CVD: Cardiovascular Disease; FBS: Fetal Bovine Serum; FFA: Free Fatty Acids; Fis-1: Mitochondrial Fission 1 Protein; HbA1c: Glycated Hemoglobin A1C; HO-1: Heme Oxygenase 1; HO-2: Heme Oxygenase 2; HDL: High Density Lipoprotein; HFD: High Fat Diet; LDL: Low Density Lipoprotein; MetS: Metabolic Syndrome; MFN-1: Mitofusin 1; MFN-2: Mitofusin 2; NADPH: Dihydronicotinamide-Adenine Dinucleotide Phosphate; NAFLD: Non-Alcoholic Fatty Liver Disease; NASH: Non-Alcoholic Steato-Hepatitis; NOV: Nephroblastoma Overexpressed; NRF2: Nuclear Factor (erythroid-derived)-like 2; OPA- 1: Optic Atrophy 1; OX-HDL: Oxidized HDL; OX-LDL: Oxidized LDL; PMSF: Phenylmethylsulfonyl Fluoride; ROS: Reactive Oxygen Species; T2DM: Type 2 Diabetes Mellitus; TQ: Thymoquinone.

According to the World Health Organization, annually over 2 million people die worldwide from the complications of excessive body fat. An altered adipose tissue function is characterized by an impaired lipid buffering capacity and subsequently by a systemic lipid over flow and ectopic lipid accumulation in several insulin sensitive peripheral tissues such as skeletal muscle, liver, pancreas, heart and kidneys [1,2]. This obesity trend is followed in men and women, both having a similar pattern, being around 40-45% obese in middle age around 35% obese when younger [3]. The ectopic deposition of triglycerides triggers a series of cardiometabolic perturbations, which are grouped into a diagnosis of metabolic syndrome (MetS). This disorder is not only associated with a higher risk of appearance of type 2 diabetes and cardiovascular events but impacts the liver [4]. Recent data suggest that nonalcoholic fatty liver disease (NAFLD), considered the hepatic manifestation of the MetS, precedes the development of MetS [5]. NAFLD is associated with a number of metabolic diseases including diabetes mellitus, obesity and hypertension. In a five-year retrospective review, individuals with NAFLD had a higher incidence of impaired fasting glucose and type 2 diabetes mellitus (T2DM) compared with NAFLD-free controls [6]. In the last few decades, a higher frequency of obesity, T2DM, and MetS have occurred as a result of various dietary changes [7]. Furthermore, individuals with NAFLD have a higher probability of liver failure and, eventually, cirrhosis [8-10]. Epidemiological results suggest that insulin resistance is a common pathogenic factor for all these obesity-related conditions and that it can be both reversed and prevented by a healthy lifestyle and a wholesome diet [11]. In this regard, beneficial effects have been reported for curcumin and Resveratrol [12] which increase the antioxidant gene and heme oxygenase-1 (HO-1). Resveratrol upregulates HO-1 expression, NAD(P)H, quinone oxidoreductase 1, through activation of nuclear factor (erythroid-derived)-like 2 (Nrf2) target genes. Resveratrol as well as other HO-1 inducers prevent CVD [13]. Importantly, HO-1 induction is regulated by levels of glucose; while glucose deprivation induces HO-1 gene expression [14], elevated levels of glucose suppress HO-1 gene expression [15,16]. HO-1 levels is affected by obesity and glucose levels (reviewed in [3,17]).

Thymoquinone, present in Nigella sativa (NS), has been proposed, based on its anti-oxidant properties, as a protective factor against several metabolic diseases. Nigella sativa Linn. (family Ranunculaceae), commonly known as black seed or black cumin, is an herbal plant that has been cultivated for thousands of years in the Middle and South East Asia. Black cumin seed is composed of fixed (stable) and essential (volatile). The Essential oil extracted from black cumin contains a rich volatile fraction comprising Thymoquinone (TQ) and Thymohydroquinone (THQ) [18,19]. Thymoquinone (TQ) is the main pharmacologically active compound of NS and is thought responsible for many therapeutic properties, including anti-inflammatory, antioxidant and anti-hyperglycemic effects. The protective effect of TQ is related to its ability to scavenge reactive oxygen species (ROS), including superoxide and hydroxyl free radicals [20], to block lipid peroxidation and to enhance levels of antioxidant enzymes [21,22]. The aim of this study was to demonstrate the effects of black seed oil, with a high content of TQ, on the metabolic profile, including adipose-mediated release in inflammatory adipokines such as NOV, mitochondrial biogenesis, LDL, Ox-HDL and hepatic steatosis in a murine model of obesity.

Animal protocols

Eight-week-old C57B16 male mice were fed western diets with 51% fat content while control mice fed regular diets, high fat diets (Harlan, Teklad Lab animal diets, Indianapolis, IN) (HFD) for 20 weeks. Mice were divided into three treatment groups of five animals each as follows: group 1) Lean, group 2) HFD, group 3) HFD treated for the last 8 weeks with HFD treated for the last 8 weeks with black seed-cold press oil formulation containing thymoquinone (TQ) between 3-3.1% obtained from TriNutra Israel. Formulation of TQ oil is as follows; TQ 3.14 %, p-Cymene, 1.24%, Carvacrol 0.08%, FFA 1,29%, Oleic Acid 21.53%, palmitic acid 11.31%, linoleic acid 57.44%, other fatty acid 1.98% and TPGS, 0.8%. TQ oil was mixed into the HFD food and made into pellets using a mixer. At the end of the experiment, mice were euthanized, assessed for total body weight, fat content and liver fibrosis. All animal experiments followed the NYMC IACUC institutionally approved protocol in accordance with NIH guidelines.

Fasting blood glucose, glucose tolerance testing

Fasting blood glucose and glucose tolerance were measured from tail blood following a 6 h fast. Blood pressure was measured by the tail-cuff method using the CODA tail-cuff System (Kent Scientific, CT, Torrington) as we previously described [23-25].

Determination of oxygen consumption

The C57 mice groups were allowed to acclimatize in the oxygen consumption chambers over a three-week period. Adaptation periods for the three-week duration were executed in two-hour increments, three times a week. The Oxylet gas analyzer and air flow unit (Oxylet, Panlab-Bioseb, Vitrolles, France) were used to determine mouse oxygen consumption (VO2). Each mouse was placed individually in the machine and VO2, VCO2 and respiratory quotient (RQ) was calculated as VCO2/VO2. The data for VO2 are expressed as the consumed oxygen per Kilogram body weight per minute (ml/kg/min) [23-25].

Measurement of HO activity

Liver microsomal HO activity was assayed by the method of Abraham et al. in which liver tissues was homogenates in phosphate buffer, pH 7.8, 0.1 mM EDTA and 1mM PMSF. HO activity was measured in presence of 20 uM heme, glucose 6 phosphate (G-6P), glucose 6 phosphate dehydrogenase (G6PDH), NADPH, at 37°C for 60 minutes. Bilirubin, the product of HO degradation was extracted with chloroform, spin down and leave overnight in the freezer. Samples defrost; spin the samples for 20 minutes and with pasture pipets remove the lower layer which has chloroform. Bilirubin concentration in chloroform determined spectrophotometrically (Perkin-Elmer (Norwalk, CT) Dual UV/VIS Beam Spectrophotometer) using the difference in absorbance at wavelength from λ 460 to λ 530 nm with an absorption coefficient of 40 mM^-1 cm^-1.

Western blot analysis

For protein expression analyses, liver tissues were lysed in RIPA lysis buffer supplemented with protease and phosphatase inhibitors (CompleteTM Mini and PhosSTOPTM, Roche Diagnostics, Indianapolis, IN) Frozen mouse adipose tissue was ground under liquid nitrogen and suspended in homogenization buffer (comprising mmol/L :10 phosphate buffer, 250 sucrose, 1.0 EDTA, 0.1 PMSF and 0.1%v/v tergitol, pH 7.5). For In vitro Western blot analysis pelleted cells were lysed and HO-1, HO-2, OPA1, MFN1, MFN2 and NOV proteins were measured. Protein detection was carried out using a secondary infrared fluorescent dye conjugated antibody absorbing at both 800 nm and 700 nm. The blots were visualized using an Odyssey Infrared Imaging Scanner (Li-Cor Science Tec) and quantified by densitometric analysis performed after normalization with β-actin. Results were expressed as arbitrary units (AU).

Histopathological examination of liver tissue

Liver samples from each experimental group were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin wax, and sectioned (6 μm thick). The main liver histopathological features commonly described in NAFLD including steatosis, inflammation, hepatocyte ballooning, and fibrosis were scored according to the NAFLD histologic activity score (NASH) system, and lipid droplet analysis was performed as previously described [26].

Cell culture and adipocyte cell differentiation

3T3-L1 murine pre-adipocytes, were purchased from American Type Culture Collection (Rockville, MD, USA). After thawing, 3T3- L1 cells were resuspended in DMEM, supplemented with 10% heat inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic solution (Invitrogen). The medium was replaced with adipogenic medium, and the cells were cultured for an additional 6 days. Differentiating 3T3-L1 pre-adipocytes were treated for 6 days with 3% TQ (2, 4, 6 M).

Oil red O staining

Staining was performed using 0.21% Oil Red O in 100% isopropanol (Sigma-Aldrich, St. Louis, MO, USA). Briefly, adipocytes were fixed in 10% formaldehyde, stained with Oil Red O for 10 minutes, rinsed with 60% isopropanol (Sigma-Aldrich), and the Oil Red O eluted by adding 100% isopropanol for 10 minutes and the optical density (OD) measured at 490 nm, for 0.5 sec reading.

Statistical analysis

Data are expressed as means ± S.E.M. Bonferroni’s post -test analysis for multiple comparisons was used to calculate the significance of mean value differences using one-way analysis of variance. The null hypothesis was rejected at p<0.05.

Effects of TQ on body weight, blood pressure, fasting blood glucose and oxygen consumption

We examined the effect of TQ in mice fed a HFD for 20 weeks (Figures 1 and 2). Blood pressure and fasting blood glucose levels were increased in mice fed a HFD as compared to control animals (Figure 1A and 1B). TQ reduced blood pressure and fasting blood glucose levels in mice fed a HFD. Mice on a HFD displayed a decrease in VO2 consumption. In contrast, TQ produced a significant (p<0.05) increase in oxygen consumption (Figure 1C). As shown in Figure 2, weight of the HFD group was increased (p<0.05) compared to Lean, but no difference occurred between the HFD and TQ groups.

Figure 1: Effect of TQ on blood pressure, blood glucose, oxygen consumption and body weight=Effect of TQ on (A)blood pressure,(B) blood glucose, (C)oxygen consumption and (D)body weight. Results are mean +/- SE n=6, *p<0.05 vs lean mice, #p<0.05 vs HFD mice.

Figure 2: Effect of TQ on oil droplets formation in 3T3 adipocytes. We measured the effect of 3% TQ administration on adipogenesis. Daily supplementation of TQ was effective on adipogenesis suppression at 6 days. TQ treatment showed a significant (p<0.05) reduction of lipid droplets formation in 3T3 adipocytes. (n=4), #p<0.05 vs. control, ** p<0.05 vs. control.

Effect of TQ on adipogenesis in vitro

TQ decreased large lipid droplet content in differentiated adipocytes compared with differentiated control cells (p< 0.05) (Figure 3A and 3B). Furthermore, TQ3 3% decreases oil lipid accumulation seen clearly between differentiation cell and cells treatment with TQ 3% at 6 M, suggesting that TQ decreased adipocyte terminal differentiation preventing the conversion of small “healthy” adipocytes to large adipocytes. TQ decreased lipid content in a dose-dependent manner (Figure 3B). Sardana and Kappas reported that the increase in HO-1 mRNA and protein are several orders of magnitude higher than the increase in liver HO activity [27], therefore, we measured the consequence of TQ treatment on liver HO activity and generation of bilirubin anti-oxidant effect. Since HO-1 converts heme to equimolar amounts of CO and bilirubin (20), we measured HO activity by formation of bilirubin. HO activity in control liver tissues was 0.81 ± 0.16 nmol bilirubin formed/mg protein/hour and decreased to 0.49 ± 0.12 nmol bilirubin formed/mg protein/hour in high fat liver (p<0.05) (Figure 3B-3F). The stimulatory effect of 3%TQ on HO-1 protein was associated with an increase in HO activity (Figure 3G) to 0.78 ± 0.12 nmol bilirubin/mg/hour (p<0.05).

Figure 3: Effect of TQ administration on levels of HO-1, Fis1, MFN2, NOV and pIR972 in adipose tissue on Lean, HFD and HFD +3% TQ. Representative western blots; (A) and densitometry analysis of (B) pIR972, (C) MFN2, (D) NOV, (E) Fis1, (F) HO-1 of Lean, HFD and HFD +3% TQ. Results are mean ± SE, n=6, *p<0.05 vs. Lean, #p<0.05 vs. HFD (G) HO activity in control, HF untreated and HF-treated with 3%TQ treated mice. HO activity was determined and results are mean±SE, n= 3, *p<0.05 vs. control, # p<0.05 vs. HF mice..

Effects of TQ on protein expression in adipose tissue

Western blot analysis of fat tissue showed significant differences in protein expression levels of pIR972, HO-1, Fis-1, Mfn2 and NOV in obese mice compared to control mice. Untreated obese animals exhibited a significant (p<0.05) decrease in insulin receptor phosphorylation levels and HO-1 when compared to age-matched lean mice. TQ increased both pIR₉₇₂ mitochondrial fusion protein and HO-1 levels in obese mice (Figure 4A-4E). A HFD resulted in a decrease in Mfn2 (p<0.05) and an increase in FIS-1 a fission protein (p<0.05). TQ treatment reversed the negative effect on mitochondrial protein as seen by the increased in the levels of MFN2 (p<0.05) and decreased FIS-1 (p<0.05) compared to HF mice (Figure 4B and 4D). As seen in Figure 4D, levels of adipose tissue derived NOV, a pro-inflammatory protein in lean group are significantly (p<0.05) lower than in the HFD group. As shown in Figure 4C, TQ treatment decreased NOV protein expression compared to mice fed a HFD alone.

Figure 4:Haematoxylin-eosin staining of liver of lean (A), HFD (B) and HFD treated with 3% TQ (C) mice. Graphs summarize the morphometrical analysis of liver lipid droplet diameter (F) and Fat percentage (G). * p< 0.05 versus lean; # p< 0.05 versus HFD. Bar 50

Discussion

TQ is an active component of TriNutra’s™ Nigella seed oil and is considered responsible for most of the latter therapeutic potential. The plant Nigella sativa (N. sativa) has been used throughout the world in various traditional systems of medicine as a therapy for many different ailments and conditions. The key finding of the present study highlights the hepato-protective effects of TQ in a rodent model of NAFLD. TQ administration for 8-weeks reduced hepatic fat accumulation preventing the development of NASH and liver fibrosis in 36-week study of obese mice. NAFLD affects ~ 25% of the adult population and is the most common cause of chronic liver disease in the Western World. Concomitantly it is associated with obesity, type II diabetes and hyperlipidemia, and may serve as a marker of increased morbidity and mortality from cardiovascular disease. While the mechanism of NAFLD has not been elucidated, it is manifest as tissue injury as a result of fat accumulation. In this process oxidative stress results in mitochondrial damage [28,29] and tissue dysfunction manifest as hepatocellular oxidative damage leading to hepatic inflammation (non-alcoholic steatohepatitis). Hepatic dysfunction leads to fibrosis followed by cirrhosis, liver failure and hepatocellular carcinoma. Treatment of obese mice with TQ improves mitochondrial function in adipose and hepatic steatosis by decreasing levels of adipocyte derived NOV, and increasing, an antioxidant gene, HO-1 expression, resulting in increased mitochondrial biogenesis, function, and fusion potential, leading to an improvement in oxidative stress and inflammation in obese mice. The following key findings substantiate this conclusion. A HFD increased the expression of the genes regulating mitochondrial fission in mice, while concomitantly reducing the expression of the genes responsible for mitochondrial quality control and fusion processes in adipose and hepatic tissue. We further investigated whether TQ treatment positively affect signaling proteins. TQ positively increased HO-1 and insulin receptor phosphorylation in liver adipose tissue (Figures 7 and 8). Similar effect is seen in heart and kidney signaling protein (data not shown). A HFD enhanced FFA generation and increased mitochondrial dysfunction and ROS levels [30,31]. Mitochondrial dysfunction results in a decrease in beta oxidation in the liver allowing fat to accumulate resulting in a “fatty liver” [26,32,33]. TQ reduced mitochondrial fission potential and normalized an enhanced expression of mitochondrial fusion-associated genes in mice fed a HFD. TQ is a natural antioxidant and hypoglycemic compound that may prove advantageous therapeutically when compared to the high cost and the adverse effects of pharmacological drugs.

Figure 8: Schematic representation of TQ effects on reprogram adipocyte phenotype from naïve state to healthy adipocyte that increase activity of HO- 1, mitochondrial proteins and insulin receptor phosphorylation. TQ treatment change the quality of fat from sick fat, adiposopathy, to healthy fat, i.e., metabolically active, that express more mitochondrial signaling, increase oxygen consumption and increase insulin receptor phosphorylation.

NOV expression in obese mice was increased when compared to lean mice, the levels of NOV in HFD-fed mice treated with TQ were lower than mice fed a HFD alone. Increased NOV levels are linked to increased levels of inflammatory cytokines which deleteriously affect insulin signaling, resulting in insulin resistance and eventually obesity [34,35]. In contrast, downregulation of NOV is associated with a reduction in adipose tissue deposition and inflammatory cytokines, as well as enhanced insulin sensitivity in obese mice [23,26].

Figures 4 and 5 showed that TQ improved hepatic steatosis, fibrosis and metabolic balance in obese mice. More importantly, ingestion of TQ in HF mice led to a reversal of this trend and a resultant increase in both the level and activity of HO-1, which strongly suggests a role for HO-1 and HO activity in the antioxidant and anti-inflammatory effect of TQ. Other report in agreement with our finding that induction of HO-1 suppresses adiposity and diabetes [36,37]. Further, sex-depends effect of HO-1 in adipose is well described [38], in which expression of HO-1 in adipose tissue may have a greater protective role in female as compared to male [38].

Further, HO-1 is considered a novel target for the treatment of hypertension and obesity [39]. More importantly beneficial effect of HO-1 is seen in human liver transplant biopsies; subjects with higher HO-1 levels showed decreased hepatocellular damage and improved outcomes [40].

Additionally, it appears that the increase of HO-1 levels, decrease in pro-inflammatory NOV expression and the normalization of mitochondrial function rescue liver function in obese mice. The beneficial effects of TQ on hepatic protein expression suggest an antisteatosis effect that prevents disease progression to steatohepatitis in our animal model support the effect on decrease fasting glucose and oxygen consumption.

It appears that TQ was capable of reprogramming the adipocyte phenotype by regulating energy gene and mitochondrial function and HO-1 expression, leading to an increase in “healthy”, i.e. small, adipocytes and a decrease in large adipocyte qualitatively and in terminal differentiation as evidence suggesting that increase in activity as evidence of increase in oxygen consumption, may maintain healthier adipocytes in obese mice. This occurred without body weight change, further, TQ improved the metabolic profile of obese mice by lowering fasting glucose, BP and hypertension, and increasing oxygen consumption compared to non-treated obese mice. One plausible explanation for body weight remaining unchanged could be the direct effect of TQ on adipocyte hyperplasia. This supports the hypothesis that the expansion of adipocytes may lead to an increased number of adipocytes of smaller size; smaller adipocytes are considered to be “healthy”, insulin-sensitive adipocyte cells that are capable of producing adiponectin [41,42]. There is a tight link exists between adipocyte hypertrophy and inflammation; followed by a reduction in adipocyte size leading to amelioration of metabolic functions [43-46]. In our current study we show that TQ decreased lipid content. In agreement with our in vivo results and previously published reports, the increase of HO-1 levels in adipocytes turns large unhealthy adipocytes into small healthy insulin-sensitive adipocytes [47]. In addition, the decrease in pro-inflammatory adipocyte NOV expression, the increase of HO-1 levels and the increased levels of insulin receptor phosphorylation in adipose tissue lead to the normalization of mitochondrial function and a reversal of adipocyte phenotype from an inflammatory to a healthy functional status. Together, these results clearly indicate that activation of the HO-1 antioxidant response is crucial to the beneficial effects of TQ on mitochondrial biogenesis and on the reduction of fission and increase of fusion-associated processes in both adipose and hepatic tissue.

Importantly, in obesity, it is well established that there is association of elevation in HDL and OxHDL in obese animals. Obese mice treated with TQ demonstrated a significant decrease in LDL and OX-LDL levels. LDL oxidation, as well as HDL oxidation, is critical in the development of atherosclerosis and NAFLD has many features in common with cardiovascular disease, including lipid accumulation, macrophage activation and infiltration, and inflammation [48-50]. The activation of Kupffer cells by OX-LDL leads to a rapid release of various inflammatory mediators and signaling molecules such as cytokines, ROS, proteases, and lipid mediators that contribute to hepatic inflammation [51]. Fundamentally, TQ ingestion that result in decrease in OX-LDL and inflammatory molecule, NOV and increase in mitochondrial biogenesis and attenuates liver steatosis and NASH will contribute to an increase in insulin sensitivity and organ protection, indicates the potential of this nutraceutical approach to prevent disease progression in an animal model of metabolic syndrome. TQ intervention that contributes to lower blood pressure, fasting glucose may be beneficial to obese and non-obese subjects, may involve the increase of HO-1. HO-1 induction shown to lower blood pressure in hypertensive and obese animal models [52-55]. The beneficial effects of TQ in the pathogenesis of NAFLD in a murine model of obesity offer a portal into therapeutic approaches to the treatment of this and other obesity-related diseases.

Acknowledgment

This work was supported by National Institutes of Health grant HL139561 (NGA). All authors had full access to the data and take responsibility for its integrity. All authors have read and agree with the manuscript as written. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

References

1. Smith U (2015) Abdominal obesity: a marker of ectopic fat accumulation. J Clin Invest 125: 1790-1792.
2. Byrne CD (2013) Ectopic fat, insulin resistance and non-alcoholic fatty liver disease. Proc Nutr Soc 72: 412-419.
3. Abraham NG, Kappas A (2008) Pharmacological and clinical aspects of heme oxygenase. Pharmacol Rev 60: 79-127.
4. Rosselli M, Lotersztajn S, Vizzutti F, Arena U, Pinzani M, et al. (2014) The metabolic syndrome and chronic liver disease. Curr Pharm Des 20: 5010-5024.
5. Lonardo A, Ballestri S, Marchesini G, Angulo P, Loria P (2015) Nonalcoholic fatty liver disease: a precursor of the metabolic syndrome. Dig Liver Dis 47: 181-190.
6. Dowman JK, Tomlinson JW, Newsome PN (2010) Pathogenesis of non-alcoholic fatty liver disease. QJM 103: 71-83.
7. Kushner RF, Kahan S (2018) Introduction: The State of Obesity in 2017. Med Clin North Am 102: 1-11.
8. Krahenbuhl L, Lang C, Ludes S, Seiler C, Schafer M, et al. (2003) Reduced hepatic glycogen stores in patients with liver cirrhosis. Liver Int 23: 101-109.
9. Estall JL, Ruas JL, Choi CS, Laznik D, Badman M, et al. (2009) PGC-1alpha negatively regulates hepatic FGF21 expression by modulating the heme/Rev-Erb(alpha) axis. Proc Natl Acad Sci U S A 106: 22510-22515.
10. Adams LA, Waters OR, Knuiman MW, Elliott RR, Olynyk JK (2009) NAFLD as a risk factor for the development of diabetes and the metabolic syndrome: an eleven-year follow-up study. Am J Gastroenterol 104: 861-867.
11. Kahn SE, Hull RL, Utzschneider KM (2006) Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature 444: 840-846.
12. Abraham NG, Junge JM, Drummond GS (2016) Translational Significance of Heme Oxygenase in Obesity and Metabolic Syndrome. Trends Pharmacol Sci 37: 17-36.
13. Barbagallo I, Galvano F, Frigiola A, Cappello F, Riccioni G, et al. (2013) Potential therapeutic effects of natural heme oxygenase-1 inducers in cardiovascular diseases. Antioxid Redox Signal 18: 507-521.
14. Chang SH, Barbosa-Tessmann I, Chen C, Kilberg MS, Agarwal A (2002) Glucose deprivation induces heme oxygenase-1 gene expression by a pathway independent of the unfolded protein response. J Biol Chem 277: 1933-1940.
15. Chang SH, Garcia J, Melendez JA, Kilberg MS, Agarwal A (2003) Haem oxygenase 1 gene induction by glucose deprivation is mediated by reactive oxygen species via the mitochondrial electron-transport chain. Biochem J 371: 877-885.
16. Abraham NG, Kushida T, McClung J, Weiss M, Quan S, et al. (2003) Heme oxygenase-1 attenuates glucose-mediated cell growth arrest and apoptosis in human microvessel endothelial cells. Circ Res 93: 507-514.
17. Abraham NG, Kappas A (2005) Heme oxygenase and the cardiovascular-renal system. Free Radic Biol Med 39: 1-25.
18. Ansari AA, Hassan S, Kenne L, Atta UR, Wehler T (1988) Structural studies on a saponin isolated from Nigella sativa. Phytochemistry 27: 3977-3979.
19. Singh S, Das SS, Singh G, Schuff C, de Lampasona MP, et al. (2014) Composition, in vitro antioxidant and antimicrobial activities of essential oil and oleoresins obtained from black cumin seeds (Nigella sativa L.). Biomed Res Int 2014: 918209.
20. Badary OA, Taha RA, Gamal el-Din AM, Abdel-Wahab MH (2003) Thymoquinone is a potent superoxide anion scavenger. Drug Chem Toxicol 26: 87-98.
21. Al Wafai RJ (2013) Nigella sativa and thymoquinone suppress cyclooxygenase-2 and oxidative stress in pancreatic tissue of streptozotocin-induced diabetic rats. Pancreas 42: 841-849.
22. Kanter M, Coskun O, Korkmaz A, Oter S (2004) Effects of Nigella sativa on oxidative stress and beta-cell damage in streptozotocin-induced diabetic rats. Anat Rec A Discov Mol Cell Evol Biol 279: 685-691.
23. Schragenheim J, Bellner L, Cao J, Singh SP, Bamshad D, et al. (2018) EET enhances renal function in obese mice resulting in restoration of HO-1 -Mfn1/2 signaling, and decrease in hypertension through inhibition of sodium chloride co-transporter. Prostaglandins Other Lipid Mediat 137: 30-39.
24. Singh SP, Schragenheim J, Cao J, Falck JR, Abraham NG, et al. (2016) PGC-1 alpha regulates HO-1 expression, mitochondrial dynamics and biogenesis: Role of epoxyeicosatrienoic acid. Prostaglandins Other Lipid Mediat 125: 8-18.
25. Singh SP, McClung JA, Bellner L, Cao J, Waldman M, et al. (2018) CYP-450 epoxygenase derived epoxyeicosatrienoic acid contribute to reversal of heart failure in obesity-induced diabetic cardiomyopathy via PGC-1 alpha activation. Cardiovasc Pharm Open Access 7: 233.
26. Sacerdoti D, Singh SP, Schragenheim J, Bellner L, Vanella L, et al. (2018) Development of NASH in Obese Mice is Confounded by Adipose Tissue Increase in Inflammatory NOV and Oxidative Stress. Int J Hepatol 2018: 3484107.
27. Sardana MK, Kappas A (1987) Dual control mechanism for heme oxygenase: tin(IV)-protoporphyrin potently inhibits enzyme activity while markedly increasing content of enzyme protein in liver. Proc Natl Acad Sci U S A 84: 2464-2468.
28. Singh SP, Grant I, Meissner A, Kappas A, Abraham NG (2017) Ablation of adipose-HO-1 expression increases white fat over beige fat through inhibition of mitochondrial fusion and of PGC1alpha in female mice. Horm Mol Biol Clin Investig 31: 1.
29. Cao J, Singh SP, McClung J, Joseph G, Vanella L, et al. (2017) EET intervention on Wnt1, NOV and HO-1 signaling prevents obesity-induced cardiomyopathy in obese mice. Am J Physiol Heart Circ Physiol 313: H368-H380.
30. Alcala M, Calderon-Dominguez M, Bustos E, Ramos P, Casals N, et al. (2017) Increased inflammation, oxidative stress and mitochondrial respiration in brown adipose tissue from obese mice. Sci Rep 7: 16082.
31. Sears B, Perry M (2015) The role of fatty acids in insulin resistance. Lipids Health Dis 14: 121.
32. Serviddio G, Bellanti F, Vendemiale G (2013) Free radical biology for medicine: learning from nonalcoholic fatty liver disease. Free Radic Biol Med 65: 952-968.
33. Spahis S, Delvin E, Borys JM, Levy E (2017) Oxidative Stress as a Critical Factor in Nonalcoholic Fatty Liver Disease Pathogenesis. Antioxid Redox Signal 26: 519-541.
34. Martinerie C, Garcia M, Do TT, Antoine B, Moldes M, et al. (2016) NOV/CCN3: A New Adipocytokine Involved in Obesity-Associated Insulin Resistance. Diabetes 65: 2502-2515.
35. Pakradouni J, Le GW, Calmel C, Antoine B, Villard E, et al. (2013) Plasma NOV/CCN3 levels are closely associated with obesity in patients with metabolic disorders. PLoS One 8: e66788.
36. Ndisang JF, Jadhav A (2009) Up-regulating the hemeoxygenase system enhances insulin sensitivity and improves glucose metabolism in insulin-resistant diabetes in Goto-Kakizaki rats. Endocrinology 150: 2627-2636.
37. Ndisang JF, Lane N, Jadhav A (2009) Upregulation of the heme oxygenase system ameliorates postprandial and fasting hyperglycemia in type 2 diabetes. Am J Physiol Endocrinol Metab 296: E1029-E1041.
38. Hosick PA, Weeks MF, Hankins MW, Moore KH, Stec DE (2017) Sex-dependent effects of HO-1 Deletion from adipocytes in mice. Int J Mol Sci 18: E611.
39. Hosick PA, Stec DE (2012) Heme oxygenase, a novel target for the treatment of hypertension and obesity? Am J Physiol Regul Integr Comp Physiol 302: R207-R214.
40. Zhang M, Nakamura K, Kageyama S, Lawal AO, Gong KW, et al. (2018) Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion injury. JCI Insight 3: 120596.
41. Vanella L, Sodhi K, Kim DH, Puri N, Maheshwari M, et al. (2013) Increased heme-oxygenase 1 expression decreases adipocyte differentiation and lipid accumulation in mesenchymal stem cells via upregulation of the canonical Wnt signaling cascade. Stem Cell Res Ther 4: 28.
42. Sun K, Kusminski CM, Scherer PE (2011) Adipose tissue remodeling and obesity. J Clin Invest 121: 2094-2101.
43. Waldman M, Bellner L, Vanella L, Schragenheim J, Sodhi K, et al. (2016) Epoxyeicosatrienoic acids regulate adipocyte differentiation of mouse 3T3 cells, via PGC-1alpha activation, which is required for HO-1 expression and increased mitochondrial function. Stem Cells Dev 25: 1084-1094.
44. Rutkowski JM, Stern JH, Scherer PE (2015) The cell biology of fat expansion. J Cell Biol 208: 501-512.
45. Peterson SJ, Vanella L, Bialczak A, Schragenheim J, Li M, et al. (2016) Oxidized HDL and isoprostane exert a potent adipogenic effect on stem cells: where in the lineage? Cell Stem Cells Regen Med 2: 2472-6990.
46. Liu L, Puri N, Raffaele M, Schragenheim J, Singh SP, et al. (2018) Ablation of soluble epoxide hydrolase reprogram white fat to beige-like fat through an increase in mitochondrial integrity, HO-1 -adiponectin in vitro and in vivo. Prostaglandins Other Lipid Mediat 138: 1-8.
47. Abraham NG, Sodhi K, Silvis AM, Vanella L, Favero G, et al. (2014) CYP2J2 targeting to endothelial cells attenuates adiposity and vascular dysfunction in mice fed a high-fat diet by reprogramming adipocyte phenotype. Hypertension 64: 1352-1361.
48. Ampuero J, Ranchal I, Gallego-Duran R, Pareja MJ, Del Campo JA, et al. (2016) Oxidized low-density lipoprotein antibodies/high-density lipoprotein cholesterol ratio is linked to advanced non-alcoholic fatty liver disease lean patients. J Gastroenterol Hepatol 31: 1611-1618.
49. Kaikkonen JE, Kresanov P, Ahotupa M, Jula A, Mikkila V, et al. (2016) Longitudinal study of circulating oxidized LDL and HDL and fatty liver: the Cardiovascular Risk in Young Finns Study. Free Radic Res 50: 396-404.
50. Peterson SJ, Vanella L, Gotlinger K, Jiang H, Bialczak A, et al. (2016) Oxidized HDL is a potent inducer of adipogenesis and causes activation of the Ang-II and 20-HETE systems in human obese females. Prostaglandins Other Lipid Mediat 123: 68-77.
51. Walenbergh SM, Koek GH, Bieghs V, Shiri-Sverdlov R (2013) Non-alcoholic steatohepatitis: the role of oxidized low-density lipoproteins. J Hepatol 58: 801-810.
52. Vera T, Kelsen S, Yanes LL, Reckelhoff JF, Stec DE (2007)HO-1 induction lowers blood pressure and superoxide production in the renal medulla of angiotensin II hypertensive mice. Am J Physiol Regul Integr Comp Physiol 292: R1472-R1478.
53. Vera T, Kelsen S, Stec DE (2008) Kidney-specific induction of heme oxygenase-1 prevents angiotensin II hypertension. Hypertension 52: 660-665.
54. Cao J, Peterson SJ, Sodhi K, Vanella L, Barbagallo I, et al. (2012) Heme oxygenase gene targeting to adipocytes attenuates adiposity and vascular dysfunction in mice fed a high-fat diet. Hypertension 60: 467-475.
55. Burgess A, Li M, Vanella L, Kim DH, Rezzani R, et al. (2010) Adipocyte heme oxygenase-1 induction attenuates metabolic syndrome in both male and female obese mice. Hypertension 56: 1124-1130

Citation: Licari M, Raffaele M, Rosman ZF, Schragenheim J, Bellner L, et al. (2019) Beneficial Effects of Thymoquinone on Metabolic Function and Fatty Liver in a Murine Model of Obesity. J Nutr Food Sci 9: 751. 

Copyright: © 2019 Licari M, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.