International Journal of Molecular Sciences Article Thymoquinone and Curcumin Defeat Aging-Associated Oxidative Alterations Induced by D-Galactose in Rats’ Brain and Heart Ali H. El-Far 1,* , Yaser H. A. Elewa 2,3,* , Elsayeda-Zeinab A. Abdelfattah 4, Abdel-Wahab A. Alsenosy 1 , Mustafa S. Atta 5 , Khalid M. Abou-Zeid 4, Soad K. Al Jaouni 6, Shaker A. Mousa 7 and Ahmed E. Noreldin 8 1 Department of Biochemistry, Faculty of Veterinary Medicine, Damanhour University, Damanhour 22511, Egypt; [email protected] 2 Department of Histology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt 3 Laboratory of Anatomy, Faculty of Veterinary Medicine, Basic Veterinary Sciences, Hokkaido University, Sapporo 060-0818, Japan 4 Animal Care Unit, Medical Research Institute, Alexandria University, Alexandria 21544, Egypt; [email protected] (E.-Z.A.A.); [email protected] (K.M.A.-Z.) 5 Department of Physiology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh 33516, Egypt; [email protected] 6 Department of Hematology/Pediatric Oncology, Yousef Abdulatif Jameel Scientific Chair of Prophetic Medicine Application, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia; [email protected] 7 Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Rensselaer, NY 12144, USA; [email protected] Citation: El-Far, A.H.; Elewa, Y.H.A.; 8 Histology and Cytology Department, Faculty of Veterinary Medicine, Damanhour University, Abdelfattah, E.-Z.A.; Alsenosy, Damanhour 22511, Egypt; [email protected] A.-W.A.; Atta, M.S.; Abou-Zeid, K.M.; * Correspondence: [email protected] (A.H.E.-F.); [email protected] (Y.H.A.E.) Al Jaouni, S.K.; Mousa, S.A.; Noreldin, A.E. Thymoquinone and Curcumin Abstract: D-galactose (D-gal) administration causes oxidative disorder and is widely utilized in Defeat Aging-Associated Oxidative aging animal models. Therefore, we subcutaneously injected D-gal at 200 mg/kg BW dose to assess Alterations Induced by D-Galactose the potential preventive effect of thymoquinone (TQ) and curcumin (Cur) against the oxidative in Rats’ Brain and Heart. Int. J. Mol. alterations induced by D-gal. Other than the control, vehicle, and D-gal groups, the TQ and Cur Sci. 2021, 22, 6839. https://doi.org/ treated groups were orally supplemented at 20 mg/kg BW of each alone or combined. TQ and Cur 10.3390/ijms22136839 effectively suppressed the oxidative alterations induced by D-gal in brain and heart tissues. The TQ Academic Editors: David Vauzour and Cur combination significantly decreased the elevated necrosis in the brain and heart by D-gal. and Cristina Angeloni It significantly reduced brain caspase 3, calbindin, and calcium-binding adapter molecule 1 (IBA1), heart caspase 3, and BCL2. Expression of mRNA of the brain and heart TP53, p21, Bax, and CASP-3 Received: 25 May 2021 were significantly downregulated in the TQ and Cur combination group along with upregulation of Accepted: 18 June 2021 BCL2 in comparison with the D-gal group. Data suggested that the TQ and Cur combination is a Published: 25 June 2021 promising approach in aging prevention. Publisher’s Note: MDPI stays neutral Keywords: thymoquinone; curcumin; D-galactose; oxidative stress; anti-aging with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction Aging is a deteriorative process that occurs mainly due to oxidative stress, leading to numerous oxidative stress-associated diseases because of the accumulation of reactive Copyright: © 2021 by the authors. oxygen species (ROS) and reduced antioxidant capability [1,2]. A D-galactose (D-gal)- Licensee MDPI, Basel, Switzerland. induced aging model is a commonly utilized model to investigate anti-aging drugs [3]. This article is an open access article When D-gal accumulates in the body, it can react with the free amines of amino acids in distributed under the terms and proteins and peptides, forming advanced glycation end products (AGEs) [4]. Consequently, conditions of the Creative Commons AGEs interact with specific receptors (RAGE) in many cell types and induce the activation Attribution (CC BY) license (https:// of the downstream nuclear factor kappa-B (NF-κB), and other signaling pathways, resulting creativecommons.org/licenses/by/ in ROS generation, which could accelerate the aging process [4,5]. The elevated ROS and 4.0/). Int. J. Mol. Sci. 2021, 22, 6839. https://doi.org/10.3390/ijms22136839 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 22 activation of the downstream nuclear factor kappa-B (NF-κB), and other signaling path- ways, resulting in ROS generation, which could accelerate the aging process [4,5]. The elevated ROS and reactive nitrogen species (RNS), including superoxide anion and nitric oxide, lead to cellular damages in protein, lipid, and DNA that are able to favor the devel- opment of different diseases, including tumors, neurodegenerative disorders, aging, and an inflammatory processes [6–9]. Int. J. Mol. Sci. 2021, 22, 6839 Natural compounds act as preventive antioxidant agents against different age-asso-2 of 22 ciated alterations [10]. Thymoquinone (TQ) is an active compound of Nigella sativa seeds with diverse biological activities such as antioxidant, antitoxic, anti-inflammatory, antidi- abetic, and anticancer activities (Figure 1) [11–15]. TQ showed a positive elevation in liver reactive nitrogen species (RNS), including superoxide anion and nitric oxide, lead to cellular glutathione levels and enhancement of total oxidant status of blood in a rat model with damages in protein, lipid, and DNA that are able to favor the development of different carbon tetrachloride-induced hepatotoxicity [16]. Also, TQ protects cardiac muscles diseases, including tumors, neurodegenerative disorders, aging, and an inflammatory against diabetic oxidative stress by upregulation of nuclear factor-erythroid-2-related fac- processes [6–9]. tor 2 (Nrf2), which improved the antioxidant potential of the cardiac muscles and allevi- Natural compounds act as preventive antioxidant agents against different age- ated the inflammatory process [12]. Moreover, TQ alleviates the testicular damage in dia- associated alterations [10]. Thymoquinone (TQ) is an active compound of Nigella sativa seedsbetic rats with through diverse its biological powerful activities antioxidant such and as antioxidant, hypoglycemic antitoxic, effects anti-inflammatory,[11]. Additionally, antidiabetic,TQ shows a regenerative and anticancer potential activities for (Figure treating1)[ damaged11–15]. TQ periph showederal anerves positive [17]. elevation in liverCurcumin glutathione (Cur) levels is anda yellow enhancement pigment obtained of total oxidant from Curcuma status of longa blood, commonly in a rat model used withas a spice carbon and tetrachloride-induced food-coloring agent hepatotoxicity (Figure 1). It [has16]. preventive Also, TQ protects or putative cardiac therapeutic muscles againstproperties diabetic because oxidative of its anti-inflammatory, stress by upregulation antioxidant, of nuclear anti-aging, factor-erythroid-2-related and anticancer poten- factor 2tial (Nrf2), [18–24]. which improved the antioxidant potential of the cardiac muscles and alleviated the inflammatoryFor the antioxidant process potential [12]. Moreover, of both TQTQ alleviates and Cur, thewe testicularinvestigated damage their inanti-aging diabetic ratspotential through either its alone powerful or in antioxidant combination and against hypoglycemic the oxidative effects alterations [11]. Additionally, in rats’ brains TQ showsand hearts a regenerative induced by potential the D-gal-aging for treating model. damaged peripheral nerves [17]. FigureFigure 1.1. ChemicalChemical Structure.Structure. ((AA)) thymoquinonethymoquinone (PubChem(PubChem CID;CID; 10281).10281). ((BB)) curcumincurcumin (PubChem(PubChem CID;CID; 969516).969516). 2. ResultsCurcumin (Cur) is a yellow pigment obtained from Curcuma longa, commonly used as a spice and food-coloring agent (Figure1). It has preventive or putative therapeu- 2.1. Biochemical Parameters tic properties because of its anti-inflammatory, antioxidant, anti-aging, and anticancer potentialFigure [18 2– 24revealed]. no significant changes in serum glucose and creatinine levels and alanineFor aminotransferase the antioxidant potential (ALT, EC of 2.6.1.2) both TQactivity and Cur,between we investigatedall groups. In their contrast, anti-aging rats in potentialthe D-gal+TQ+Cur either alone group or in exhibited combination a significant against the reduction oxidative in alterations aspartatein aminotransferase rats’ brains and hearts(AST, EC induced 2.6.1.1) by (p the < 0.05) D-gal-aging activity model.and urea (p < 0.05), and uric acid (p < 0.001) level com- pared with the D-gal. D-gal+TQ and D-gal+Cur groups also revealed a marked lower uric 2.acid Results than the control, vehicle, and D-gal groups. 2.1. Biochemical Parameters Figure2 revealed no significant changes in serum glucose and creatinine levels and alanine aminotransferase (ALT, EC 2.6.1.2) activity between all groups. In contrast, rats in the D-gal+TQ+Cur group exhibited a significant reduction in aspartate aminotransferase (AST, EC 2.6.1.1) (p < 0.05) activity and urea (p < 0.05), and uric acid (p < 0.001) level compared with the D-gal. D-gal+TQ and D-gal+Cur groups also revealed a marked lower uric acid than the control, vehicle, and D-gal groups. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 22 Int. J. Mol. Sci. 2021, 22, 6839 3 of 22 Figure 2. Serum biochemical parameters. (A) glucose (mg/dL). (B) ALT (U/L). (C) AST (U/L). (D) Figureurea (mg/dL).2. Serum (biochemicalE) creatinine parameters. (mg/dL). (F(A)) uric glucose acid (mg/dL). ( DataB) ALT were (U/L). analyzed (C) AST with (U/L). one-way (D) ureaANOVA, (mg/dL). followed (E) creatinine by Tukey’s (mg/dL). multiple (F) comparison uric acid (mg/dL). test. * p <Data 0.05, were ** p < analyzed 0.01, and with *** p <one-way 0.001 vs. ANOVA,control. followed+ p < 0.05, by++ Tukey’sp < 0.01, multiple and +++ comparisonp < 0.001 vs.