Introduction

Antineoplastic drugs could increase life expectancy during the management of different types of malignancy. However, the associated cardiovascular complications could be serious and become an important issue. ¹ These disorders highly challenge the usage of cyclophosphamide (CYC), which is used in the treatment of systemic lupus erythematous, leukemia, and lymphomas. CYC-induced cardiotoxicity is still poorly understood and requires additional research. Exploring the different pathways involved in such toxicity is so important to protect the myocardium. Acrolein and phosphoramide mustard are the two major metabolites released from CYC metabolism that form covalent bonds with DNA and protein, causing apoptosis.^2,3

CYC administration for a long period and in high doses leads to marked heart damage, cardiac muscle failure, and arrhythmias. ⁴ Oxidative stress is followed by direct endothelial cell damage, which is more susceptible to CYC toxicity than other cells. The released reactive oxygen species (ROS) cause membrane lipid peroxidation and disturbances in normal cell functions followed by cell death^5–9 and stimulate different signaling molecules like nuclear factor kappa-B (NF-kB), which regulates the stimulation of pro-inflammatory cytokines like tumor necrosis factor α (TNFα) and interleukin ILβ (IL1β). Furthermore, CYC has been shown to suppress several antioxidant defense mechanisms.^10,11

One of the most essential pathways in mediating different drug-induced injuries is the inflammasome/caspase1/interleukin1β pathway. The inflammasome composes a macromolecular protein complex that regulates the activation of caspase1 and the maturation of pro-inflammatory cytokines such as IL1β and IL18. This pathway is highly involved in CYC-induced cardiac injury.^12–15 Recently, 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) have become the essential treatment for dyslipidemia. Statins are mainly effective in decreasing both low-density lipoprotein cholesterol and, to a lesser extent, triglyceride levels. The anti-inflammatory and antioxidant properties of statins have been proposed not only to treat significant cardiovascular diseases but also to diminish others that are not affected by cholesterol levels, such as stroke, rheumatoid arthritis, infection, and transplant rejection.^16,17

Statins are lipid-lowering drugs with anti-oxidant properties, scavenging oxygen radicals and potentially maintaining the endothelial barrier. Low-dose statins promote the activation of endothelial nitric oxide synthase (eNOS) and thus increase endothelial nitric oxide (NO) production. eNOS-derived NO induces pericyte recruitment with subsequent stabilization of angiogenic vessels, indicating vascular normalization. Endothelial cells release NO, which is effective for some biological activities, including vascular integrity and relaxation.^16,17

Nitric oxide (NO) has an essential role in mediating cardiac ischemic preconditioning (IPC).

Previously, it was detected that cardioprotection was lost when nitric oxide synthase (NOS) inhibitors were administered, and the expression of inducible NO synthase (iNOS) increased in cardiac myocytes. Nitro- ω-L-arginine (L.NNA) is one of these inhibitors, and it was used to inhibit the ameliorating effect of eNOS and evaluate the effect of the protective drug in the absence of eNOS action.^16,17

Keeping the heart against CYC’s harmful effects is highly required, especially after the administration of CYC in high doses. Moreover, CYC toxicity is associated with marked oxidative stress, inflammation, metabolic disturbances, and apoptosis. ¹⁸ Therefore, the present study aimed to evaluate the protective effect of SIM on CYC-induced cardiotoxicity based on its effectiveness as a metabolic regulator, antioxidant, anti-inflammatory, and anti-apoptotic drug, with further investigations of the involved mechanisms in mediating this effect and impact on the inflammasome/caspase1/interleukin1β pathway and the role of eNOS.

Materials and methods

Animals and experimental design

Thirty-six male Wistar albino rats weighing about 200–220 g were involved in the current model. Animals were purchased from the Animal Research Centre, Giza, Egypt. The rodents were kept in a suitable standard housing condition (3rats/cage), fed on chow and tap water, and left for 2 weeks to acclimatize. The ethical standards of the study were approved by the Faculty of Medicine, Minia University, Egypt. Approval No. 21:3/2021. Animal experiments were repeated several times to ensure reproducibility.

SIM and L-NNA were emulsified in 1% carboxymethylcellulose immediately before administration, while CYC was dissolved in saline.

Rats were randomized into 4 groups (n = 9)

Group I is the control group that received the vehicle only (carboxymethylcellulose) orally for 5 days and i.p. saline on the 4th and 5th days.

Group II is the CYC given group. It was administered carboxymethylcellulose orally for 5 days and CYC (150 mg/kg/day) i.p. ¹⁹ on the 4th and 5th days.

Group III (the CYC+SIM group) was given SIM (10 mg/kg/day) orally for 5 days ¹⁸ and CYC (150 mg/kg/day) i.p. ¹⁹ on the 4th and 5th days.

Group IV (the CYC+SIM+L-NNA group) received L-NNA (25 mg/kg/day) , ²⁰ SIM (10 mg/kg/day) ¹⁸ orally for 5 days, and CYC (150 mg/kg/day) i.p. ¹⁹ on the 4th and 5th days.

Preparation of samples

At the end of the experiment, each rat was anesthetized by urethane hydrochloride (125 mg/kg) i.p. injection. Blood was collected from the abdominal aorta and centrifuged at 4000 rpm for 15 minutes (JanetzkiT30 centrifuge, Germany). Each heart was excised, washed with saline, and weighed. A longitudinal section was obtained, fixed in 10% formalin, and embedded in paraffin for the histopathological examination. The samples of heart homogenate were centrifuged at 3000 rpm for 20 min and the supernatant was stored at−80°C till used (Glas-Col homogenizer, USA).

Chemicals

The SIM and Nitro- ω-L-arginine (L-NNA) powder forms were obtained from Sigma Aldrich Co., USA. CYC was purchased from Baxter Company (Germany). Total antioxidant capacity (TAC) and superoxide dismutase (SOD) kits were from Biodiagnostic Co., Egypt (Cataloge no. TA 25 13 and SD 25 21, respectively). The polyclonal rabbit/anti-rat eNOS antibody, biotinylated goat anti-rabbit secondary antibody, and IL1β ELISA kit were from Thermo Fisher Scientific (Fermont, CA, USA) (Cataloge no. BMS630). The rabbit polyclonal anti-caspase3 antibody was from Neo Markers Co. CA. The mouse monoclonal anti-β-actin antibody was from Sigma-Aldrich Co., USA. Alkaline phosphatase-tagged anti-rabbit or anti-mouse secondary antibodies were from Sigma-Aldrich Co., USA. ELISA kits of creatine kinase-MB (CK-MB), troponin I, and lactate dehydrogenase (LDH) were purchased from My BioSource Co., USA (Cataloge no. MBS2515061, MBS727624, and MBS269777, respectively). NLRP3 Inflammasome, caspase 1, tumor necrosis factor-alpha (TNFα), and interleukin 1β (IL1β) ELISA kits were from MyBioSource, USA (Cataloge no. MBS2706815, MBS765838, MBS2507393, and MBS#824956, respectively).

Measurements

The available commercial kits were used for measuring serum CK-MB, troponin I, and LDH according to the manufacturers' instructions (Cataloge no. MBS2515061, MBS727624, and MBS269777, respectively).

Cardiac tissue lipid peroxidation was evaluated using a thiobarbituric acid reacting substance that was expressed as the equivalent of MDA, using 1, 1, 3, 3-tetra methoxy propane as a standard. The data was analysed and expressed as nmol/g tissue. ²¹

Cardiac tissue SOD quantification depended on its ability to suppress phenazine methosulphate-mediated reduction of nitro blue tetrazolium dye. The results were presented as unit/g tissue. ²²

GSH was measured depending on the binding of the sulfhydryl group with Ellman’s reagent; a yellow color was formed and detected calorimetrically at 405 nm by the Beckman DU-64 UV/VIS spectrophotometer, USA as nmol/g tissue. ²³

The available commercial colorimetric kit was used to evaluate the serum TAC.

The evaluation was performed according to the manufacturer’s instructions and data sheet of the ELISA kits. NLRP3 Inflammasome, caspase 1, tumor necrosis factor-alpha (TNFα), and IL1β Elisa kits were from MyBioSource, USA (Cataloge no. MBS2706815, MBS765838, MBS2507393, and MBS#824956 respectively).

Histopathological examination

Five µm serial sections were prepared and stained with hematoxylin and eosin. Cardiac tissue alterations were assessed in ten non-overlapping high-power fields of each rat’s slide and evaluated in a blinded manner using an Olympus microscope from Japan to grade heart damage as follows:

Grading of the cardiac lesion was based on the degree of vascular congestion, hemorrhage, hydropic change, inflammatory infiltrate, absence of muscle striation, fatty degeneration, and necrosis. According to the degree of structural abnormalities, the severity of the lesions was rated semi-quantitatively as follows: Normal was a score of 0, mild alterations were a score of 1, moderate changes were a score of 2, and severe alterations were a score of 3. ²⁴

Immunohistochemistry of eNOS

Sections of cardiac tissue were prepared at 4 μm thickness on positively charged slides, de-paraffinized with xylene, rehydrated through graded ethanol, treated with 3% hydrogen peroxide for about 30 minutes to quench endogenous peroxidase, then rinsed in a PBS solution followed by citrate buffer (pH 6.0) treatment for antigen retrieval by boiling in a microwave. After cooling at room temperature, they were washed in a PBS solution, incubated with the eNOS primary antibody (7 ml ready to use) overnight in a humidity chamber, rinsed with a PBS solution, and treated with the secondary antibody for 30 minutes. After washing the sections in PBS, they were treated with the streptavidin-biotin complex reagent. By adding 3, 3-di amino benzidine tetrahydrochloride, a brown color was generated, then rinsed in distilled water, stained with hematoxylin, dehydrated, cleared by xylene, and covered.

An Olympus light microscope was used to assess positive cytoplasmic staining for eNOS. Each slide was evaluated and graded in a completely unbiased manner.

The intensity and distribution patterns of eNOS immunoexpression were evaluated semi-quantitatively and scored as follows: 0 indicates no staining, 1 indicates mild positivity, 2 indicates moderate positivity, and 3 indicates high positivity. ²⁵

Detection of apoptosis by measuring caspase3 using Western blotting

For the quantification of caspase3 in cardiac tissue samples, the western blotting technique was carried out.^20,26 The cardiac tissue was homogenized before being separated on a 10% SDS–PAGE gel. A semi-dry blotter was used to transfer protein bands to a nitrocellulose membrane (Bio-Rad). The blot was blocked and probed with rabbit polyclonal anti-caspase3 or mouse monoclonal anti-actin antibodies overnight at 4°C. After that, the secondary antibodies were incubated for 1 hour at room temperature with alkaline phosphatase-tagged anti-rabbit or anti-mouse antibodies. The colorimetric detection technique of 5-Bromo-4-chloro-3^′-polyphosphate and nitro-blue tetrazolium (BCIP/NBT) was used to examine the blots (Sigma-Aldrich Co., USA). Image-J and GraphPad Prism-6 software tools were used to examine protein bands.

Statistical analysis

One-way ANOVA and Tukey’s multiple comparison test were used. Results were displayed as means ± SEM. GraphPad Prism software (version 6) was used for analysis. p-value less than 0.05 was set for significance.

Results

Effect of simvastatin on heart weights and serum cardiac enzymes

Heart weights and the measured serum cardiac enzymes (troponin I, CK-MB, LDH) were significantly increased in the CYC given group compared to the control group. However, the CYC+SIM group showed a significant decrease in these enzymes relative to the CYC group. Interestingly, the ameliorative effect of SIM was reversed by co-administration of L-NNA (Table 1).

Effect of simvastatin on oxidative stress parameters

Cardiac MDA showed a significant elevation and cardiac SOD, GSH, and serum TAC showed a significant suppression in the CYC and the CYC+SIM+L-NNA groups relative to the control group. This action was significantly reversed by the co-administration of SIM (10 mg/kg/day) compared to the CYC group (Table 2).

Effect of simvastatin on cardiac tissue NLRP3 inflammasome, caspase1, TNFα and IL1β

Cardiac tissue NLRP3inflammasome, TNFα, caspase1, and IL1β were significantly increased in the CYC and CYC+SIM+L-NNA groups relative to the control group. Interestingly, the co-administration of SIM (10 mg/kg/day) significantly suppressed these parameters compared to the CYC given group (Table 3).

Histopathological examination of cardiac tissue

In Figure 1(a), the control group displayed normal myocardium with the integrity of myocardial cells without inflammatory cell infiltration. In the CYC-induced cardiotoxicity and CYC+ SIM+L-NNA groups, Figure 1(b) and (d) showed areas of myocardial necrosis, congested blood vessels, inflammatory infiltrate, and haemorrhage. In Figure 1(c), the CYC+SIM group revealed an obvious improvement with less inflammatory infiltrate and mild necrosis.

The control group was scored as 0. The CYC and CYC+SIM+L-NNA groups were scored as 3. The CYC+SIM group was scored as 1 (Figure 1(e)).

Assessment of eNOS immunoexpression

Negative expressions of eNOS in the CYC (Figure 2(b)) and the CYC+SIM+L-NNA groups (Figure 2(d)) were noticed. However, there was a positive immunoexpression in the CYC+SIM group (Figure 2(c)).

Significant decreases in eNOS immunoexpression in the CYC and the CYC+SIM+L-NNA groups were noticed in comparison to the CYC+SIM and the control groups. However, a significant elevation of its immunoexpression was noticed in the CYC+SIM group relative to the CYC group. (Figure 2(e))

Evaluation of caspase3 levels in the cardiac tissue using western blotting

Caspase3 was significantly increased in the CYC and the CYC+SIM+L-NNA groups relative to the control group, but the co-administration of CYC+SIM showed significant suppression of its level in comparison to the CYC-induced cardiotoxicity group (Figure 3).

Discussion

Cardiotoxicity of CYC is still a great challenge that affects its clinical use. Finding new ameliorative agents to diminish this deleterious adverse effect and enhance the CYC’s effectiveness is mandatory.^2,3 This model aimed to detect the potential cardiopreserving role of SIM against CYC-induced cardiotoxicity and explore the different mechanisms involved in this protection. CYC administration had significant increases in cardiac enzymes with toxic features on studying the histopathology. Moreover, heart weight, caspase-3, MDA, NLRP3inflammasome, caspase-1, TNFα, and IL1β levels were elevated. However, results detected significant decreases in antioxidant parameters and eNOS.

Oxidative stress has a great role in mediating CYC toxicity, followed by membrane lipid peroxidation, disturbances of the cell membrane, disturbed oxidant/antioxidant states inside the cell, DNA damage, and cell death. ² On the other hand, different antioxidant enzymes act as the first line of defense against oxidative stress and protect the cell components from harmful oxidative damage.^3,27 SOD is a critical antioxidant enzyme that detoxifies superoxide free radicals into hydrogen peroxide. Catalase acts on hydrogen peroxide, converting it into H₂O and O₂, while glutathione peroxidase forms the oxidized glutathione and reduces hydrogen peroxide to H₂O. Furthermore, GSH is an important antioxidant molecule and a cofactor in several antioxidant processes.^4,28

The production of acrolein is responsible for initiating the release of ROS that interferes with the cardiac tissue antioxidant defense system associated with increased lipid peroxidation of the cell membrane and significant depletion of antioxidant molecules. ⁴ Our findings revealed a significant increase in MDA, the most important parameter in detecting oxidative stress, and significant decreases in GSH, SOD, and TAC, which is consistent with other models.^29–31,12

Inflammasomes are a type of protein complex that is formed to enhance immune responses and cause cellular damage. Several inflammasomes were described, including NLRP3, NLRP1, AIM2, and NLRC4. The inflammasome comprises the sensor molecule NLRP3, the adaptor protein ASC, and pro-caspase-1. The NLRP3 protein contains a pyrin domain (PYD), and the ASC protein harbors PYD and CARD domains. Upon activation, the NLRP3 protein interacts with ASC via PYD, and the CARD domain of ASC recruits the CARD domain of pro-caspase-1 to form the NLRP3–ASC–pro-caspase-1 complex, also named the NLRP3 inflammasome. The AIM2 (absent in melanoma 2) inflammasome, which senses cytosolic DNA through its C-terminal HIN200 domain, can recruit pro-caspase-1 via ASC to form the AIM2–ASC–pro-caspase-1 complex.^32–35 Unlike NLRP3 and AIM2, the NLRP1 protein contains both PYD and CARD domains, which interact directly with pro-caspase-1 without the adaptor protein ASC, but the presence of ASC could enhance NLRP1-mediated caspase-1 activation. Ca²⁺ signaling is required for NLRP3 inflammasome activation, and Inositol 1,4,5-trisphosphate can interact with its receptor IP3R on the endoplasmic reticulum and promote Ca²⁺ mobilization and NLRP3 inflammasome activation. The assembly of inflammasome protein triggers the proteolytic cleavage of procaspase-1 into active caspase-1, which converts the pro-IL-1β and pro-IL-18 into mature IL-18 and IL-1β. They are considered potent pro-inflammatory mediators in many immune reactions, including innate immune cells to the site of infection and modulation of adaptive immune cells.^32–35 We can conclude that the generation of ROS and the release of free radicals are essential contributing factors in stimulating the Inflammasome/caspase 1/IL1β signaling cascade. NLRP3 activation forms a complex that regulates the activation of caspase1 and promotes the production of other essential inflammatory cytokines.³³ Moreover, TNFα and NF-κB are triggering factors in stimulating this pathway.

The apoptotic process is under the control of several caspases with excessive release of pro-inflammatory mediators that have a critical role in mediating CYC-induced heart injury. An imbalance between apoptotic and anti-apoptotic factors initiates programmed cell death and the apoptotic process. ³² The Inflammasome/caspase 1/IL1β pathway is a major contributing factor in mediating cardiac injury, edema, inflammation, hypertrophy, and cardiac cell death, especially in CYC-induced cardiotoxicity.^34,35

Our model showed significant increases in the NLRP3 inflammasome (an apoptotic and inflammatory mediator), caspase-1 (an apoptotic marker), and IL1 β (a pro-inflammatory cytokine) in the CYC group. In addition, TNFα increased in cardiac tissue, which is a critical pro-inflammatory cytokine that causes further release of other inflammatory mediators, interleukins, and apoptotic factors. Besides that, there is a significant elevation of the caspase-3 level, indicating the occurrence of apoptosis. This is due to the excessive production of ROS that stimulates different inflammatory mediators in cardiac tissue followed by direct DNA destruction and the induction of programmed cell death. These results are in accordance with earlier research.^35–37

CYC cytotoxicity is associated with hemorrhage, edema, inflammation, and necrosis. The same was found in the histopathological features of our study. Furthermore, there are marked increases in cardiac enzymes in the CYC group, indicating cardiac injury relative to the control group. This can be caused by the excessive release of ROS, leading to direct myocardial injury, damage of the myocardium, and efflux of the cell components. ¹⁸ These findings are consistent with previous research.^3,4,20,38

In general, mechanisms of CYC-induced cardiac damage encompass disturbance of the oxidant/antioxidant state inside the cells followed by cardiomyocyte inflammation, nuclear splitting, programmed cell death, vacuolization, and swelling of the cardiomyocytes with alteration in the signaling of different pathways. CYC-induced oxidative stress is accompanied by a decrease in GSH, glucose-6-phosphate dehydrogenase, glutathione peroxidase, and SOD activities, and the total antioxidant status is highly affected, which is in accordance with our findings. ³⁸ Furthermore, CYC toxicity is associated with metabolic disorders caused by oxidative stress and membrane lipid peroxidation, including lipid profile disturbances, hyperlipidemia, and marked elevations in cholesterol. ¹⁹ So we chose a potent member of the anti-hyperlipidemic family (statins) and it could regulate metabolic processes, antioxidant, anti-inflammatory, and anti-apoptotic effects; SIM.

Statins are 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors usually used for the treatment of hypercholesterolemia. Moreover, it enhances eNOS activation and increases NO release. ³⁸ Previous studies found that SIM could ameliorate myocardial hypertrophy in isoproterenol-given rats, the culture of cardiomyocytes, diabetic cardiomyopathy, and in a rat model of abdominal aortic constriction due to its ability to suppress inflammation, apoptosis, and oxidative stress.^39–41 The initial pathogenesis of CYC cardiotoxicity depends on stimulating oxidative stress. SIM has a potent antioxidant effect, ameliorating the released free radicals, accompanied by prevention of the apoptotic signaling process. This was evaluated in the current model that showed a significant decrease in MDA and increases in GSH, SOD, and TAC in accordance with others.^39,40 In addition, prevention of oxidative stress is followed by a potent anti-apoptotic effect of SIM detected by measuring caspase-3 immune expression, and our histopathological data found a significant decrease in caspase-3 immune expression. Furthermore, the action of SIM depends mainly on eNOS stimulation, which leads to vasoprotection.^16,39 Up-regulation of eNOS by SIM is a mandatory process for delivering normal blood flow to different organs and keeping the vascular endothelium. The essential role of eNOS was evaluated by the co-administration of L-NNA, which acts as a nitric oxide synthase inhibitor, especially acting on eNOS, affecting mainly vascular endothelium. Blocking of eNOS by L-NNA results in deterioration of cardiac function with oxidative stress, inflammation, and apoptosis and reverses the effect of SIM.^16,39,40 This method was used previously in different studies to explore the action of the protective drug and its effect on eNOS. Muhammad and his colleagues found that SIM could ameliorate 5FU cardiotoxicity via enhancing eNOS action and decreasing apoptotic signaling; caspase-3. ⁴¹ In addition, Wang and his colleagues found that SIM could inhibit NLRP3 inflammasome activation and ameliorate lung injury in hyperoxia-induced bronchopulmonary dysplasia via the KLF2-mediated mechanism, depending on previous inhibition of oxidative stress followed by prevention of inflammation. ⁴² These suppressing effects of SIM on different harmful pathways involved in CYC cardiotoxicity finally lead to a significant decrease and normalization of all cardiac enzymes and improvement of the histopathological features. Our model is the first step in studying the different mechanisms involved in mediating the cardioprotective effect of SIM on CYC-induced heart damage in patients receiving CYC.

Conclusion

Simvastatin (10 mg/kg/day) ameliorated cyclophosphamide-induced cardiotoxicity. This is most probably due to its effects as a metabolic regulator (anti-hyperlipidemic effect), anti-oxidant, anti-apoptotic, and anti-inflammatory with the stimulation of endothelial nitric oxide release and modulation of the inflammasome/caspase1/interleukin1β pathway. Further studies are needed to evaluate its role in patients receiving cyclophosphamide chemotherapy.

ORCID iDs

Marwa MM Refaie https://orcid.org/0000-0003-0216-5020

Nermeen N Welson https://orcid.org/0000-0001-7854-2086

Walaa Yehia Abdelzaher https://orcid.org/0000-0001-6511-1797

References

• 1 Mahipa P, Pawar RS, . Nephroprotective effect of Murraya koenigii on cyclophosphamide induced nephrotoxicity in rats. Asian Pac J Trop Med 2017; 10: 808–812.
• 2 Fouad AA, Qutub HO, Al-Melhim WN, . Punicalagin alleviates hepatotoxicity in rats challenged with cyclophosphamide. Environ Toxicol Pharmacol 2016; 45: 158–162.
• 3 Avci H, Epikmen ET, Ipek E, , et al Protective effects of silymarin and curcumin on cyclophosphamide-induced cardiotoxicity. Exp Toxicol Pathol 2017; 69: 317–327.
• 4 Omole JG, Ayoka OA, Alabi QK, , et al. Protective Effect of Kolaviron on Cyclophosphamide-Induced Cardiac Toxicity in Rats. J Evid Based Integr Med 2018; 23: 2156587218757649.
• 5 Hughes FM, Vivar NP, Kennis JG, , et al. Inflammasomes are important mediators of cyclophosphamide-induced bladder inflammation. Am J Physiol Renal Physiol 2014; 306(3): F299–F308.
• 6 Mahmoud AM, . Hesperidin protects against cyclophosphamide-induced hepatotoxicity by upregulation of PPARgamma and abrogation of oxidative stress and inflammation. Can J Physiol Pharmacol 2014; 92: 717–724.
• 7 Nafees S, Rashid S, Ali N, , et al. Rutin ameliorates cyclophosphamide induced oxidative stress and inflammation in Wistar rats: role of NFkappaB/MAPK pathway. Chem Biol Interact 2015; 231: 98–107.
• 8 Refaie MMM, El-hussieny M, Bayoumi AMA, , et al. Mechanisms mediating the cardioprotective effect of carvedilol in cadmium induced cardiotoxicity. Role of eNOS and HO1/Nrf2 pathway. Environ Toxicol Pharmacol 2019; 70: 103198.
• 9 El-Kashef DH, . Role of venlafaxine in prevention of cyclophosphamide-induced lung toxicity and airway hyperactivity in rats. Environ Toxicol Pharmacol 2018; 58: 70–76.
• 10 Ghareeb MA, Sobeh M, El-Maadawy WH, , et al. Chemical Profiling of Polyphenolics in Eucalyptus globulus and Evaluation of Its Hepato-Renal Protective Potential Against Cyclophosphamide Induced Toxicity in Mice. Antioxidants 2019; 8(9): 415.
• 11 Aladaileh SH, Abukhalil MH, Saghir SAM, , et al. Galangin Activates Nrf2 Signaling and Attenuates Oxidative Damage, Inflammation, and Apoptosis in a Rat Model of Cyclophosphamide-Induced Hepatotoxicity. Biomolecules 2019; 9(8): 346.
• 12 Komolafe OA, Arayombo BE, Abiodun AA, , et al. Immunohistochemical and histological evaluations of cyclophosphamide-induced acute cardiotoxicity in wistar rats: The role of turmeric extract. Morphologie 2020; 104(345): 133–142.
• 13 Mansour DF, Saleh DO, Mostafa RE, . Genistein ameliorates cyclophosphamide—induced hepatotoxicity by modulation of oxidative stress and inflammatory mediators. Open Access Macedonian J Med Sci 2017; 5: 836–843.
• 14 Schroder K, Zhou R, Tschopp J, . The NLRP3 inflammasome: A sensor for metabolic danger. Science 2010; 327: 296–300.
• 15 Rajamäki K, Lappalainen J, Öörni K, , et al. Cholesterol crystals activate the NLRP3 inflammasome in human macrophages: A novel link between cholesterol metabolism and inflammation. PLoS One 2010; 5: e11765.
• 16 Chen Y, Zhang S, Peng G, , et al. Endothelial NO synthase and reactive oxygen species mediated effect of simvastatin on vessel structure and function: pleiotropic and dose-dependent effect on tumor vascular stabilization. Int J Oncol 2013; 42(4): 1325–1336.
• 17 Habibey R, Ajami M, Ebrahimi SA, , et al. Nitric oxide and renal protection in morphine-dependent rats. Free Radic Biol Med 2010; 49(6): 1109–1118.
• 18 Hughes FM, McKeithan P, Ellett J, , et al. Simvastatin suppresses cyclophosphamide-induced changes in urodynamics and bladder inflammation. Urology 2013; 81(1): 209.e9.
• 19 Ray S, Chowdhury P, Pandit B, , et al. Exploring the antiperoxidative potential of morin on cyclophosphamide and flutamide-induced lipid peroxidation and changes in cholesterol profile in rabbit model. Acta Poloniae Pharmaceutica 2010; 67(1): 35–44.
• 20 Refaie MMM, Shehata S, El-Hussieny M, , et al. Role of ATP-Sensitive Potassium Channel (KATP) and eNOS in Mediating the Protective Effect of Nicorandil in Cyclophosphamide-Induced Cardiotoxicity. Cardiovasc Toxicol 2019; 20(1): 71–81.
• 21 Buege JA, Aust SD, . Microsomal lipid peroxidation. Methods Enzymol 1978; 52: 302–310.
• 22 Nishikimi M, Appaji N, Yagi K, . The occurrence of superoxide anion in the reaction of reduced phenazine methosul-fate and molecular oxygen. Biochem Biophysical Res Communications 1972; 46: 849–854.
• 23 Moron MS, Depierre JW, Mannervik B, . Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et Biophysica Acta 1979; 582(1): 67–78.
• 24 El-Agamy DS, Abo-Haded HM, Elkablawy MA, . Cardioprotective effects of sitagliptin against doxorubicin-induced cardiotoxicity in rats. Experimental Biology Med 2016; 241: 1577–1587.
• 25 Ӧzen -A, Ergűn E, Ӧztas¸ E, , et al. Immunohistochemical expression of nitric oxide synthase enzymes (iNOS, eNOS, nNOS) in the estrual and luteal phases of the sexual cycle in the cow oviduct. Anat Histol Embryol 2013; 42: 384–393.
• 26 Ewees MG, Messiha BAS, Abdel-Bakky MS, , et al. Tempol, a superoxide dismutase mimetic agent, reduces cisplatin-induced nephrotoxicity in rats. Drug Chem Toxicol 2018; 42: 1–8.
• 27 Iqubal A, Sharma S, Najmi AK, , et al. Nerolidol ameliorates cyclophosphamide-induced oxidative stress, neuroinflammation and cognitive dysfunction: Plausible role of Nrf2 and NF- κB. Life Sci 2019; 11: 116867.
• 28 Kitamura Y, Ushio S, Sumiyoshi Y, , et al. N-Acetylcysteine Attenuates the Anxiety-Like Behavior and Spatial Cognition Impairment Induced by Doxorubicin and Cyclophosphamide Combination Treatment in Rats. Pharmacology 2021; 106(5–6): 286–293.
• 29 El-Agamy DS, Elkablawy MA, Abo-Haded HM, . Modulation of cyclophosphamide-induced cardiotoxicity by methyl palmitate. Cancer Chemother Pharmacol 2017; 79(2): 399–409.
• 30 Hassanein EHM, Abd El-Ghafar OAM, Ahmed MA, , et al. Edaravone and Acetovanillone Upregulate Nrf2 and PI3K/Akt/mTOR Signaling and Prevent Cyclophosphamide Cardiotoxicity in Rats. Drug Design. Development Therapy 2020; 14: 5275–5288.
• 31 Abd El-Ghafar OAM, Hassanein EHM, Sayed AM, , et al. Acetovanillone prevents cyclophosphamide-induced acute lung injury by modulating PI3K/Akt/mTOR and Nrf2 signaling in rats. Phytotherapy Res 2021; 35(8): 4499–4510.
• 32 Yang Y, Wang H, Kouadir M, , et al. Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors. Cell Death Disease 2019; 10: 128.
• 33 He Y, Hara H, Núñez G, . Mechanism and regulation of NLRP3 inflammasome activation. Trends in Biochem Sci 2016; 41(12): 1012–1021.
• 34 Zhu J, Wu S, Hu S, , et al. NLRP3 inflammasome expression in peripheral blood monocytes of coronary heart disease patients and its modulation by rosuvastatin. Molecular Med Reports 2019; 20: 1826–1836.
• 35 Buccione C, Fragale A, Polverino F, , et al. Role of interferon regulatory factor 1 in governing Treg depletion, Th1 polarization, inflammasome activation and antitumor efficacy of cyclophosphamide. Int J Cancer 2018; 142(5): 976–987.
• 36 Gunes S, Sahinturk V, Uslu S, , et al. Protective effects of selenium on cyclophosphamide-induced oxidative stress and kidney injury. Biological Trace Element Res 2018; 185: 116–123.
• 37 Abd-ElRaouf A, Nada AS, Mohammed NEA, , et al. Low dose gamma irradiation attenuates cyclophosphamide-induced cardiotoxicity in rats: role of NF-kappaB signaling pathway. International J Radiation Biology 2021; 97(5): 632–641.
• 38 Ayza MA, Zewdie KA, Tesfaye BA, , et al. The Role of Antioxidants in Ameliorating Cyclophosphamide-Induced Cardiotoxicity. Oxid Med Cell Longev 2020; 2020: 4965171.
• 39 Cheng W, Ho W, Chang C, , et al. Simvastatin induces a central hypotensive effect via Ras-mediated signalling to cause eNOS up-regulation. British J Pharmacology 2013; 170(4): 847–858.
• 40 Al-Rasheed NM, Hasan IH, Al-Amin MA, , et al. Simvastatin Ameliorates Diabetic Cardiomyopathy by Attenuating Oxidative Stress and Inflammation in Rats. Oxidative Medicine and Cellular Longevity. 2017; 2017: 1092015.
• 41 Muhammad RN, Sallam N, El-Abhar HS, . Activated ROCK/Akt/eNOS and ET-1/ERK pathways in 5-fluorouracil-induced cardiotoxicity: modulation by simvastatin. Sci Rep 2020; 10(1): 146931469.
• 42 Wang X, Huo R, Liang Z, , et al. Simvastatin Inhibits NLRP3 Inflammasome Activation and Ameliorates Lung Injury in Hyperoxia-Induced Bronchopulmonary Dysplasia via the KLF2-Mediated Mechanism. Oxid Med Cell Longev 2022; 2022: 8336070.