Effect of thymoquinone on micronucleus frequency and proinflammatory cytokine secretion in rat liver on high-fat cholesterol diet

Increased catabolism of arginine by arginase is increasingly viewed as an important pathophysiological factor in cardiovascular disease, including atherosclerosis induced by high cholesterol diets. Whereas previous studies have focused primarily on effects of high cholesterol diets on arginase expression and arginine metabolism in specific blood vessels, there is no information regarding the impact of lipid diets on arginase activity or arginine bioavailability at a systemic level. We, therefore, evaluated the effects of high fat (HF) and high fat-high cholesterol (HC) diets on arginase activity in plasma and tissues and on global arginine bioavailability (defined as the ratio of plasma arginine to ornithine + citrulline) in apoE−/− and wild-type C57BL/6J mice. HC and HF diets led to reduced global arginine bioavailability in both strains. The HC diet resulted in significantly elevated plasma arginase in both strains, but the HF diet increased plasma arginase only in apoE−/− mice. Elevated plasma arginase activity correlated closely with increased alanine aminotransferase levels, indicating that liver damage was primarily responsible for elevated plasma arginase. The HC diet, which promotes atherogenesis, also resulted in increased arginase activity and expression of the type II isozyme of arginase in multiple tissues of apoE−/− mice only. These results raise the possibility that systemic changes in arginase activity and global arginine bioavailability may be contributing factors in the initiation and/or progression of cardiovascular disease.

Objective: To clarify the potential therapeutic effects of thymoquinone (TQ) on pancreatic cancer and its gemcitabine (GEM) sensitivity.Methods: The expression levels of hypoxia inducible factor-1α (HIF-1α), collagens (COL1A1, COL3A1, and COL5A1), and transforming growth factor-β1 (TGFβ1) in pancreatic cancer and para-carcinoma tissues were compared using immunohistochemical methods, and their relationships with TNM staging were analyzed. The effects of TQ on apoptosis, migration, invasion, and GEM sensitivity of pancreatic cancer cells were assessed using in vitro and in vivo experiments. Western blot and immunohistochemistry were used to detect the expression levels of HIF-1α, extracellular matrix (ECM) production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins.Results: The expression levels of HIF-1α, COL1A1, COL3A1, COL5A1, and TGFβ1 in pancreatic cancer tissues were significantly higher than those in para-carcinoma tissues and correlated with TNM staging (p < 0.05). TQ and GEM administration inhibited the migration and invasion of the human pancreatic cancer cell line PANC-1 and promoted the apoptosis of PANC-1 cells. The combination of TQ and GEM was more effective than GEM alone. Western blot analysis showed that the expression levels of HIF-1α, ECM production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins were significantly decreased when TQ was used to treat PANC-1 cells (p < 0.05), and the expression levels of these proteins in the TQ + GEM group were significantly more decreased than those in the GEM group. Overexpression or knockdown of HIF-1α in PANC-1 cells showed the same effects as those induced by TQ administration. In vivo experiments showed that in PANC-1 tumor-bearing mice, tumor volume and tumor weight in mice treated with GEM and TQ were significantly lower than those in control or GEM-treated mice, whereas cell apoptosis was significantly increased (p < 0.05). Western blot and immunohistochemistry results showed that the levels of HIF-1α, ECM production pathway-related proteins, and TGFβ/Smad signaling pathway-related proteins in the GEM + TQ treatment group were further decreased compared to the control group or the GEM treatment group (p < 0.05).Conclusion: In pancreatic cancer cells, TQ can promote apoptosis, inhibit migration, invasion, and metastasis, and enhance the sensitivity to GEM. The underlying mechanism may involve the regulation of ECM production through the TGFβ/Smad pathway, in which HIF-1α plays a key role.

The aim was to investigate whether thymoquinone (TQ) attenuates hyperglycemia-induced insulin resistance in experimental type 2 diabetes. Type 2 diabetes mellitus (T2DM) was induced by injection of streptozotocin (STZ, 40mg/kg) in high fat diet (HFD) rats. The levels of glucose, insulin, area under curve (AUC) of glucose, lipid profile parameters, homeostasis model assessment of insulin resistance (HOMA-IR), peroxisome proliferator-activated receptor-γ (PPARγ), and dipeptidyl peptidase peptidase-IV (DPP-IV) were evaluated in HFD + STZ-induced type 2 diabetic rats. TQ treatment significantly reduced elevated levels of glucose, AUC of glucose, insulin, and DPP-IV in diabetic-treated groups. In addition, TQ treatment significantly reduced high levels of triglycerides (TG) and cholesterols (total, low-density and very low-density lipoprotein) accompanied by significant augmentation in high-density lipoprotein (HDL) levels in diabetic-treated groups. However, TQ treatment significantly improved insulin sensitivity in diabetic-treated groups, which was confirmed by increased level of PPARγ and decreased level of HOMA-IR. Molecular docking of TQ exhibited substantial binding affinity with PPARγ and DPP-IV target proteins, which is supported by in vivo results. These results demonstrate that TQ attenuates hyperglycemia-induced insulin resistance by counteracting hyperinsulinemia, improving lipid profile, insulin sensitivity, and inhibiting DPP-IV. PRACTICAL APPLICATIONS: T2DM results in relentless hyperglycemia which eventually progress to a state of insulin resistance. TQ is an active principle compound found in Nigella sative seed, having myriad of traditional medicinal values. Administration of TQ significantly prevented hyperglycemia, hyperinsulinemia, hyperlipidemia, insulin resistance, and inhibited DPP-IV in experimental type 2 diabetes. The in vivo results are also supported by molecular docking study of PPARγ and DPP-IV target proteins. Thus, we hypothesize that TQ can be used as an alternative natural drug in the management of hyperglycemia-induced insulin resistance in T2DM.

Nonalcoholic steatohepatitis (NAFLD) is a progressive form of liver disease that leads to advanced fibrosis. The present study was designed to assess the hepatoprotective effect of thymoquinone (TQ) on liver functions, insulin resistance, and PPAR-γ expression in NAFLD. Rats were divided into two main groups: one fed with normal rat chow diet and the other with high-fat high-cholesterol diet group for 6 weeks. Every group was subdivided into three subgroups (n = 8): treated with saline, low dose TQ (10 mg/kg), high dose TQ (20 mg/kg). High fat high cholesterol diet caused marked liver damage as noted in histopathology and significant increase in liver index, liver enzymes. There was significant increase in the insulin resistance, serum cholesterol, triglyceride, PPAR-γ gene overexpression with significant decrease in HDL. Additionally, oxidative stress increased by measuring MDA associated with significant decrease in serum total antioxidant capacity. As markers of inflammation, hepatic TNF-α was significantly increased with decrease in IL10. Further, there was increase in BAX protein with decrease in Bcl as compared to control group. This model of 6 weeks high-fat high-cholesterol diet showed minimal fibrosis as noticed by increase MMP2 and Masson trichrome satin. Co-treatment with TQ improved all previous parameters. High dose was more effective, although mostly non-statistically significant. TQ may have a promising agent to improve hepatic steatosis, oxidative stress; inflammatory, apoptotic status, fibrosis and so prevent liver damage in patients with NAFLD. Although PPAR-γ was significantly under-expressed by TQ, insulin resistance was improved significantly suggesting a role of liver damage.

蒟蒻纖維對高脂肪飲食相關大腸癌危險因子之調節作用-臨床、動物及初步細胞研究

Thymoquinone-rich fraction nanoemulsion (TQRFNE) decreases Aβ40 and Aβ42 levels by modulating APP processing, up-regulating IDE and LRP1, and down-regulating BACE1 and RAGE in response to high fat/cholesterol diet-induced rats

High-fat diet (HFD) is a major problem causing neuronal damage. Thymoquinone (TQ) could regulate oxidative stress and the inflammatory process. Hence, the present study elucidated the significant role of TQ on oxidative stress, inflammation, as well as morphological changes in the cerebellum of rats with HFD. Rats were divided into three groups as (1) control, (2) saturated HFD for eight weeks and (3) HFD supplementation (four weeks) followed by TQ 300 mg/kg/day treated (four weeks). After treatment, blood samples were collected to measure oxidative stress markers glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and inflammatory cytokines. Furthermore, neuronal morphological changes were also observed in the cerebellum of the rats. HFD rats show higher body weight (286.5 ± 7.4 g) as compared with the control group (224.67 ± 1.78 g). TQ treatment significantly (p < 0.05) lowered the body weight (225.83 ± 13.15 g). TQ produced a significant (p < 0.05) reduction in cholesterol, triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL). The antioxidative enzymes significantly reduced in HFD rats (GSH, 1.46 ± 0.36 mol/L and SOD, 99.13 ± 5.41 µmol/mL) as compared with the control group (GSH, 6.25 ± 0.36 mol/L and SOD, 159.67 ± 10.67 µmol/mL). MDA was increased significantly in HFD rats (2.05 ± 0.25 nmol/L) compared to the control group (0.695 ± 0.11 nmol/L). Surprisingly, treatment with TQ could improve the level of GSH, MDA, and SOD. TQ treatment significantly (p < 0.05) reduced the inflammatory markers as compared with HFD alone. TQ treatment minimizes neuronal damage as well as reduces inflammation and improves antioxidant enzymes. TQ can be considered as a promising agent in preventing the neuronal morphological changes in the cerebellum of obese populations.

High-fat diet (HFD) is a major problem causing neuronal damage. However, the mechanisms and potential therapeutic targets have not been evaluated yet. Thymoquinone (TQ) could regulate oxidative stress and inflammatory process. Hence, the present study elucidated the significant role of TQ on oxidative stress, inflammation as well as morphological changes in the cerebellum of rats with HFD. The present study establishes the role of TQ on oxidative stress, anti-inflammatory process and morphological changes in the cerebellum following high-fat diet supplementation in rats. Rats were divided into three groups as (1) Control, (2) HFD (6 mg/day) for eight weeks and (3) HFD supplementation (4 weeks) followed TQ 300 mg/kg/day treated (4 weeks). After treatment, body weight and serum cholesterol profile were measured. Also, blood samples were collected to measure oxidative stress markers glutathione (GSH), superoxide dismutase (SOD) and inflammatory cytokines. Furthermore, neuronal morphological changes were also observed in the cerebellum of the rats. HFD rats show higher in body weight as compared with the control group. TQ treatment significantly (p<0.05) lowers the body weight with significant (p<0.05) reduction in cholesterol, triglycerides, high-density lipoprotein (HDL) and low-density lipoprotein (LDL). The anti-oxidative enzymes significantly reduced in HFD rats as compared with the control group. Surprisingly, treatment with TQ could improve the level of GSH and SOD. TQ treatment significantly (p<0.05) reduced the inflammatory markers as compared with HFD alone. The histological study revealed that neuronal damage was prevented in the cerebellum following treatment of TQ. TQ treatment minimizes neuronal damage as well as reduces inflammation and improves anti-oxidant enzymes.

Both lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity, a biomarker of inflammation, and concentration of its primary associated lipoprotein, LDL, are correlated with adverse coronary outcomes. We previously reported a quantitative trait locus (QTL) corresponding to HSA2p24.3-p23.2 with pleiotropic effects on Lp-PLA(2) activity and LDL-cholesterol (LDL-C) concentration in baboons fed a basal diet. Here, our goal was to locate pleiotropic QTLs influencing both traits in the same baboons fed a high-cholesterol, high-fat (HCHF) diet, and to assess whether shared genetic effects on these traits differ between diets. We assayed Lp-PLA(2) activity and LDL-C concentration in 683 baboons fed the HCHF diet. We used a bivariate maximum likelihood-based variance components approach in whole-genome linkage screens to locate a QTL [logarithm of odds (LOD) = 3.13, genome-wide P = 0.019] corresponding to HSA19q12-q13.2 with pleiotropic effects on Lp-PLA(2) activity and LDL-C levels in the HCHF diet. We additionally found significant evidence of genetic variance in response to diet for Lp-PLA(2) activity (P = 0.0017) and for LDL-C concentration (P = 0.00001), revealing a contribution of genotype-by-diet interaction to covariation in these two traits. We conclude that the pleiotropic QTLs detected at 2p24.3-p23.2 and 19q12-q13.2 on the basal and HCHF diets, respectively, exert diet-specific effects on covariation in Lp-PLA(2) activity and LDL-C concentration.

The current study investigated the mitigating effects of thymoquinone (TQ) against high-fat diet (HFD)-mediated brain injury, cognitive and memory impairment, and the underlying mechanisms. Twenty-four adult male Wistar rats were divided into four groups of six rats each. Rats were fed HFD for 12 weeks to induce obesity. On the 9th week, TQ was administered orally to obese rats for four weeks. The effects of TQ were estimated by neurobehavioral testing, biochemical analysis, DNA damage, molecular docking, and histopathological examination of brains and visceral fat. TQ reduced body weight, body weight gain and adipocyte size, improved hyperlipidemia, and normalized the levels of leptin and adiponectin. TQ significantly attenuated the increase in HbA1c percent and insulin resistance. TQ decreased the accumulation of amyloid-β and tau proteins and improved the levels of neurotransmitters in the brains of obese rats. TQ-treated obese rats showed improved thickening of the pyramidal cell layer in the hippocampus and improved cognitive function and memory impairments. Molecular docking analysis indicated that TQ exhibited a marked affinity for inhibiting binding sites of tau and amyloid-β proteins. Furthermore, TQ controlled oxidative stress and enhanced the Nrf2 expression in the pyramidal cell layer and the activity of HO-1, SOD, and CAT in the brain. The restoration of redox balance by TQ was associated with normalization of inflammatory indicators and alleviation of DNA damage in the brains of HFD-treated animals. These changes contributed to the normalization of mitochondrial apoptotic pathway mediators (p53, Bcl-2, Bax, and caspase-3) and maintained the histological structure of the hippocampus. In conclusion, TQ attenuated brain injury, cognitive impairment, and memory deficit with improvement of body weight gain and metabolic status in obese rats through interrelated biological processes, including regulation of redox balance, inflammatory response, neurotransmitter equilibrium, and regression of DNA injury and apoptosis.

ABSTRACTWomen with obesity are more likely to have a complicated reproductive life. Insulin resistance and metabolic dysfunction are associated with obesity. Thymoquinone (TQ) is a well-known antioxidant, considered to be an AMPK-activator. The goal of this work was to investigate the ability of TQ to improve fertility and lactation and clarify the possible mechanism. Female C57BL/6 mice were subjected to High Fat Diet (HFD) supplemented with TQ (10% pmm) and TQ (20% pmm). Histopathological examination was conducted on mammary and ovarian samples. Metabolic and oxidant status was evaluated, and qRT-PCR analysis was performed to verify AMPK/PGC1α/SIRT1 metabolic pathway activity. The present study reports positive effects of TQ on ovarian metabolic function in a dose-dependent manner. TQ showed its positive effects on mammary gland metabolic function at lower dose. This is the first study that indicates these dose related impacts of TQ.Abbreviations: AKT1: serine-threonine protein kinase 1; AMPK: 5′ AMP-activated protein kinase; CAT: catalase; CON: control; FBS: fasting blood sugar; GLUT1: glucose transporter 1; GSH: reduced glutathione; GSSG: Glutathione disulfide; HE: hematoxylin and eosin stains; HDL: high-density lipoprotein; HFD: high fat diet; IL-6: interleukin-6; K18: keratin 18; LD: lactation day; LDL: low-density lipoprotein; LKB1: serine-threonine liver kinase B1; MDA: malondialdehyde; mTOR: the mammalian target of rapamycin; NAD: nicotinamide adenine dinucleotide; NADH: nicotinamide adenine dinucleotide phosphate; NS: nigella sativa; PBS: phosphate-buffered saline; PGC1α: peroxisome proliferator-activated receptor gamma coactivator 1-alpha; SIRT1: sirtuin 1; SOD: superoxide dismutase; T-AOC: total antioxidants; TFAM: transcription factor A mitochondrial; TG: triglycerides; TNF-α: tumor necrosis factor-α; TQ: thymoquinone; TQ10: high fat diet + thymoquinone 10% ppm; TQ20: high fat diet + thymoquinone 20% ppm; UCP2: uncoupling Protein 2.

1. Ferret on a high fat diet accumulated large amounts of fat in its liver and had blood acetoacetate and beta-hydroxybutyrate concentrations 250 and 375% of those in control animals. 2. The high fat diet alone increased ferret liver microsomal 7-ethoxyresorufin O-deethylase (EROD) activity by 90%, but had no effect on 7-methoxy-, 7-pentoxy-, or 7-benzyloxy-resorufin, O-dealkylase activities. Administration of 3-methylcholanthrene (MC) increased only liver EROD activity, by 5- to 6-fold, in ferret on both high fat and control diets. Induction of EROD, but not MROD activity, in ferret on the high fat diet indicates that P4501A1, but not P4501A2, is induced. 3. Activation of 3H-paracetamol, measured by covalent tissue binding to ferret liver microsomal fractions, was increased three-fold in ferret on the high fat diet, nine-fold by MC administered to ferret on a control diet, and 13-fold by MC given to ferret on the high fat diet. Similar results were obtained with activation of the cooked-food amine, Glu-P-1, by ferret liver microsomes. 4. Western blots with antibodies to rat liver P450s showed that ferret liver contains proteins orthologous with rat liver P4504A1 and bifunctional protein. However, whereas clofibrate, similar to high fat diets, induced these two proteins in rat liver, no increase of these proteins occurred in liver of ferret fed a high fat diet. Western blots also showed that ferret liver contains no P4501A1 or 1A2, and although these two proteins were induced by MC, no induction occurred when ferret was fed the high fat diet alone. Ferret liver microsomes also contain a protein recognized by rat anti-P4502E1 but of a lower molecular weight. 5. Immunosorbent (ELISA) analyses of ferret liver for P4501A1 and 4A1 showed that the high fat diet increased a protein orthologous to rat P4501A1 but did not increase any protein orthologous to rat P4504A1. 6. These findings indicate that the high fat diet does not induce ferret liver bifunctional protein or P4504A1 enzyme protein, but may enhance liver P4501A1 and 1A2 activities through the hyperketonaemia resulting from the high dietary fat. The conflicting P450 results, namely Glu-P-1 activation but no MROD activity for P4501A2, high EROD activity and ELISA quantification of P4501A1, but no positive Western blot, are probably due to differences in substrate specificity and immunological characteristics between rat and ferret enzymes.

High-fat diet is one of the most imperative risk factors for cardiovascular disorders. Thymoquinone (TQ) is one of the active pharmacological components of Nigella sativa (black cumin). Salvia officinalis L. (sage) has been demonstrated to have diverse pharmacological actions. The main objective of this study was to determine the effects of sage and TQ combination on hyperglycemia, oxidative stress, blood pressure, and lipid profile in rats fed with a high-fat diet (HFD). Wistar male rats were divided into five groups; normal diet (ND) and HFD, in which rats were fed with a normal diet or HFD for 10 weeks, respectively. In HFD + sage group, animals were administered sage essential oil (0.052 ml/kg) orally along with HFD. In HFD + TQ group, rats were administered TQ (50 mg/kg) orally with HFD. In HF + sage + TQ group, animals received sage + TQ along with HFD. Blood glucose (BGL) and Fast serum insulin (FSI) levels, oral glucose tolerance test, blood pressure, liver function tests, plasma, and hepatic oxidative stress markers, antioxidant enzymes, and glutathione content, and lipid profile were measured. Sage and TQ combination decreased the final body weight, weight gain, BGL, FSI, and Homeostasis Model Assessment-Insulin Resistance (HOMA-IR). The combination also lowered systolic and diastolic arterial pressures and liver function enzymes. The combination deterred lipid peroxidation, advanced protein oxidation product, and nitric oxide amplification, as well as restoring the superoxide dismutase, catalase activities, and glutathione content in plasma and hepatic tissue. Sage and TQ combination reduced the plasma total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL) levels and amplified high-density lipoprotein (HDL). The results of the current study verified that sage essential oil, together with TQ exhibited hypoglycemic, hypolipidemic, and antioxidant actions and thus could be a valuable addition to diabetes management.

Relationships of plasma and hepatic variables with rates of plasma low-density lipoprotein apolipoprotein B metabolism in baboons fed low- and high-fat diets

Lowering serum cholesterol level by feeding a 40% ethanol-eluted fraction from HP-20 resin treated with hot water extract of adzuki beans ( Vigna angularis) to rats fed a high-fat cholesterol diet