Pharmacognosy Magazine

: 2016  |  Volume : 12  |  Issue : 45  |  Page : 76--81

Protective mechanisms of thymoquinone on methotrexate-induced intestinal toxicity in rats

Azza A El-Sheikh1, Mohamed A Morsy2, Azza H Hamouda3,  
1 Department of Pharmacology, Faculty of Medicine, Minia University, El-Minia 61511, Egypt
2 Department of Pharmacology, Faculty of Medicine, Minia University, El-Minia 61511, Egypt; Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa 31982, Saudi Arabia
3 Department of Histology, Faculty of Medicine, Minia University, El-Minia 61511, Egypt

Correspondence Address:
Mohamed A Morsy
Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, 31982 Al-Ahsa, Saudi Arabia


Background: Intestinal toxicity is a serious side effect in methotrexate (MTX) chemotherapy. Objective: To investigate the mechanisms by which the anticancer drug MTX-induced intestinal damage could be prevented by thymoquinone (TQ), an active ingredient of Nigella sativa. Materials and Methods: TQ was given orally for 10 days, and MTX toxicity was induced at the end of day 3 of the experiment, with or without TQ pretreatment. Results: MTX caused intestinal damage, represented by distortion in normal intestinal histological structure, with significant oxidative stress, exhibited as decrease in reduced glutathione concentration and catalase activity, along with significant increase in malondialdehyde level compared to control group. MTX also caused nitrosative stress evident by increased intestinal nitric oxide (NO) level, with up-regulation of inducible NO synthase expression shown in immunohistochemical staining. Furthermore, MTX caused inflammatory effects as evident by up-regulation of intestinal necrosis factor-kappa beta and cyclooxygenase-2 expressions, which were confirmed by increased intestinal tumor necrosis factor-alpha level via enzyme-linked immunosorbent assay. Moreover, MTX caused apoptotic effect, as it up-regulated intestinal caspase 3 expression. Concomitant TQ significantly reversed the MTX-induced intestinal toxic effects by reversing intestinal microscopic damage, as well as significantly improving oxidative/nitrosative stress, inflammatory and apoptotic markers tested compared to MTX alone. Conclusion: TQ may possess beneficial intestinal protective effects as an adjuvant co-drug against MTX intestinal toxicity during cancer chemotherapy. TQ protection is conferred via antioxidant, anti-nitrosative, anti-inflammatory, and anti-apoptotic mechanisms.

How to cite this article:
El-Sheikh AA, Morsy MA, Hamouda AH. Protective mechanisms of thymoquinone on methotrexate-induced intestinal toxicity in rats.Phcog Mag 2016;12:76-81

How to cite this URL:
El-Sheikh AA, Morsy MA, Hamouda AH. Protective mechanisms of thymoquinone on methotrexate-induced intestinal toxicity in rats. Phcog Mag [serial online] 2016 [cited 2022 Aug 19 ];12:76-81
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Full Text


Methotrexate induces oxidative and nitrosative stress in intestinal tissues Methotrexate also initiates inflammatory and apoptotic intestinal injury Thymoquinone co-administration ameliorates methotrexate-induced intestinal toxicity Thymoquinone has antioxidative, anti-nitrosative, anti-inflammatory, and anti-apoptotic mechanisms.



For more than 60 years, methotrexate (MTX) has been successfully used in treating various malignancies and autoimmune disorders.[1] Being a structural analog of folic acid, MTX inhibits folate metabolism, via interference with dihydrofolate reductase, which leads to suppression of synthesis of nucleic acid precursors; purine and pyrimidine. Unfortunately, the curative potential of MTX is sometimes accompanied by morbid multi-organ toxicity.[2] MTX may induce intestinal toxicity that might end fatally even at low MTX dosages used in the treatment of rheumatoid arthritis.[3] The mechanisms contributing to intestinal toxicity are independent of folate metabolism and involve modifying cellular metabolic processes through altering antioxidant, anti-inflammatory, and apoptotic pathways.[1] Several attempts have been made to ameliorate MTX-induced intestinal damage.[4],[5],[6],[7] However, the outcomes were not completely satisfactory.

Thymoquinone (TQ; 2-isopropyl-5-methyl-1,4-benzoquinone) is the major bioactive component of the black seed Nigella sativa (family Ranunculaceae), which is considered a miracle healing herb in the middle and far East for a wide range of diseases.[8] This is due to the potent antioxidant, anti-inflammatory, and anti-apoptotic effects of TQ. Indeed, TQ has been shown to protect against experimental colitis.[9] In addition, TQ ameliorates inflammatory response to intestinal obstruction.[10] Interestingly, at higher doses, TQ might possess pro-oxidant cytotoxic effects that might explain its potential anticancer activity.[11] In combination with MTX, TQ was able to ameliorate MTX-induced testicular damage.[12] The aim of the current study is to investigate the possible intestinal protective effect of TQ against MTX-induced toxicity in rats and explore the mechanisms involved.

 Materials and Methods


TQ was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA), MTX from Minapharm Pharmaceuticals (Egypt), and tumor necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay (ELISA) kit from Wkea Med Supplies Corporation (China). Kits for examining reduced glutathione (GSH) and catalase were purchased from Biodiagnostic (Giza, Egypt). The ready-to-use inducible nitric oxide (NO) synthase (iNOS), nuclear factor-κB (NF-κB/p65), cyclooxygenase-2 (COX-2), and caspase 3 rabbit polyclonal antibodies were purchased from Thermo Fisher Scientific Inc./Lab Vision (Fremont, CA, USA).

Experimental design

Thirty-two adult male rats of 180–220 g weight were purchased from the National Research Center (Giza, Egypt). All animal care and experimental procedures were in accordance with European (EU) directive 2010/63/EU. Rats were housed as four rats per cage in the standard animal facility during the whole experiment, where they had free access to commercial laboratory chow and tap water. Animals were left to acclimatize for 2 weeks before the start of the experiment. Afterward, animals were weighed and divided into four groups (n = 8 each). TQ-treated group received a single daily oral dose of 10 mg/kg/day TQ by gastric gavage for ten consecutive days.[13] MTX-treated group received a single i.p. dose of 20 mg/kg MTX [14] at the end of day 3 of the experiment. Combined MTX/TQ-treated group received both MTX and TQ treatments as previously indicated. Untreated group served as control.

Sample preparation

After 7 days of MTX injection, total body weights of rats were recorded at the end of the experiment. Rats were sacrificed and venous blood samples were collected from the jugular vein, centrifuged at 5000 rpm for 15 min. Serum was then collected and stored at −80°C until used. The small intestine was removed and the jejunoileal segment (10 cm from the Treitz ligament till 10 cm from ileocecal junction) was divided into two sections. One jejunoileal section had its mucosa scraped off, snap frozen in liquid nitrogen, and kept at −80°C until further use. The second section was fixed in 10% formalin and embedded in paraffin for histopathological and immunohistochemical examinations. Mucosal samples were homogenized in 20% w/v ice-cold phosphate buffer (0.01 M, pH 7.4). The homogenate was centrifuged at 3000 rpm for 20 min and the supernatant was aliquoted to avoid sample thawing and refreezing, and was kept at −80°C until used.

Evaluation of intestinal tissue oxidative stress markers

Biochemical oxidative stress markers were determined in intestinal mucosal tissue homogenate, where GSH concentration, catalase activity, and lipid peroxide content were evaluated. Spectrophotometric kits were used for the assessment of GSH level and catalase activity and the results were expressed as μmol/g tissue and unit/g tissue, respectively. Tissue content of lipid peroxides was determined by biochemical assessment of thiobarbituric acid reacting substance through spectrophotometric measurement of color at 535 nm.[15] The results were expressed as equivalents of malondialdehyde (MDA) in tissue homogenate in nmol/g tissue.

Assessment of nitrosative stress marker and tumor necrosis factor-alpha in intestinal tissue homogenate

For the assessment of nitrosative stress in rat intestinal mucosal homogenate, the stable oxidation end products of NO, nitrite, and nitrate were used as an index of NO production, as NO has a half-life of only a few seconds, being readily oxidized to nitrite then to nitrate. The method used was based on Griess reaction,[16] which depends on conversion of nitrate into nitrite by copperized cadmium granules, then measuring the total nitrites spectrophotometrically at 540 nm. Results were expressed as nmol/100 mg tissue. TNF-α was determined according to ELISA kit manufacturer's instructions.

Histopathological and immunohistochemical examination

Five µm thick paraffin sections of intestinal specimens were prepared and then routinely stained with hematoxylin and eosin dyes. Stained slides were microscopically analyzed using light microscopy. For immunohistochemical staining, sections were fixed at 65°C for 1 h. Trilogy pretreatment (deparaffinization, rehydration, and antigen unmasking) was used to enhance standardization of the pretreatment step and produce more consistent results. The sections were incubated with ready-to-use rabbit polyclonal antibodies against iNOS, NF-κB, COX-2, and caspase 3. After applying the antibodies, slides were incubated overnight at 4°C followed by 20 min of PolyHRP enzyme conjugation. Afterward, diaminobenzidine chromogen was applied for 2 min, and then rinsed, followed by counterstaining with Mayer hematoxylin before examination under the light microscope. Using image J 1.41 (freeware;, the immunopositive cells and total number of cells in a field was calculated.[17] Results were the average of counting three sections from each rat and were expressed as percent of immunopositive cells compared to total number of cells.

Statistical analysis

The data were analyzed by one-way ANOVA followed by Dunnett Multiple Comparison Test. The values are represented as means ± standard error of mean. All statistical analyses were done using GraphPad Prism version 5.00 (San Diego, CA, USA). The differences were considered significant when P < 0.05.


Effect of thymoquinone on intestinal histopathology in methotrexate-treated rats

Intestinal histopathological examination revealed that control and TQ groups had normal structure of villi and crypts [Figure 1]a and [Figure 1]b, respectively]. MTX-treated group, on the other hand, presented with degenerated villi and flattened crypts, with focal loss of intestinal epithelial cell lining [Figure 1]c. Treatment with MTX/TQ improved intestinal histology, with only mild shortening of villi [Figure 1]d.{Figure 1}

Effect of thymoquinone on oxidative stress markers in methotrexate-treated rat intestine

GSH concentration, catalase activity, and MDA level were determined as markers of oxidative stress in intestinal mucosa. MTX-treated group showed a significant decrease in GSH concentration and catalase activity compared with untreated control [Table 1]. Concomitant treatment with MTX and TQ increased intestinal GSH and catalase values to levels statistically higher than the MTX-treated group. On the other hand, intestinal MDA levels increased in the MTX-treated group compared to control. This increase was reversed by combined treatment with MTX/TQ which showed significantly lower levels of MDA compared to the group treated with MTX alone.{Table 1}

Effect of thymoquinone on nitrosative stress markers in methotrexate-treated rat intestine

Total nitrite/nitrate levels, as well as expression of iNOS, were assessed as indicators of nitrosative stress. Total nitrite/nitrate levels increased in the MTX-treated group compared to control, which was reversed in MTX/TQ-treated group, which showed significantly lower levels compared to the group treated with MTX alone [Table 1]. Similarly, TQ-treated group did not show any significant difference in iNOS intestinal expression [Figure 2]b compared to control [Figure 2]a, while MTX treatment caused a significant increase in expression [Figure 2]c. Pretreatment with TQ prior to MTX significantly decreased iNOS intestinal expression compared to MTX alone [Figure 2]d. The significance was calculated through semi-quantitative analysis of the results of iNOS expression [Figure 2]e.{Figure 2}

Effect of thymoquinone on inflammatory markers in methotrexate-treated rat intestine

To evaluate the effect of TQ on inflammatory pathways, the level of the pro-inflammatory cytokine; TNF-α, in intestinal mucosa, as well as intestinal expression of NF-κB and COX-2 were evaluated. The level of TNF-α in MTX-treated group significantly increased compared to control group, while in MTX/RES group, TNF-α significantly decreased compared to rats treated with MTX alone [Figure 3]. Expression of NF-κB and COX-2 was visualized via immunohistochemical staining [Figure 2]; right panel and [Figure 4]; left panel, respectively] and semi-quantitative analysis was further performed to evaluate the degree of significance [Figure 2]j and [Figure 4]e, respectively]. Expression of both NF-κB and COX-2 in TQ-treated group [Figure 2]g and [Figure 4]b, respectively] was comparable to background expression in control groups [Figure 2]f and [Figure 4]a, respectively]. To the contrary, the MTX-treated group showed significantly higher expression of NF-κB and COX-2 [Figure 2]h and [Figure 4]c, respectively] compared to respective controls. Pretreatment with TQ prior to administration of MTX caused a significant decrease in expression of both markers compared to MTX alone [Figure 2]i and [Figure 4]4, respectively]. The significance was calculated through semi-quantitative analysis of the results of caspase 3 expression [Figure 4]j.{Figure 3}{Figure 4}

Effect of thymoquinone on expression of caspase 3 as marker of apoptosis in methotrexate-treated rat intestine

Immunostaining of rat intestine using caspase 3 antibody [Figure 4]; right panel] was performed as a marker of apoptosis. Both control and TQ groups showed minimal caspase 3 expression [Figure 4]f and [Figure 4]g, respectively]. In the MTX-treated group [Figure 4]h, however, caspase 3 was significantly up-regulated, especially in the epithelial lining the villi. This expression was significantly reversed in the MTX/TQ group [Figure 4]i.


Using MTX may be accompanied by damage of intestinal mucous membranes and mucositis, which may limit the patients' ability to endure treatment, hinder their nutritional status, and might even end fatally.[3] Following MTX administration, mucositis is initiated by the generation of reactive oxygen species as a response to MTX-induced DNA and non-DNA damage, followed by up-regulation of transcription factors including NF-κB and subsequent activation of genes of pro-inflammatory cytokines producing proteins, such as TNF-α, which cause direct tissue damage and promote apoptosis.[18] In concurrence with this model of mucositis, the present study demonstrated that MTX increased intestinal mucosa oxidative/nitrosative stress markers, up-regulated NF-κB and COX-2, increased TNF-α level, and induced apoptosis as illustrated by the induction of caspase 3 expression.

Few studies reported that the main constituent of Nigella sativa; TQ, may confer protection against MTX toxicity. One study reported that TQ has a protective effect against MTX-induced testicular toxicity.[12] Another study, in a different animal model than the present study, suggested that TQ protects against MTX-induced renal damage.[19] Here, we proved that TQ provides intestinal protective activity against MTX-induced mucositis. The vast majority of studies showing alimentary tract protection of TQ were conducted on the stomach.[20],[21],[22],[23] Only few studies focusing on such protection was carried out on the intestine, as in experimental acetic acid-induced colitis,[9] trinitrobenzene sulfonic acid-induced colitis,[24] and intestinal obstruction.[10]

In the current study, we explored the mechanisms involved in TQ-conferred intestinal protection and found that they include reversal of oxidative and nitrosative stress, down-regulation of inflammatory markers as NF-κB and COX-2, as well as inhibition of apoptosis. The antioxidant characteristics of TQ have been previously reported in different organs, as in the forebrain,[25] kidney [26],[27] heart,[28] prostate,[29] and liver.[30],[31] TQ was also known to modulate NO level and iNOS expression in different tissues.[26],[32] The anti-inflammatory/anti-apoptotic effects of TQ have also been documented in several previous studies.[30],[33]

Recently, a review [34] about interaction among oxidative, nitrosative, inflammatory, and apoptotic pathways showed how complicated it is to determine if the relationship among these pathways is a cause or a consequence of one another. Still, we hypothesize that the powerful antioxidant activity of TQ prevents MTX-induced oxidative stress from initiating intestinal damage, which, in turn, prevent the activation of TNF-α/NF-κB/COX-2 inflammatory pathway, as well as the subsequent triggering of automated cell death; apoptosis.

The protective effect of TQ against MTX-induced toxicity raises the question whether it confers similar protection to cancer cells, decreasing MTX chemotherapeutic efficacy. TQ has been reported to possess potential anticancer activity by itself on intestinal tumor development in Msh2loxP/loxP Villin-Cre mice [35] and on several tumor cells, including human hepatocellular carcinoma,[36] breast cancer,[37] and lung cancer.[38] In addition, TQ showed synergistic effects when given prior to conventional anticancer drugs as cisplatin [39] and doxorubicin.[40] Still, further studies are necessary to confirm lack of interference or possible synergism between TQ and MTX on tumor cells.


The main constituent of Nigella sativa; TQ, confers intestinal protection against MTX-induced mucositis. The mechanisms involved include interference with MTX-induced oxidative/nitrosative stress, inflammation, and apoptosis.

Financial support and sponsorship

This study was supported in part by Deanship of Scientific Research (DSR, Grant no. 150201), King Faisal University, Saudi Arabia.

Conflicts of interest

There are no conflicts of interest.


1Neradil J, Pavlasova G, Veselska R. New mechanisms for an old drug; DHFR- and non-DHFR-mediated effects of methotrexate in cancer cells. Klin Onkol 2012;25 Suppl 2:2S87-92.
2Morsy MA, Ibrahim SA, Amin EF, Kamel MY, Rifaai RA, Hassan MK. Curcumin ameliorates methotrexate-induced nephrotoxicity in rats. Adv Pharmacol Sci 2013;2013:387071.
3Tsukada T, Nakano T, Miyata T, Sasaki S. Life-threatening gastrointestinal mucosal necrosis during methotrexate treatment for rheumatoid arthritis. Case Rep Gastroenterol 2013;7:470-5.
4Chen C, Tian L, Zhang M, Sun Q, Zhang X, Li X, et al. Protective effect of amifostine on high-dose methotrexate-induced small intestinal mucositis in mice. Dig Dis Sci 2013;58:3134-43.
5Kolli VK, Kanakasabapathy I, Faith M, Ramamoorthy H, Isaac B, Natarajan K, et al . A preclinical study on the protective effect of melatonin against methotrexate-induced small intestinal damage: Effect mediated by attenuation of nitrosative stress, protein tyrosine nitration, and PARP activation. Cancer Chemother Pharmacol 2013;71:1209-18.
6Koppelmann T, Pollak Y, Mogilner J, Bejar J, Coran AG, Sukhotnik I. Dietary L-arginine supplementation reduces methotrexate-induced intestinal mucosal injury in rat. BMC Gastroenterol 2012;12:41.
7Sugiyama A, Kimura H, Ogawa S, Yokota K, Takeuchi T. Effects of polyphenols from seed shells of Japanese horse chestnut (Aesculus turbinata BLUME) on methotrexate-induced intestinal injury in rats. J Vet Med Sci 2011;73:673-8.
8Ahmad A, Husain A, Mujeeb M, Khan SA, Najmi AK, Siddique NA, et al. A review on therapeutic potential of Nigella sativa : A miracle herb. Asian Pac J Trop Biomed 2013;3:337-52.
9Mahgoub AA. Thymoquinone protects against experimental colitis in rats. Toxicol Lett 2003;143:133-43.
10Kapan M, Tekin R, Onder A, Firat U, Evliyaoglu O, Taskesen F, et al. Thymoquinone ameliorates bacterial translocation and inflammatory response in rats with intestinal obstruction. Int J Surg 2012;10:484-8.
11Zubair H, Khan HY, Sohail A, Azim S, Ullah MF, Ahmad A, et al. Redox cycling of endogenous copper by thymoquinone leads to ROS-mediated DNA breakage and consequent cell death: Putative anticancer mechanism of antioxidants. Cell Death Dis 2013;4:e660.
12Gökçe A, Oktar S, Koc A, Yonden Z. Protective effects of thymoquinone against methotrexate-induced testicular injury. Hum Exp Toxicol 2011;30:897-903.
13Awad AS, Kamel R, Sherief MA. Effect of thymoquinone on hepatorenal dysfunction and alteration of CYP3A1 and spermidine/spermine N-1-acetyl-transferase gene expression induced by renal ischaemia-reperfusion in rats. J Pharm Pharmacol 2011;63:1037-42.
14Ibrahim MA, El-Sheikh AA, Khalaf HM, Abdelrahman AM. Protective effect of peroxisome proliferator activator receptor (PPAR)-alpha and-gamma ligands against methotrexate-induced nephrotoxicity. Immunopharmacol Immunotoxicol 2014;36:130-7.
15Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol 1978;52:302-10.
16Sastry KV, Moudgal RP, Mohan J, Tyagi JS, Rao GS. Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy. Anal Biochem 2002;306:79-82.
17El-Sheikh AA, Morsy MA, Al-Taher AY. Multi-drug resistance protein (Mrp) 3 may be involved in resveratrol protection against methotrexate-induced testicular damage. Life Sci 2014;119:40-6.
18Keefe DM. Gastrointestinal mucositis: A new biological model. Support Care Cancer 2004;12:6-9.
19Budancamanak M, Kanter M, Demirel A, Ocakci A, Uysal H, Karakaya C. Protective effects of thymoquinone and methotrexate on the renal injury in collagen-induced arthritis. Arch Toxicol 2006;80:768-76.
20El-Abhar HS, Abdallah DM, Saleh S. Gastroprotective activity of Nigella sativa oil and its constituent, thymoquinone, against gastric mucosal injury induced by ischaemia/reperfusion in rats. J Ethnopharmacol 2003;84:251-8.
21Magdy MA, Hanan el-A, Nabila el-M. Thymoquinone: Novel gastroprotective mechanisms. Eur J Pharmacol 2012;697:126-31.
22Kanter M, Demir H, Karakaya C, Ozbek H. Gastroprotective activity of Nigella sativa L oil and its constituent, thymoquinone against acute alcohol-induced gastric mucosal injury in rats. World J Gastroenterol 2005;11:6662-6.
23Abdelwahab SI, Sheikh BY, Taha MM, How CW, Abdullah R, Yagoub U, et al. Thymoquinone-loaded nanostructured lipid carriers: Preparation, gastroprotection, in vitro toxicity, and pharmacokinetic properties after extravascular administration. Int J Nanomedicine 2013;8:2163-72.
24Juhás S, Cikos S, Czikková S, Veselá J, Il'ková G, Hájek T, et al. Effects of borneol and thymoquinone on TNBS-induced colitis in mice. Folia Biol (Praha) 2008;54:1-7.
25Al-Majed AA, Al-Omar FA, Nagi MN. Neuroprotective effects of thymoquinone against transient forebrain ischemia in the rat hippocampus. Eur J Pharmacol 2006;543:40-7.
26Khattab MM, Nagi MN. Thymoquinone supplementation attenuates hypertension and renal damage in nitric oxide deficient hypertensive rats. Phytother Res 2007;21:410-4.
27Sayed-Ahmed MM, Nagi MN. Thymoquinone supplementation prevents the development of gentamicin-induced acute renal toxicity in rats. Clin Exp Pharmacol Physiol 2007;34:399-405.
28Nagi MN, Al-Shabanah OA, Hafez MM, Sayed-Ahmed MM. Thymoquinone supplementation attenuates cyclophosphamide-induced cardiotoxicity in rats. J Biochem Mol Toxicol 2011;25:135-42.
29Rifaioglu MM, Nacar A, Yuksel R, Yonden Z, Karcioglu M, Zorba OU, et al. Antioxidative and anti-inflammatory effect of thymoquinone in an acute Pseudomonas prostatitis rat model. Urol Int 2013;91:474-81.
30Abd El-Ghany RM, Sharaf NM, Kassem LA, Mahran LG, Heikal OA. Thymoquinone triggers anti-apoptotic signaling targeting death ligand and apoptotic regulators in a model of hepatic ischemia reperfusion injury. Drug Discov Ther 2009;3:296-306.
31Aycan IÖ, Tüfek A, Tokgöz O, Evliyaoglu O, Firat U, Kavak GÖ, et al. Thymoquinone treatment against acetaminophen-induced hepatotoxicity in rats. Int J Surg 2014;12:213-8.
32Ahlatci A, Kuzhan A, Taysi S, Demirtas OC, Alkis HE, Tarakcioglu M, et al. Radiation-modifying abilities of Nigella sativa and thymoquinone on radiation-induced nitrosative stress in the brain tissue. Phytomedicine 2014;21:740-4.
33El-Khouly D, El-Bakly WM, Awad AS, El-Mesallamy HO, El-Demerdash E. Thymoquinone blocks lung injury and fibrosis by attenuating bleomycin-induced oxidative stress and activation of nuclear factor Kappa-B in rats. Toxicology 2012;302:106-13.
34Taye A, El-Sheikh AA. Lectin-like oxidized low-density lipoprotein receptor 1 pathways. Eur J Clin Invest 2013;43:740-5.
35Kortüm B, Campregher C, Lang M, Khare V, Pinter M, Evstatiev R, et al. Mesalazine and thymoquinone attenuate intestinal tumour development in Msh2loxP/loxP Villin-Cre mice. Gut 2015;64:1905-12.
36Ashour AE, Abd-Allah AR, Korashy HM, Attia SM, Alzahrani AZ, Saquib Q, et al. Thymoquinone suppression of the human hepatocellular carcinoma cell growth involves inhibition of IL-8 expression, elevated levels of TRAIL receptors, oxidative stress and apoptosis. Mol Cell Biochem 2014;389:85-98.
37Abukhader MM. Thymoquinone in the clinical treatment of cancer: Fact or fiction? Pharmacogn Rev 2013;7:117-20.
38Yang J, Kuang XR, Lv PT, Yan XX. Thymoquinone inhibits proliferation and invasion of human nonsmall-cell lung cancer cells via ERK pathway. Tumour Biol 2015;36:259-69.
39Nessa MU, Beale P, Chan C, Yu JQ, Huq F. Synergism from combinations of cisplatin and oxaliplatin with quercetin and thymoquinone in human ovarian tumour models. Anticancer Res 2011;31:3789-97.
40Effenberger-Neidnicht K, Schobert R. Combinatorial effects of thymoquinone on the anti-cancer activity of doxorubicin. Cancer Chemother Pharmacol 2011;67:867-74.