ 6; *p50.05, treated versus untreated cells.
change (Figure 4C). These results suggest that black seed induces apo A-I gene expression in part by increasing PPARa/ RXRa levels. To determine if PPARa is required for induction of apo A-I secretion by black seed extract, we knockout PPARa expression using siRNA (Figure 4D). The addition of 5 and 10 mM PPARa siRNA inhibited PPARa expression from 488 ± 31 A.U. to 98 ± 18 and 92 ± 25 A.U., respectively (p50.0001 and p50.0001, respectively) Next, HepG2 cells were transfected with either control or PPARa siRNA and treated with
either solvent (dH2O) or a 105-fold dilution of black seed for 24 h. As a control, cells were also treated with the PPARa agonist fenofibrate (100 mM). In cells treated with the control siRNA (Figure 4E), black seed treatment induced apo A-I secretion 2.8-fold (p50.0002) while fenofibrate increased apo A-I secretion 1.5-fold (p50.01). In contrast, cells transfected with the PPARa siRNA, black seed treatment increased apo A-I protein levels 1.5-fold (p50.03, relative to solvent-treated cells) and fenofibrate induced apo A-I protein levels 1.1-fold (N.S., relative to solvent-treated cells) These
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Figure 4. The effect of black seed extract on PPARa and RXRa expression. HepG2 cells were either left untreated or treated with a 105-fold dilution of black seed extract for 24 h and PPARa (A), RXRa (B), and GAPDH (C) expressions were measured by Western blot. Black seed induced PPARa and RXRa expressions but GAPDH levels did not change. N ¼ 3; *p50.05, treated versus untreated cells. (D) HepG2 cells were transfected with 5 and 10 mM PPAR a siRNA for 72 h and PPAR a levels were measured by Western blot. N ¼ 3; *p50.0001 versus control cells. (E) HepG2 cells were transfected with the control or PPARa siRNA and after 72 h, treated with or without black seed (105-fold dilution) or fenofibrate in SFM for 24 h. Apo A-I levels were measured by Western blot in the conditioned medium. Treatment with the control siRNA had no effect on the ability of black seed to induce apo A-I synthesis. In contrast, the addition of the PPAR a siRNA prevented most but not all the abilities of black seed to induce apo A-I protein secretion. N ¼ 3; *p50.0002 and p50.01 in cells treated with black seed and fenofibrate, respectively, versus control cells; p50.02 and N.S. in cells treated with black seed and fenofibrate, respectively, versus control cells; yp50.02 in black seed treated cells versus fenofibrate-treated cells.
results suggest that PPARa modulates some but not all the effects of black seed on apo A-I gene expression.
Discussion Black seed extracts have been shown to regulate lipid metabolism in several animal studies (Dahri et al., 2005; Meral et al., 2001). The present study shows that ethanol extracts of black seed increase apo A-I levels of conditioned media from HepG2 cells by several fold (Figure 1). The apo A-I inducing activity was also observed in a concentrated volatile oil extract after steam extraction and after trypsin digestion (Figure 2) suggesting that the active components are heat resistant and probably not a protein. The increase in apo A-I protein was accompanied by increased apo A-I mRNA and promoter activity (Figure 3). Induction of apo A-I promoter activity required a region of the apo A-I gene promoter (325 to 189) that contains a PPARa/RXRa binding site (Figure 3). Furthermore, treatment of HepG2
cells with black seed extract induced both PPARa and RXRa expression (Figure 4). Intestinal cells synthesize apo A-I and are an important contributor to plasma HDL levels. They also regulate apo A-I gene expression in a PPARa-dependent manner (Colin et al., 2013). Therefore, we examined the effect of black seed extract on apo A-I expression in Caco-2 intestinal cells (Figure 3). Black seed extract induced apo A-I secretion and apo A-I mRNA levels by approximately 2-fold (Figure 3). These results suggest that black seed induces apo A-I gene expression in hepatocytes as well as intestinal cells. The contents of ethanol black seed extracts have been elucidated to some extent and they have been shown to contain numerous compounds that have anti-oxidant and anti-inflammatory properties. Some of the effects of black seed, especially on cell growth and inhibition of lipid peroxidation, have been attributed to its thymoquinone content. However, thymoquinone had no effect on apo A-I levels in the present study (Figure 1), although other
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anti-oxidants such as a-tocopherol and ascorbic acid have been previously demonstrated to suppress apo A-I gene expression in HepG2 cells (Mooradian et al., 2006). Treatment with black seed extract increased PPARa expression 9-fold and RXRa expression 2.4-fold suggesting that black seed may increase expression of genes involved in fatty acid oxidation (Figure 4). Likewise, the volatile oil and ethanol extracts of black seeds may contain endogenous RXRa and/or PPARa ligands that would further increase activity of these nuclear receptors. PPARa has a large ligand binding pocket that can be occupied by a wide variety of fatty acids or arachidonic acid metabolites (Xu et al., 2001) that serve as endogenous ligands for the receptor. Several natural compounds have been shown to stimulate PPARa activity (Devchand et al., 1996; Xu et al., 2001). Alternatively, black seed extract treatment may promote fatty acid oxidation and the generation of endogenous PPARa ligands, including acyl-Co A’s and enoyl-CoA’s (Hostetler et al., 2005). The precise identity of the active ingredient in black seed extracts responsible for the apo A-I induction is not known. The available data suggest that it is a lipophilic compound that is resistant to heat and trypsin digestion. Over 46 compounds have been identified in the volatile oil from black seed, including thymol, thymoquinone, 4-terpineol, longifolene-(V4), and p-cymene, among others (Ahmad and Beg, 2013). Alcohol extracts from black seed contain thymoquinone as well as several fatty acids, primarily linoleic and palmitic acid. However, it is unlikely to be one of the common dietary fatty acids since previous studies in the same hepatic cell line did not reveal apo A-I induction by a number of saturated, unsaturated, monounsaturated, and trans-fatty acids such as stearic, myristic, palmitic, oleic, linoleic, linolenic, elaidic, linolelaidic, and linolenelaidic acid (Haas et al., 2004). We are currently fractionating the extracts to identify the active components.
Conclusions Our studies show that ethanol and lipid extracts prepared from black seed are potent inducers of apo A-I gene expression, in part by enhancing PPARa/RXRa expression. These results suggest that black seed may have beneficial effects in treating dyslipidemia and coronary heart disease.
Declaration of interest This work was supported in part by a Dean’s Fund Research Award from the University of Florida-Jacksonville to E. Naem. The authors have no conflicts to declare. The authors are responsible for the contents and writing of the paper.
References Ahmad S, Beg ZH. (2013). Elucidation of mechanisms of actions of thymoquinone-enriched methanol and volatile oil extracts from Nigella sativa against cardiovascular risk parameters in experimental hyperlipidemia. Lipids Health Disease 12:86. AIM-HIGH Investigators, Boden WE, Probstfield JL, Anderson T, et al. (2011). Niacin in patients with low HDL cholesterol levels receiving intensive statin therapy. New Engl J Med 365:2255–67.
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Al-Jenoobi FI, Al-Thukair AA, Abbas FA, et al. (2010). Effect of black seed on dextromethorphan O- and N-demethylation in human liver microsomes and healthy human subjects. Drug Metabol Lett 4: 51–5. Atta-ur-Rahman, Malik S, Hasan SS, et al. (1995). Nigellidine, a new indazole alkaloid from the seeds of Nigella sativa. Tetrahedron Lett 36:1993–6. Burtis M, Bucar F. (2000). Antioxidant activity of Nigella sativa essential oil. Phytother Res 14:323–8. Cerda A, Hirata MH, Hirata RD. (2012). Molecular mechanisms underlying statin effects on genes involved in the reverse cholesterol transport. Drug Metabol Drug Interact 27:101–11. Chomczynski P, Sacchi N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 132:6–13. Colin S, Briand O, Touche V, et al. (2013). Activation of intestinal peroxisome proliferator-activated receptor-a increases high-density lipoprotein production. Eur Heart J 34:2566–74. Dahri AH, Chandiol AM, Rahoo AA, Memon RA. (2005). Effect of Nigella sativa (kalonji) on serum cholesterol of albino rats. J Ayub Med Coll, Abbottabad 17:72–4. Devchand PR, Keller H, Peters JM, et al. (1996). The PPARalphaleukotriene B4 pathway to inflammatory control. Nature 384:39–43. Fararh KM, Atoji Y, Shimizu Y, Takewaki T. (2002). Insulinotropic properties of Nigella sativa oil in streptozotocin plus nicotinamide diabetic hamster. Res Vet Sci 73:279–82. Ghosh RK, Ghosh SM. (2012). Current status of CETP inhibitors in the treatment of hyperlipidemia: An update. Curr Clin Pharm 7: 102–10. Gordon T, Castelli WP, Hjotland MC, et al. (1977). High density lipoprotein as a protective factor against coronary heart disease. The Framingham study. Am J Med 62:707–14. Gorman CM, Moffat LF, Howard BH. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2:1044–51. Haas MJ, Horani MH, Wong NC, Mooradian AD. (2004). Induction of the apolipoprotein AI promoter by Sp1 is repressed by saturated fatty acids. Metabolism 53:1342–8. Haas MJ, Mooradian AD. (2010a). Therapeutic interventions to enhance apolipoprotein A-I-mediated cardioprotection. Drugs 70: 805–21. Haas MJ, Mooradian AD. (2010b). Regulation of high-density lipoprotein by inflammatory cytokines: Establishing links between immune dysfunction and cardiovascular disease. Diab Metabol Res Rev 26: 90–9. Hachem SB, Mooradian AD. (2006). Familial dyslipidaemias: An overview of genetics, pathophysiology and management. Drugs 66: 1949–69. Herbomel P, Bourachot B, Yaniv M. (1984). Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell 39:653–62. Hossain M, Tsujita M, Gonzalez F, Yokoyama S. (2008). Effects of fibrate drugs on expression of ABCA1 and HDL biogenesis in hepatocytes. J Cardiovas Pharm 51:258–66. Hostetler HA, Petrescu AD, Kier AB, Schroeder F. (2005). Peroxisome proliferator-activated receptor a interacts with high affinity and is conformationally responsive to endogenous ligands. J Biol Chem 280: 18667–82. Houghton PJ, Zarka R, de las Heras B, Hoult JR. (1995). Fixed oil of Nigella sativa and derived thymoquinone inhibit eicosanoid generation in leukocytes and membrane lipid peroxidation. Planta Med 61: 33–6. Meral I, Yener Z, Kahraman T, Mert N. (2001). Effect of Nigella sativa on glucose concentration, lipid peroxidation, anti-oxidant defense system and liver damage in experimentally-induced diabetic rabbits. J Vet Med A: Phys Path Clin Med 48:593–9. Mooradian AD. (2003). Cardiovascular disease in type 2 diabetes mellitus: Current management guidelines. Arch Int Med 163: 33–40. Mooradian AD, Haas MJ, Wadud K. (2006). Ascorbic acid and alphatocopherol down-regulate apolipoprotein A-I gene expression in HepG2 and Caco-2 cell lines. Metabolism 55:159–67. Mooradian AD, Haas MJ, Wehmeier KR, Wong NC. (2008). Obesityrelated changes in high-density lipoprotein metabolism. Obesity (Silver Spring) 16:1152–60.
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Mossman T. (1983). Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Meth 65:55–63. Sayed MD. (1980). Traditional medicine in health care. J Ethnopharmacol 2:19–22. Smith PK, Krohn RI, Hermanson GT, et al. (1985). Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85.
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Worthen DR, Ghosheh O, Crooks PA. (1998). The in vitro anti-tumor activity of some crude and purified components of black seed. Anticancer Res 18:1527–32. Xu HE, Lambert MH, Montana VG, et al. (2001). Structural determinations of ligand binding selectivity between the peroxisome proliferator-activated receptors. Proc Nat Acad Sci USA 98: 13919–24.
ORIGINAL PAPER
Induction of apolipoprotein A-I gene expression by black seed (Nigella sativa) extracts Michael J. Haas1, Luisa M. Onstead-Haas1, Emad Naem1, Norman C. W. Wong2, and Arshag D. Mooradian1 Pharmaceutical Biology Downloaded from informahealthcare.com by National University of Singapore on 05/30/14 For personal use only.
1
Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Florida, Jacksonville, FL, USA and 2Departments of Medicine and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada Abstract
Keywords
Context: Black seed [Nigella sativa L. (Ranunculaceae)] has been shown in animal models to lower serum cholesterol levels. Objectives: In order to determine if extracts from black seed have any effects on high-density lipoprotein (HDL), we characterized the effects of black seed extract on apolipoprotein A-I (apo A-I) gene expression, the primary protein component of HDL. Materials and methods: Hepatocytes (HepG2) and intestinal cells (Caco-2) were treated with black seed extracts, and Apo A-I, peroxisome proliferator-activated receptor a (PPARa), and retinoid-x-receptor a (RXRa) were measured by Western blot analysis. Apo A-I mRNA levels were measured by quantitative real-time polymerase chain reaction and apo A-I gene transcription was measured by transient transfection of apo A-I reporter plasmids. Results: Extracts from black seeds significantly increased hepatic and intestinal apo A-I secretion, as well as apo A-I mRNA and gene promoter activity. This effect required a PPARa binding site in the apo A-I gene promoter. Treatment of the extract with either heat or trypsin had no effect on its ability to induce apo A-I secretion. Treatment with black seed extract induced PPARa expression 9-fold and RXRa expression 2.5-fold. Furthermore, the addition of PPARa siRNA but not a control siRNA prevented some but not all the positive effects of black seed on apo A-I secretion. Discussion: Black seed extract is a potent inducer of apo A-I gene expression, presumably by enhancing PPARa/RXRa expression. Conclusions: We conclude that black seed may have beneficial effects in treating dyslipidemia and coronary heart disease.
Black seed, cardiovascular disease, HDL
Introduction Black seed [Nigella sativa L. (Ranunculaceae)] has been used as an herbal remedy for various ailments for centuries in countries such as India and Pakistan as well as throughout the Middle East and the Mediterranean countries (Sayed, 1980). Recent studies have shown that black seed has antitumorigenic effects, due in part to its thymoquinone content, as well as antioxidative, anti-inflammatory, and glucose and lipid modulating effects (Burtis & Bucar, 2000; Houghton et al., 1995; Worthen et al., 1998). As to the latter, black seed has been shown to lower total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL) in rats fed a diet rich in black seed (Dahri et al., 2005; Meral et al., 2001). Several studies have demonstrated that high-density lipoprotein (HDL) levels are inversely correlated with the risk of developing coronary artery disease (CAD) (Gordon et al., Correspondence: Michael J. Haas, Ph.D., Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, University of Florida Jacksonville, 653-1 West 8th Street L14, Jacksonville, FL 32209, USA. Tel: +1 904 244 8619. Fax: +1 904 244 5650. E-mail: [email protected]
History Received 21 October 2013 Revised 10 December 2013 Accepted 22 December 2013 Published online 17 March 2014
1977; Mooradian, 2003). HDL and its primary protein component apolipoprotein A-I (apo A-I) have several antiatherogenic properties which may influence the development of CAD (Haas & Mooradian, 2010a,b; Hachem & Mooradian, 2006). First, HDL participates in the process of reversecholesterol transport, by which cholesterol is transported from peripheral sources such as lipid-laden macrophage cells to the liver where it is either converted to bile acids or directly excreted in the feces (Mooradian et al., 2008). Second, in healthy individuals, HDL has antioxidative, anti-inflammatory, and antithrombotic properties that are thought to decrease macrophage activation, vascular reactivity and thrombus formation (Haas & Mooradian, 2010a,b). Although exercise, weight loss, and dietary modulation can raise HDL levels, the changes are modest and difficult to maintain. Certain drugs such as fibrates and some statins have been shown to induce apo A-I mRNA levels and gene transcription, but only modestly (Cerda et al., 2012; Hachem & Mooradian, 2006). Niacin and cholesterol-ester transfer protein (CETP) inhibitors have been shown to have robust positive effects on plasma HDL levels but they have not been documented to reduce cardiovascular risk in all studies
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(AIM-HIGH Investigators, 2011; Ghosh & Ghosh, 2012). The recent Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides: Impact on Global Health Outcomes (AIM-HIGH) trial showed no benefit of extended release niacin on cardiovascular outcomes (AIM-HIGH Investigators, 2011). Therefore, there remains a need for drugs or other small molecule compounds that elevate apo A-I and HDL significantly. Since black seed has lipid-modulating effects as well as antioxidant and anti-inflammatory properties, we examined the effects of various extracts prepared from the seed on apo A-I gene expression in hepatocyte and intestinal cell lines.
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Materials and methods Lipofectamine and Trizol were purchased from Life Technologies (Carlsbad, CA). 14C-Chloramphenicol was obtained from PerkinElmer (Waltham, MA). Fenofibrate was purchased from Sigma Chemical Company (St. Louis, MO). Plasmid purification kit was purchased from Qiagen (Valencia, CA). An anti-human apo A-I antibody (178422) and an antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (MAB374) were from EMD Millipore (Billerica, MA). Antibodies to retinoid-x-receptor a (RXRa) (D-20) and peroxisome proliferator-activated receptor a (PPARa) (H-98), and control siRNA and human PPARa siRNA were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An antibody to human albumin (109-4133) was purchased from Rockland Chemical (Gilbertsville, PA). Secondary antibodies prepared in goats and conjugated to horseradish peroxidase (HRP) were purchased from Southern Biotech (Birmingham, AL). Tissue culture media, media supplements, trypsin, and fetal bovine serum (FBS) were purchased from BioWittaker (Walkersville, MD). Immobilized TPCK trypsin was purchased from Pierce Biotechnology (Rockford, IL). All other chemicals were purchased from Fisher Scientific (Pittsburg, PA) or Sigma Chemical Company. Cell culture HepG2 and Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA). HepG2 cells were maintained in Dulbecco’s Modified Eagle’s Medium containing 10% FBS and penicillin and streptomycin (100 units/ml and 100 mg/ml, respectively). Caco-2 cells were maintained in Eagle’s Minimal Essential Medium containing 10% FBS, non-essential amino acids, sodium pyruvate, and 100 units/ml penicillin and 100 mg/ml streptomycin. The cells were housed in a humidified incubator at 37 C with an atmosphere of 95% air and 5% CO2. Cell viability was measured via mitochondrial dehyrodrogenase activity using 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (Mossman, 1983). Black seed extract preparation Two methods were used to prepare black seed extracts – ethanol extraction and volatile oil extraction. Ethanol extractions were performed essentially as previously described (Al-Jenoobi et al., 2010) by shaking 5 g of seeds in 25 ml of
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100% molecular biology-grade ethanol for 96 h. The ethanol was then evaporated and the residue was dissolved in 300 ml of dimethylsulfoxide (DMSO). Volatile oil extracts were prepared by grinding 20 g of black seed, which was mixed with 100 ml of distilled water and distilled in a Bialetti espresso maker (Bioletti Industrie Spa, Brescia, Italy) (Fararh et al., 2002). After distillation, the liquid was allowed to cool and the aqueous and lipid phases were allowed to separate. The pH of each phase was 6.8 and the amount of protein in each phase was measured using the bicinchoninic acid (BCA) method (Smith et al., 1985). Both the aqueous and lipid fractions were tested for activity. Black seed extracts contains several fixed oils including arachidic acid and eicosadienoic acid (Houghton et al., 1995). They also contain thymoquinone, dithymoquinone, p-cymene, carvacrol, t-anethole, 4-terpineol, and longifoline (Burtis & Bucar, 2000) and several alkaloids including nigellicine, nigellidine, and the isoquinoline nigellimine (Atta-ur-Rahman et al., 1995). In most experiments, the cells were cultured in serum-free medium (SFM) and treated with either a solvent (dH2O or ethanol, where appropriate) or the indicated dilution of black seed. Western blotting Apo A-I and albumin were measured in conditioned medium by Western blots. Briefly, the protein content of each media sample was determined using the BCA method and 5 mg of protein per lane was fractionated by electrophoresis on a 10% sodium dodecylsulfate (SDS) polyacrylamide gel. After electrophoresis, the protein was transferred to nitrocellulose (GE Healthcare, Pittsburg, PA) and after blocking for 2 h in phosphate buffered saline-Tween 20 (PBST) (10 mM Na2HPO4, 2 mM KH2PO4, 150 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) containing 5% non-fat dry milk, the antihuman apo A-I primary antibody (1:1000) or anti-human albumin primary antibody (1:2000) was added to the membrane and incubated overnight at 4 C. After washing in PBST, the membrane was incubated with the goat-anti-rabbit HRP secondary antibody (1:5000). After washing in PBST, the membrane was incubated in enhanced chemiluminescence (ECL) reagent for the detection of the signal by autoradiography. PPARa, RXRa, and GAPDH expression was measured in control and black seed extract-treated cells by Western blot. After 24 h of treatment, the media were removed and the cells were washed three times with ice-cold PBS and lysed in 300 ml of sample buffer [50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-Cl) (pH 7.4), 1% SDS, and 1 mM ethylenediamine tetraacetic acid]. After brief sonication to reduce the viscosity of the samples, particulates were removed by centrifugation at 10 000 g for 10 min at 4 C. Protein content of each sample was measured using the BCA assay and 50 mg of protein was loaded onto a 10% SDS polyacrylamide gel. After electrophoresis, the samples were transferred to nitrocellulose, which was then blocked in Trisbuffered saline [50 mM Tris-Cl (pH 7.4), 150 mM NaCl] containing 0.1% Tween 20 (TBST) and 10% newborn calf serum (NCS). The primary antibodies were diluted in TBST/ 10% NCS (PPARa, 1:200; RXRa, 1:200; GAPDH, 1:1000)
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and incubated with the membranes overnight at 4 C. After washing in TBST, the secondary antibodies were diluted in TBST/10% NCS (1:5000) and added to the membranes for 1 h at room temperature. After washing, the membranes were incubated with the ECL reagent and images were captured on film.
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Real-time quantitative polymerase chain reaction (PCR) Total RNA was extracted from HepG2 cells treated with black seed extract for 24 h as previously described (Chomczynski & Sacchi, 1987). Reverse transcription was performed with 2 mg of total RNA and random hexamers with AMV reverse transcriptase (Promega, Madison, WI). The cDNA was then used for PCR with primers for apo A-I (forward, 50 -AGC TTG CTG AAG GTG GAG GT-30 ; reverse, 50 -ATC GAG TGA AGG ACC TGG C-30 ), albumin (forward, 50 -TTG CAT GAG AAA ACG CCA GTA-30 ; reverse, 50 -GTC GCC TGT TCA CCA AGG A-30 ), and GAPDH (forward, 50 -CTC TCT GCT CCT CCT GTT CGA C-30 ; reverse, 50 -TGA GCG ATG TGG CTC GGC T-30 ). Amplification was performed with SYBRgreen dye (Bio-Rad, Hercules, CA) at 95 C for 1 min, 55 C for 1 min, and 72 C for 1 min for 40 cycles. Melt curve analysis was performed for each amplification to verify that only one product was formed in each well. Relative changes in RNA levels were calculated using the DDCt method using GAPDH as the control. Transient transfection analysis of apo A-I promoter activity HepG2 cells were transfected with 1 mg of the apo A-I reporter plasmid pAI.474.CAT and 1 mg of the control plasmid pCMV.SPORT.b-gal, to normalize transfection efficiency, using Lipofectamine. After 24 h, the cells were treated with a 105-fold dilution of black seed extract for 24 h, and assayed for chloramphenicol acetyltransferase (CAT) (Gorman et al., 1982) and b-galactosidase activity (Herbomel et al., 1984). To identify the black seed-responsive element in the apo A-I gene promoter, a series of apo A-I deletion constructs were used. The plasmids pAI.474.CAT, pAI.425.CAT, pAI.375.CAT, pAI.325.CAT, pAI.186.CAT, pAI.170.CAT, pAI.144.CAT, and pAI.46.CAT contain 474, 425, 375, 325, 186, 170, 144, and 46 bp of the 50 -flanking region of the apo A-I gene promoter. The plasmid pCMV.SPORT.b-gal was cotransfected with each apo A-I construct in order to normalize the transfection efficiency. Inhibition of PPARa expression using siRNA HepG2 cells were transfected with either control siRNA or siRNA targeting PPARa with lipofectamine, and after 72 h, treated with either a solvent of black seed (105-fold dilution) for 24 h. Apo A-I levels were measured in the conditioned medium by Western blot as described above. Data analysis Values are expressed as the mean ± the standard deviation (SD). Statistical significance was assessed using Student’s
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t-test for independent variables and a one-way analysis of variance. p Values less than 0.05 were considered significant.
Results The effect of black seed extract on apo A-I secretion HepG2 cells were treated with 109, 108, 107, 106, 105, and 104-fold dilutions of ethanol black seed extract (2.0 mg/ ml protein equivalent) for 24 h and apo A-I protein levels were measured in the conditioned medium (Figure 1). Apo A-I protein levels (Figure 1A and B) increased from 541 ± 50 arbitrary units (A.U.) in control cells to 622 ± 61, 1341 ± 156, 1250 ± 101, 1356 ± 133, and 1324 ± 127 A.U. in cells treated with 109, 108, 107, 106, 105, and 104-fold dilutions of ethanol black seed extract (N.S., p50.001, 0.003, 0.0005, 0.003, and 0.004, respectively). In contrast, albumin levels in control cells were 591 ± 50 A.U. and 588 ± 33, 584 ± 31, 583 ± 41, 604 ± 39 589 ± 33 A.U. in cells treated with 109, 108, 107, 106, 105, and 104-fold dilutions of black seed extract [all not significant (N.S.)]. Thymoquinone is a substantial component of black seed extract and has been shown to be responsible for many of the biological activities of the mixture. To determine if thymoquinone induces apo A-I protein synthesis, HepG2 cells were treated with increasing doses of thymoquinone and apo A-I levels were measured in the medium (Figure 1C and D). Apo A-I levels were 618 ± 45 A.U. and 648 ± 32, 611 ± 27, 631 ± 33, 609 ± 43, and 617 ± 55 A.U. in cells treated with 109, 108, 107, 196, and 105 M thymoquinone (D) (all N.S.) These results suggest that thymoquinone is not the component that regulates apo A-I protein secretion. The effect of aqueous and lipid portion of black seed extracts on apo A-I protein secretion In addition to ethanol black seed extracts, volatile oil and aqueous black seed extracts were prepared and tested for their ability to induce apo A-I gene expression. The aqueous fraction had significantly higher protein content per milliliter (1.1 mg/ml) than the volatile oil fraction (0.6 mg/ml) (Figure 2A). In HepG2 cells treated with the aqueous fraction, apo A-I levels in conditioned media were 235 ± 12, 254 ± 28, 249 ± 15, 234 ± 5, 241 ± 13, and 235 ± 8 A.U. in cells treated with 109, 108, 107, 106, 105, and 104-fold dilutions of aqueous black seed extract (all N.S.) (Figure 2B). In contrast, the volatile oil fraction increased apo A-I concentrations from 332 ± 43 A.U. to 391 ± 7, 789 ± 17, 973 ± 66, 903 ± 95, 675 ± 54, 493 ± 36 A.U. in cells treated with 109, 108, 107, 106, 105, and 104-fold dilutions of black seed volatile oil extract (N.S., p50.001, 0.001, 0.007, 0.0001, 0.001, and 0.008, respectively) (Figure 2C). These results suggest that a heat-resistant volatile oil from black seed extract induces apo A-I protein levels. To determine whether or not a protein or proteins in the black seed extract are involved in inducing apo A-I production, we examined the effect of trypsin digestion. Aliquots of black seed volatile oil extract were digested with TPCKtrypsin for 24 h at 37 C, at which time they were used to treat the HepG2 cells (Figure 2D). Undigested extract increased
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Figure 1. The effect of ethanol black seed extract and thymoquinone on apo A-I protein secretion. HepG2 cells were treated with the indicated amounts of black seed extract for 24 h and apo A-I and albumin levels (A) were measured by Western blot. The blots were quantified and are shown in (B). Black seed extract induced apo A-I protein synthesis but had no effect on albumin levels. HepG2 cells were treated with the indicated amounts of thymoquinone and apo A-I and albumin levels were measured in the conditioned medium by Western blot (C). The blots were quantified and are shown in (D). Apo A-I levels were normalized to albumin gene expression. Thymoquinone had no effect on apo A-I and albumin protein secretion. N ¼ 6; *p50.05, treated cells relative to untreated cells.
apo A-I protein levels from 568 ± 44 A.U. to 1565 ± 78 A.U. (p50.001, relative to control cells) and trypsin pre-treated extract increased apo A-I protein levels to 1589 ± 173 A.U. (p50.001, relative to control cells) (Figure 2D and E). These results suggest that the component or components that induced apo A-I protein secretion are in the volatile oil fraction and are resistant to heat and proteolysis. The effect of black seed extract on apo A-I mRNA and gene promoter activity To determine if the changes in apo A-I protein levels are due to changes in apo A-I mRNA, HepG2 cells were treated with a 105-fold dilution of black seed extract for 24 h and apo A-I and albumin mRNA levels were measured by quantitative real-time PCR and normalized to GAPDH mRNA levels (Figure 3). Apo A-I mRNA levels increased 1.8-fold in HepG2 cells relative to control cells (p50.001) (Figure 3A). Albumin mRNA levels were similar in each group (N.S.) (Figure 3B). To determine if black seed induces apo A-I promoter activity, HepG2 cells were transfected with the plasmids pAI.474.CAT and pCMV.SPORT.b-gal (to normalize transfection efficiency) and treated with either the solvent (DMSO) or black seed extract for 24 h (Figure 3C). Black seed extract increased apo A-I reporter gene expression from 21.1 ± 1.2% acetylation to 20.2 ± 2.2, 23.4 ± 0.4, 28.5 ± 1.1, and 31.3 ± 1.9% acetylation in cells treated with 109, 108,
107, 106, and 105-fold dilutions of black seed extract (N.S., p50.04, 0.006, 0.001, and 0.001, respectively) (Figure 3C). These results suggest that black seed induces apo A-I protein secretion by inducing apo A-I gene promoter activity and mRNA expression. Identification of a black seed response element in the apo A-I gene promoter To determine the regions of the apo A-I gene promoter that respond to black seed extract, HepG2 cells were transfected with a series of apo A-I reporter plasmids containing successive deletions of the apo A-I gene promoter. The cells were also transfected with the plasmid pCMV.SPORT.b-gal to normalize transfection efficiency. The cells were then treated with black seed extract (a 105-fold dilution) for 24 h and CAT and b-galactosidase activity were measured (Figure 3D). Black seed extract induced reporter gene expression by 1.44 ± 0.05, 1.44 ± 0.04, 1.39 ± 0.03, and 1.38 ± 0.06-fold in cells transfected with the plasmids pAI.474.CAT, pAI.425.CAT, pAI.375.CAT, and pAI.325.CAT (p50.001, 0.001, 0.003, and 0.001, respectively). However, black seedtreatment had no effect on apo A-I promoter activity in cells transfected with pAI.186.CAT (1.1 ± 0.06-fold), pAI.170.CAT (1.0 ± 0.08-fold), pAI.144.CAT (1.0 ± 0.08fold), and pAI.46.CAT (0.9 ± 0.13-fold) (all N.S.) (Figure 3D). These findings suggest that the region of the apo A-I promoter between nucleotides 325 and 186 is
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DOI: 10.3109/13880209.2013.879187
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Figure 2. The effect of aqueous and volatile oil black seed fractions and trypsin digestion on apo A-I gene expression. HepG2 cells were left untreated or were treated with the indicated dilutions of either the aqueous (A) or volatile oil (B) black seed fraction for 24 h and apo A-I levels were measured in the conditioned medium. The volatile oil fraction of the black seed extract induced apo A-I gene expression; however, the aqueous fraction had no effect. HepG2 cells were either untreated or treated with a 105-fold dilution of black seed or a 105-fold dilution of black seed digested with trypsin for 24 h. Apo A-I and albumin levels were measured by Western blot (C). (D) The blots were quantified and apo A-I and albumin expression are shown. Trypsin digestion had no effect on the ability of black seed to induce apo A-I gene expression. N ¼ 6; p50.05, trypsin-digested versus trypsinundigested extract.
essential for the induction of promoter activity in cells exposed to black seed extract. To determine if black seed extract effects apo A-I gene expression in intestinal cells, Caco-2 cells were treated with black seed extract for 24 h and apo A-I protein was measured in the conditioned medium and apo A-I and GAPDH mRNA levels were measured by real-time quantitative PCR (Figure 3E and F). Black seed induced apo A-I secretion 2-fold (from 465 ± 50 A.U. in control cells to 1048 ± 98 A.U. in cells treated with black seed extract; p50.001) (Figure 3E) and apo A-I mRNA levels 2.2-fold (p50.03) (Figure 3F). These results suggest that black seed induces apo A-I gene expression in hepatocytes as well as intestinal cells.
The effect of black seed extract on PPARa and RXRa expressions The black seed extract-responsive region in the apo A-I gene promoter between 325 and 186 contains a PPARa/RXRa binding site. PPARa and RXRa have been shown to induce apo A-I gene transcription (Hossain et al., 2008). To determine if PPARa and or RXRa expressions are regulated by exposure to black seed extract, HepG2 cells were treated with black seed extract for 24 h and PPARa and RXRa levels were measured by Western blot (Figure 4). Treatment with black seed extract increased PPARa (Figure 4A) and RXRa (Figure 4B) levels 9.0-fold (p50.001) and 2.4-fold (p50.003), respectively. In contrast, GAPDH levels did not
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Figure 3. The effect of black seed extract on apo A-I mRNA expression and apo AI promoter activity in liver and intestinal cells. HepG2 cells were either untreated or treated with a 105-fold dilution of black seed for 24 h and apo A-I, albumin, and GAPDH mRNA levels were measured by quantitative real-time PCR. Apo A-I (A) and albumin (B) mRNA levels were normalized to GAPDH mRNA. Black seed treatment induced apo A-I mRNA levels but had no effect on albumin mRNA expression. (C) HepG2 cells were transfected with the plasmids pAI.474.CAT and pCMV.SPORT.bgal and either left untreated or treated with the indicated dilution of black seed for 24 h. Treatment of black seed extract induced apo A-I promoter activity but had no effect on the CMV promoter. N ¼ 6; *p50.05, treated versus untreated cells. (D) HepG2 cells were transfected with plasmids containing successive deletions of the apo A-I gene promoter and either left untreated or treated with a 105-fold dilution of black seed extract for 24 h. The fold-change in black seed cells relative to control cells is presented for each plasmid. Induction of apo A-I promoter activity by black seed required the region between 326 and 186. N ¼ 3; *p50.05. Caco-2 cells were treated with black seed extract for 24 h and apo A-I expression was measured by Western blot (E) and apo A-I mRNA levels were measured by quantitative real-time PCR (F). Black seed induced apo A-I protein secretion and apo A-I mRNA levels. N ¼ 6; *p50.05, treated versus untreated cells.
change (Figure 4C). These results suggest that black seed induces apo A-I gene expression in part by increasing PPARa/ RXRa levels. To determine if PPARa is required for induction of apo A-I secretion by black seed extract, we knockout PPARa expression using siRNA (Figure 4D). The addition of 5 and 10 mM PPARa siRNA inhibited PPARa expression from 488 ± 31 A.U. to 98 ± 18 and 92 ± 25 A.U., respectively (p50.0001 and p50.0001, respectively) Next, HepG2 cells were transfected with either control or PPARa siRNA and treated with
either solvent (dH2O) or a 105-fold dilution of black seed for 24 h. As a control, cells were also treated with the PPARa agonist fenofibrate (100 mM). In cells treated with the control siRNA (Figure 4E), black seed treatment induced apo A-I secretion 2.8-fold (p50.0002) while fenofibrate increased apo A-I secretion 1.5-fold (p50.01). In contrast, cells transfected with the PPARa siRNA, black seed treatment increased apo A-I protein levels 1.5-fold (p50.03, relative to solvent-treated cells) and fenofibrate induced apo A-I protein levels 1.1-fold (N.S., relative to solvent-treated cells) These
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DOI: 10.3109/13880209.2013.879187
Induction of apo A-I expression by black seed extract
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Figure 4. The effect of black seed extract on PPARa and RXRa expression. HepG2 cells were either left untreated or treated with a 105-fold dilution of black seed extract for 24 h and PPARa (A), RXRa (B), and GAPDH (C) expressions were measured by Western blot. Black seed induced PPARa and RXRa expressions but GAPDH levels did not change. N ¼ 3; *p50.05, treated versus untreated cells. (D) HepG2 cells were transfected with 5 and 10 mM PPAR a siRNA for 72 h and PPAR a levels were measured by Western blot. N ¼ 3; *p50.0001 versus control cells. (E) HepG2 cells were transfected with the control or PPARa siRNA and after 72 h, treated with or without black seed (105-fold dilution) or fenofibrate in SFM for 24 h. Apo A-I levels were measured by Western blot in the conditioned medium. Treatment with the control siRNA had no effect on the ability of black seed to induce apo A-I synthesis. In contrast, the addition of the PPAR a siRNA prevented most but not all the abilities of black seed to induce apo A-I protein secretion. N ¼ 3; *p50.0002 and p50.01 in cells treated with black seed and fenofibrate, respectively, versus control cells; p50.02 and N.S. in cells treated with black seed and fenofibrate, respectively, versus control cells; yp50.02 in black seed treated cells versus fenofibrate-treated cells.
results suggest that PPARa modulates some but not all the effects of black seed on apo A-I gene expression.
Discussion Black seed extracts have been shown to regulate lipid metabolism in several animal studies (Dahri et al., 2005; Meral et al., 2001). The present study shows that ethanol extracts of black seed increase apo A-I levels of conditioned media from HepG2 cells by several fold (Figure 1). The apo A-I inducing activity was also observed in a concentrated volatile oil extract after steam extraction and after trypsin digestion (Figure 2) suggesting that the active components are heat resistant and probably not a protein. The increase in apo A-I protein was accompanied by increased apo A-I mRNA and promoter activity (Figure 3). Induction of apo A-I promoter activity required a region of the apo A-I gene promoter (325 to 189) that contains a PPARa/RXRa binding site (Figure 3). Furthermore, treatment of HepG2
cells with black seed extract induced both PPARa and RXRa expression (Figure 4). Intestinal cells synthesize apo A-I and are an important contributor to plasma HDL levels. They also regulate apo A-I gene expression in a PPARa-dependent manner (Colin et al., 2013). Therefore, we examined the effect of black seed extract on apo A-I expression in Caco-2 intestinal cells (Figure 3). Black seed extract induced apo A-I secretion and apo A-I mRNA levels by approximately 2-fold (Figure 3). These results suggest that black seed induces apo A-I gene expression in hepatocytes as well as intestinal cells. The contents of ethanol black seed extracts have been elucidated to some extent and they have been shown to contain numerous compounds that have anti-oxidant and anti-inflammatory properties. Some of the effects of black seed, especially on cell growth and inhibition of lipid peroxidation, have been attributed to its thymoquinone content. However, thymoquinone had no effect on apo A-I levels in the present study (Figure 1), although other
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M. J. Haas et al.
anti-oxidants such as a-tocopherol and ascorbic acid have been previously demonstrated to suppress apo A-I gene expression in HepG2 cells (Mooradian et al., 2006). Treatment with black seed extract increased PPARa expression 9-fold and RXRa expression 2.4-fold suggesting that black seed may increase expression of genes involved in fatty acid oxidation (Figure 4). Likewise, the volatile oil and ethanol extracts of black seeds may contain endogenous RXRa and/or PPARa ligands that would further increase activity of these nuclear receptors. PPARa has a large ligand binding pocket that can be occupied by a wide variety of fatty acids or arachidonic acid metabolites (Xu et al., 2001) that serve as endogenous ligands for the receptor. Several natural compounds have been shown to stimulate PPARa activity (Devchand et al., 1996; Xu et al., 2001). Alternatively, black seed extract treatment may promote fatty acid oxidation and the generation of endogenous PPARa ligands, including acyl-Co A’s and enoyl-CoA’s (Hostetler et al., 2005). The precise identity of the active ingredient in black seed extracts responsible for the apo A-I induction is not known. The available data suggest that it is a lipophilic compound that is resistant to heat and trypsin digestion. Over 46 compounds have been identified in the volatile oil from black seed, including thymol, thymoquinone, 4-terpineol, longifolene-(V4), and p-cymene, among others (Ahmad and Beg, 2013). Alcohol extracts from black seed contain thymoquinone as well as several fatty acids, primarily linoleic and palmitic acid. However, it is unlikely to be one of the common dietary fatty acids since previous studies in the same hepatic cell line did not reveal apo A-I induction by a number of saturated, unsaturated, monounsaturated, and trans-fatty acids such as stearic, myristic, palmitic, oleic, linoleic, linolenic, elaidic, linolelaidic, and linolenelaidic acid (Haas et al., 2004). We are currently fractionating the extracts to identify the active components.
Conclusions Our studies show that ethanol and lipid extracts prepared from black seed are potent inducers of apo A-I gene expression, in part by enhancing PPARa/RXRa expression. These results suggest that black seed may have beneficial effects in treating dyslipidemia and coronary heart disease.
Declaration of interest This work was supported in part by a Dean’s Fund Research Award from the University of Florida-Jacksonville to E. Naem. The authors have no conflicts to declare. The authors are responsible for the contents and writing of the paper.
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