Lutein protection against doxorubicin-induced liver damage in male Wistar rat is associated with inhibition of oxido-inflammatory stress and modulation of Beclin-1/mTOR activities

Lutein protection against doxorubicin-induced liver damage in male Wistar rat is associated with inhibition of oxido-inflammatory stress and modulation of Beclin-1/mTOR activities | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Lutein protection against doxorubicin-induced liver damage in male Wistar rat is associated with inhibition of oxido-inflammatory stress and modulation of Beclin-1/mTOR activities Jerome Ndudi Asiwe, Godwin D. Yovwi, Mercy Oluwalani Alawode, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4641525/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Nov, 2024 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted 15 You are reading this latest preprint version Abstract A wide range of clinical applications are reported for doxorubicin (DOX), yet both people and research animals experience substantial tissue damage. However, the protective mechanism of lutein, a natural carotenoid against doxorubicin associated liver toxicity has not been fully studied. Therefore, the aim of this study is to investigate the protective mechanism of lutein in doxorubicin-induced liver damage. Twenty male Wistar rats were randomly assigned to four groups and treated as follows: Group 1 was administered 10 ml/kg body weight of distilled water intraperitoneally for a duration of 28 days. Group 2 was administered Doxorubicin (15 mg/kg body weight) intraperitoneally for three days in a row. Group 3 was administered intraperitoneal injections of Lutein (40 mg/kg body weight) daily for 28 days, and Group 4 was administered intraperitoneal injections of Lutein (40 mg/kg body weight) daily for 25 days and three days in a row of injections of Doxorubicin (15 mg/kg body weight). Our results showed that lutein reduced levels of AST, ALT, ALP, LDH, MDA, nitrite, beclin-1, caspase-3, IL-6 as well as TNF-α against the increase caused by doxorubicin. GSH, SOD, GST, catalase, mTOR as well as Bcl-2 were markedly increased by lutein against the harmful effect of doxorubicin. Moreso, lutein restored normal histoarchitecture as well as reduced fibrosis. In conclusion, Lutein protection against doxorubicin-induced liver damage in male Wistar rat is associated with inhibition of oxidative stress, pro-inflammatory reactions and modulation of Beclin-1/mTOR activities Antioxidants Apoptosis Autophagy Doxorubicin Inflammation Lutein Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Doxorubicin (DOX), an anthracycline class of antibiotic is frequently used to treat a range of cancers [ 1 ]. Concerns over the cumulative dose-dependent hepatic damage during cancer therapy have long existed among oncologists. Nevertheless, the extensive toxicity exhibited has limited the safe usage range of this life-saving medication [ 2 ]. The negative effects of DOX that have been described includes neurotoxicity, cardiotoxicity and reproductive toxicity [ 3 ]. Reactive oxygen species (ROS), which include hydroxyl radicals and superoxide anions as well as lipid peroxide can damage tissues, which may be the source of toxicity [ 4 ]. Cheah et al. [ 5 ] have postulated that the mechanism of DOX-induced oxidative stress involves the creation of an anthracycline-iron (Fe 2+ ) free radical complex. Reactive oxygen species (ROS) levels may increase in stressful circumstances which could lead to oxidative stress, a disorder that can cause major damage to cell architecture. Also, according to Akeel and Jaleel [ 6 ], ROS can harm lipids, proteins, carbohydrates and nucleotides, which are essential building blocks of membranes, enzymes, and DNA, among other biological components. The liver, a vital metabolic organ, is vulnerable to oxidative damage as a result of ongoing exposure to cytotoxic chemicals [ 7 ]. Among the liver enzymes that are suggestive of hepatic injury are lactate dehydrogenase, alanine transaminase, aspartate transaminase, and alkaline phosphatase [ 8 ]. Immune cell activation, which results in the generation of pro-inflammatory cytokines, is nevertheless one of the numerous patho-biological processes that ultimately lead to the death of the liver cell, also known as necrosis. Moreso, Panwar et al. [ 9 ], have reported that beclin-1 and mammalian target of rapamycin (mTOR) have a major impact on liver cell homeostasis. It has been discovered that Beclin-1, an essential autophagy regulator that acts as an initiator, is involved in these processes. A potential avenue for intervention in liver damage induction is the disruption of Beclin-1-mediated autophagy dysregulation, which has been linked to several diseases [ 10 ]. Simultaneously, the mTOR pathway, a crucial regulator of cell growth and metabolism, interacts intricately with autophagy. Dysregulated mTOR signaling has been associated with tumor growth and proliferation, resulting in the multiplication, dissemination and infiltration of tumor cells into newly created healthy tissues [ 11 ]. It is interesting to note that special enzyme functions are used to phagocytize the dead cell while maintaining and repairing tissue normalcy [ 12 ]. To the best of our knowledge, no study has been published in the literature that shows lutein's impact in this capacity. Nonetheless, a number of studies have hypothesized that a new therapeutic target is, the screening of pharmacological substances that have the ability to modify these molecular actors in order to guarantee appropriate cell physiology. Lutein (C40H56O2), also known as tetraterpenoids, is found in eggs and dark green leafy vegetables like kale and spinach [ 13 , 14 ]. Lutein acts as a cytoprotector against several diseases, mostly those brought on by oxidative stress, and has antioxidant qualities. Pap et al. [ 15 ], have demonstrated that lutein's protective role involves inhibition of hydrogen peroxide-induced apoptosis, hence facilitating photoreceptor survival and development. Pereira et al. [ 16 ], found that lutein suppressed the development of atherosclerosis by lowering MDA levels and inflammatory processes. Furthermore, Kim et al. [ 17 ] discovered that the NF-κB pathway was inhibited and lutein's anti-inflammatory properties were boosted in lipopolysaccharide (LPS)-induced inflammation in macrophages [ 18 ]. While studies on lutein's protective effect against doxorubicin-induced liver damage have been scarce in literature. The current study is designed to investigate the preventive mechanism of lutein against doxorubicin-induced liver damage. Materials and methods Chemicals and reagents Naman Pharma drugs Mumbai-2 (India) provided the doxorubicin (BDRL2302YE) and Puritan pride Inc., provided the lutein supplement (B51902-00A). J.I. Baker (USA) supplied reduced glutathione (GSH) and trichloracetic acid (TCA). Randox diagnostics, provided the kits for the assays for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea, and creatinine. The sodium hydroxide was supplied by the German business Merck. All of the materials and reagents that were left were of pure grade. The Purchasing and Care of Animals In total, twenty male Wistar rats were procured in this study from Faculty of Basic Medical Science, Delta State University's animal house, Abraka. They weighed between 130 and 150 grams when they were 12 or 12 weeks old. Standard laboratory conditions, comprising 12 hours of light and 12 hours of darkness, 45–55 percent humidity and 25°C temperature, were maintained while the rats were housed. They also had unrestricted access to food and clean tap water and the animal ethics committee of the Faculty of Basic Medical Science at Delta State University, Abraka, Nigeria authorized the protocols that were used when treating the animals with approval number (RBC/FBMS/DELSU/24/303). Research design Using a randomized controlled experimental design, the study evaluated lutein's putative defense mechanism against doxorubicin-induced liver damage. Twenty male Wistar rats were randomly assigned to four groups of five animals each after a seven-day acclimatization period (n = 5). For a period of 28 days, Group 1 received intraperitoneal injections of distilled water at a rate of 10 milliliters per kilogram of body weight. For three days straight, Group 2 received intraperitoneal injections of Doxorubicin (15 mg/kg body weight). Intraperitoneal injections of lutein (40 mg/kg body weight) were given to Group 3 for a duration of 28 days, whereas Group 4 received injections of lutein (40 mg/kg body weight) every day for 25 days, followed by injections of doxorubicin (15 mg/kg body weight) three days in a row (Fig. 1 ). While the lutein dosage used in this study was taken from past research by Oyovwi et al. [ 19 ], the doxorubicin dosage was slightly altered from the study by Ahmed et al. [ 20 ]. At the completion of the investigation, the animals were weighed and had their cervical dislocations to end their lives. Sensitive scales were used to weigh the liver after it was harvest. The liver was homogenized in a cold centrifuge and the supernatant was then decanted in order to get ready for the biochemical test. More liver tissues were kept for histological analysis in 10% phosphate buffered formalin. Assay for liver enzymes The Buhl and Jackson [ 21 ] method was utilized to quantify the lactate dehydrogenase (LDH) in the serum samples, and the Reitman and Frankel [ 22 ] method was employed to evaluate the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP). Lipid peroxidation To determine the malondialdehyde (MDA) levels in the liver, a lipid peroxidation product, or TBA reactants, were calculated using the approach previously described by Ohkawa et al. [ 23 ]. Following an hour-long boil in the presence of TBA reagent, tissue samples were subjected to spectrophotometric measurements at 532 nm. Next, a chromogen in the color pink was created. The data were represented as TBARS formed/mg of protein. Measuring nitric oxide The main byproduct of nitric oxide oxidation is nitrite (NOx). Griess reagent was added to the nitrite in order to quantify the amount of NO that was present in the sample. Asiwe et al. [ 24 ] state that a spectrophotometer operating at 540 nm wavelength was used to measure color variations. Glutathione (GSH) concentration The GSH content was determined using McCord and Fridovich's [ 25 ] approach, with a few minor changes. A total of 0.2 ml of dithiobisnitrobenzoic acid (0.4 mg/ml in 1% sodium citrate) and 0.3 M of Na2HPO4·2H2O were added to the tissue sample. The absorbance was measured after the incubation period at 412 nm. Activities of antioxidant enzymes Goth [ 26 ], reported that the SOD activity was determined using reduced nitroblue tetrazolium (NBT) as the basis of the test. Beers and Sizer [ 27 ] outlined the methodology for CAT (catalase) activity, which was determined using tissue homogenate dissolved in 50 mM phosphate buffer (pH 7.0) with 30 mM H 2 O 2 as the reaction mixture. The activity was calculated by measuring the reduction in H 2 O 2 absorbance at 240 nm. For Glutathione-S-Transferase (GST) activity, McCord and Fridovich [ 25 ] reported that dinitrophenyl thioether is produced when reduced glutathione reacts with 1-chloro-2, 4-dinitro benzene (CDNB) and is detected at 340 nm. Caspase-3 and B-cell lymphoma factor-2 assessment The levels of B-cell lymphoma factor-2 (Bcl-2) and caspase-3 in the liver was measured using the ELISA method according to the manufacturer's instructions. All of the wells were combined and then each contained 100µL of the standard or sample. The incubation period lasted 90 minutes at 37°C. A 100µL working solution of biotinylated detection Ab was added to each well, and it was incubated for 60 minutes at 37°C. The solutions were taken off the plate after three washing cycles. After adding 100µL of HRP conjugate working solution, an incubation time of 30 minutes at 37°C was necessary. On top of that, the solution underwent five washing. Following a 15-minute incubation at 37°C with 90µL of substrate reagent, 50µL of stop solution was added, and the sample was read with microplate reader at 450 nm. Tumor necrosis factor alpha and interleukin-6 assay An ELISA kit (Biolegend, 2018) was used to measure the amounts of Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the supernatant of rat liver tissue according to the manufacturer's instructions. Before being used, all samples, reagents and reference solutions were brought to room temperature. A biotinylated antibody-coated microplate was filled with samples, standards, controls and a streptavidin solution. Subsequently, the microplate was incubated at room temperature for 45 minutes. Each well was filled to capacity with 100µL of TMB One-Step substrate, which was allowed to sit at room temperature for 30 minutes before the addition of the Stop solution (50µl). A log-log logistic curve-fit was utilized to ascertain the unknown sample concentrations in parts per million. A Spectramax M-5 (Molecular Devices, Sunnyvale, CA) multifunctional plate reader equipped with Softmax Pro v 5.4 (SMP 5.4) was used to get a reading at 450 nm. Assay of Beclin-1 and mammalian target of rapamycin (mTOR) activity Beclin-1 together with mTOR activities were determined by employing ELISA techniques with strict adherence to the manufacturer’s guideline. Briefly, 50 µL of each sample or standard was added to the appropriate wells. Subsequently, 50 µL of the appropriate Antibody Cocktail were added to each well. After capping the wells, the plates were shaken for an hour at room temperature at 400 rpm. Extra liquid was wiped from the plate by swishing it against clean paper towels after the final wash with wash buffer. Additionally, 100 µL of TMB Development Solution was injected to each well. For ten minutes, the light was off and the plates were shaken at 400 rpm. The signal becomes yellow in color and increases in intensity by around three times when Stop Solution is applied. After stopping the reaction with stop buffer, the end point was measured at 450 nm using a microplate reader. Histopathology The liver samples were embedded in paraffin, dried in alcohol, and then placed in 10% formalin buffered with phosphate saline. Hematoxylin and eosin were used to stain the slide after it had been split into five micrometer sections and deparaffinized. The dyed slides were seen under a light microscope and magnified 400 times before being photographed. The resulting histomicrograph's histoarchitecture was then examined for changes. After the slices were treated with Masson trichrome to check for fibrosis, pictures were taken of them at 40 and 200 times their original size. The cell nucleus was labeled in black, the cytoplasm in red, and the fibrous tissue in blue. Images at 200x magnification were taken while the sections were being inspected under a light microscope. Image-Pro Plus (Media Cybernetics, Rockville, MD, USA) was used to measure the collagen content of the liver's various regions, and the average collagen content was computed for all the regions following random selection of five different sight fields was made. Statistical test GraphPad Prism 8.3 (GraphPad software, San Diego, CA, USA) was used to conduct the statistical analysis. The data is presented as mean ± SEM. To compare groups, one-way analysis of variance (ANOVA) and the Post hoc Tukey test were employed. The standard for statistical significance was set at P < 0.05. Results Antioxidant impact of lutein on doxorubicin-induced oxidative stress The impact of lutein on doxorubicin-associated oxidative stress was evaluated in this study and as presented in Fig. 2 A-B, doxorubicin significantly causes an increase in liver MDA [F(3, 12) = 69.36, p < 0.0001, R 2 = 0.9455] and nitrite [F(3, 12) = 119.5, p < 0.0001, R 2 = 0.9676] levels when compared with control. Also, liver GSH [F(3, 12) = 46.50, p < 0.0001, R 2 = 0.9208], catalase [F(3, 12) = 22.29, p < 0.0001, R 2 = 0.8478], SOD [F(3, 12) = 89.91, p < 0.0001, R 2 = 0.9574] and GST [F(3, 12) = 21.36, p < 0.0001, R 2 = 0.8423] were significantly reduced following doxorubicin exposure when compared with control. However, lutein pretreatment significantly alleviated oxidated stress by lowering MDA and Nitrite levels as well as elevating GSH, SOD, GST and catalase levels when compared with DOX-exposed group. Lutein prevents hepatocellular membrane disruption Doxorubicin exposure have been reported to cause membrane damage leading to leakage of cytosolic components into the blood stream. As presented in Fig. 3 A-D, doxorubicin significantly caused increase in AST [F(3, 12) = 93.14, p < 0.0001, R 2 = 0.9588], ALT [F(3, 12) = 121.6, p < 0.0001, R 2 = 0.9682], ALP [F(3, 12) = 55.52, p < 0.0001, R 2 = 0.9328] and LDH [F(3, 12) = 321.6, p < 0.0001, R 2 = 0.9877] when compared with control group. However, pretreatment with lutein significantly reduced AST, ALT, ALP and LDH when compared DOX-exposed rats. Lutein prevents pro-inflammatory reactions Oxidative damage has been reported to cause inflammatory responses. Meanwhile, doxorubicin in this study, cause a significant increase in IL-6 [F(3, 12) = 41.95, p < 0.0001, R 2 = 0.9129] and TNF-α [F(3, 12) = 8.954, p = 0.0022, R 2 = 0.6912] when compared with control. However, pretreatment with lutein reduced significantly the release of these pro-inflammatory cytokines when compared with DOX-exposed animals as presented in Fig. 4 A-B. Lutein prevents against doxorubicin associated cell death In this study, we investigated the protective impact of lutein on doxorubicin-induced apoptosis and our result showed that animals exposed to doxorubicin caused significant decrease in Bcl-2 [F(3, 12) = 120.8, p < 0.0001, R 2 = 0.9680] level as well as increased in caspase-3 [F(3, 12) = 166.3, p < 0.0001, R 2 = 0.9765] level when compared with control as shown in Fig. 5 A-B. However, apoptosis was inhibited by lutein pretreatment as indicated by significant increase in Bcl-2 and a reduction in caspase-3 activities when compared with DOX-treated animals. Lutein modulates autophagy mediators Liver beclin-1 [F(3, 12) = 32.44, p < 0.0001, R 2 = 0.8902] activities was significantly increased by doxorubicin while suppressing mTOR [F(3, 12) = 49.36, p < 0.0001, R 2 = 0.9250] activation when compared with control. However, pretreatment with lutein modulated these autophagic process by significantly inhibiting beclin-1 and increased mTOR activities when compared with doxorubicin exposed animals as shown in Fig. 6 A-B. Lutein prevents liver atrophy and fibrosis As presented in Fig. 7 , doxorubicin exposure caused a significant necrosis leading to reduced liver weight [F(3, 12) = 33.85, p < 0.0001, R 2 = 0.8943]. Following lutein pretreatment, the liver weight was significantly restored comparatively to the doxorubicin group. Also, there was an increased area of fibrosis [F(3, 12) = 128.6, p < 0.0001, R 2 = 0.9698] in DOX-exposed group. However, pretreatment with lutein reduced the fibrotic area significantly as shown in Fig. 8 Effect of lutein pretreatment on liver histology As presented in Fig. 9 , eosinophilic infiltration, inflammation and liver cell shrinkage and necrosis was observed in doxorubicin exposed group. However, no significant lesion was observed in lutein pretreatment group which showed restoration of normal histoarchitecture. Discussion A wide range of clinical applications are reported for doxorubicin (DOX), yet both people and research animals experience substantial tissue damage when exposed to DOX, according to Sheibani et al. [ 28 ]. Hanumegowda and Davis, [ 29 ] and Eguchi et al. [ 30 ], have reported that the cytotoxicity of DOX is associated with the generation of acrolein during drug metabolism which resulted to lipid peroxidation, excess reactive oxygen species (ROS) production, reduced antioxidant defense system and a severe inflammatory response via neutrophil attraction. Dietary antioxidants have recently drawn more attention for their function in protecting the liver from DOX-induced toxicity due to the minimal side effects of utilization [ 31 ]. Strong antioxidant qualities and cytoprotective efficiency against a variety of diseases induced by free radicals are attributes of lutein, according to Maiuolo et al. [ 32 ]. Thus, in order to evaluate lutein's potential protective mechanism against doxorubicin-induced liver damage in male Wistar rats, this experiment was conducted. The structure and functionality of biological membranes can be harmed by the chemical process known as lipid peroxidation. It is caused by lipids being attacked by free radicals [ 33 , 34 ]. The present investigation demonstrated increased levels of hepatic malondialdehyde, a lipid peroxidation marker. This implies that doxorubicin can generate reactive oxygen species (ROS) in the liver that attack lipids affixed to the hepatic cell membrane, leading to peroxidative damage. In keeping with earlier research, animals given doxorubicin also showed noticeably higher hepatocellular nitrite levels. Belenchev et al. [ 35 ] state that the production of extremely cytotoxic peroxynitrite, which is the result of the interaction between superoxide anions and nitric oxide, mediates nitrergic damage. Asiwe et al. , [ 36 ], hypothesized that increased nitrite levels in the liver brought on by doxorubicin therapy may have caused the release of glutamate and pro-inflammatory cytokines. Thus, an environment that encouraged hepatocellular injury might have been produced. Interestingly, lutein pretreatment significantly decreased liver nitrite and MDA levels, indicating cytoprotection against lipid peroxidative damage. One of the key mechanisms underlying the cytotoxic effects of DOX is the overproduction of reactive oxygen species (ROS), which in turn causes enhanced lipid peroxidation and the consequent inhibition of the antioxidant defense system. This report's findings, which are consistent with earlier studies [ 19 , 37 ], indicated that DOX administration increased oxidative stress. This was shown in the liver tissues by a significant decrease in the levels of GST, SOD, catalase activities as well as GSH level. The overproduction of ROS mediated by doxorubicin may inactivate catalase, GST, and SOD, resulting in a decrease in their activity. Conversely, GSH depletion was previously thought to be caused by the direct conjugation of DOX and its metabolites (acrolein) with free or protein-bound SH groups, which interfered with the antioxidant functions [ 37 , 38 ]. As mentioned earlier, GSH deficiency weakens the body's defenses against free radical-induced cellular damage, which can result in necrotic cell death [ 39 ]. The pretreatment with lutein showed remarkable anti-oxidative action, correcting the poor antioxidant status in liver tissues by significantly raising the GSH level, SOD, catalase and GST activities. Notwithstanding, due to its chemical composition, lutein has a considerable scavenging activity, which is noteworthy [ 40 ]. Additionally, it has been shown that lutein can repair the cell's deficient thiol status by promoting the de novo synthesis of GSH [ 41 ]. Still, the increase in GSH after lutein injection might be part of the defense against DOX-induced liver injury. The activities of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine transaminase (ALT) were significantly elevated after doxorubicin treatment. Due to its propensity to increase cell membrane permeability and release these enzymes into the bloodstream, hepatocellular necrosis may be the cause of these consequences. Similar results were found in the serum of rats exposed to lead acetate by Asiwe et al. [ 42 ] and Asiwe et al. [ 43 ]. According to supplementary research, the principal factor responsible for liver damage in rats receiving doxorubicin is an increase in serum levels of LDH, AST, ALT, and ALP [ 20 , 42 ]. The capacity of lutein to preserve membranes may be the reason why pretreatment with lutein greatly decreased the high levels of AST, ALT, ALP, and LDH. As a biological defensive mechanism against substances that cause disease, Maheshwari et al. [ 44 ], have connected immune cell activation generated by oxidative damage to inflammatory responses. In this study, it was discovered that exposure to doxorubicin raised levels of pro-inflammatory cytokines (TNF-α and IL-6). Nevertheless, lutein pretreatment significantly reduced the levels of TNF-α and IL-6 in the liver. This is consistent with a previous study that found that lutein can prevent NF-kB activation, a protein that causes the production of inflammatory cytokines. Numerous studies have connected oxidative stress and inflammatory reactions to mitochondria-mediated cell death [ 45 ]. The primary theory is that excessive ROS production leading to DNA uncoupling releases cytochrome-C into the mitochondria, which starts the signaling cascade that ends in cell death. According to this study, doxorubicin treatment significantly reduced Bcl-2 and increased caspase-3 activity, which in turn activated the apoptotic process. But as previous studies have shown, lutein administration significantly decreased caspase-3 activity while increasing liver Bcl-2. Recent research has demonstrated that dysregulated cellular processes, specifically autophagy and the mTOR pathway, are responsible for the hepatocellular damage caused by doxorubicin. Autophagy, a cellular breakdown mechanism that exposes intracellular entities to lysosomal degradation, is essentially necessary for maintaining cellular homeostasis [ 46 ]. Many liver disorders have been linked to dysregulation of Beclin-1-mediated autophagy, an important regulator of autophagy that acts as an initiator [ 10 ]. Also, Ballesteros-Álvarez and Andersen [ 47 ], have observed intricate relationships between autophagy and the mTOR pathway, which is a crucial regulator of cell growth and metabolism. In this investigation, doxorubicin exposure significantly increased beclin-1 levels and lowered mTOR levels in the liver, indicating a dysregulated autophagic mechanism [ 10 ]. The pretreatment of lutein inhibited Beclin-1 and activated mTOR, protecting hepatocellular components and restoring normal hepatocellular activity. Upon histological inspection, the effects of doxorubicin exposure were observed, including substantial eosinophilic infiltration, inflammation, and liver cell shrinkage and necrosis. Additionally, doxorubicin therapy caused interstitial collagen fibrils to deposit and a significant infiltration of mono nuclear cells into the liver tissues which was consistent with the reports of El-Horany et al. , [ 48 ]. Furthermore, doxorubicin therapy dramatically reduced liver weight, indicating liver tissue mass necrosis and atrophy. However, following lutein pretreatment, the fibrotic area significantly reduced and the liver's histoarchitecture appeared normal. Furthermore, there was a discernible improvement in the liver's weight, indicating that liver cells were regenerating and tissue mass was being restored. Other studies have demonstrated improvements in oxidative stress and restoration of the antioxidant defense in tissues, which may account for the improved liver tissue structure brought about by lutein therapy [ 49 , 50 ]. Conclusion Finally, when doxorubicin was given to animals in this study, it was observed to be associated with oxidative stress, inflammation and apoptosis. Due to hepatocellular membrane disruption, hepatic cytosolic enzymes were able to leak out and cause significant liver damage. Moreover, the disturbance of hepatocellular homeostasis caused by doxorubicin resulted in shrinkage and aberrant histoarchitecture. Lutein pretreatment drastically corrected these alterations via modulation of Beclin-1/mTOR activities and decreased oxido-inflammatory stress. Potential limitations of the research include the lack of evidence supporting apoptotic gene profiling and the requirement for inflammatory substances such as nuclear factor kappa-B to facilitate the assessment of lutein's impact on damage caused by chemotherapy. Declarations Conflict of interest: The authors have no known conflict of interest. Ethical approval: The study was approved by the research ethics committee of Faculty of Basic Medical Sciences, Delta State University, Abraka with approval number; RBC/FBMS/DELSU/24/303. Funding: The study did not receive funding from any funding body. Author Contribution Conceptualization JNA, and VUI; data curation, writing original draft preparation GDY, JNA, MOA; review and editing TI JNA, NA; supervision JNA VUI; funding acquisition MOA, EKU, TI, GDY and VUI. All authors have read and agreed to the publishing of the manuscript. Acknowledgement: The author acknowledges Dr A.M Ajayi and Dr O.G Adebayo with sincere gratitude for the technical assistance rendered during the animal experimentation. References Rawat PS, Jaiswal A, Khurana A, Bhatti JS, Navik U (2021) Doxorubicin-induced cardiotoxicity: An update on the molecular mechanism and novel therapeutic strategies for effective management. 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Cite Share Download PDF Status: Published Journal Publication published 30 Nov, 2024 Read the published version in Naunyn-Schmiedeberg's Archives of Pharmacology → Version 1 posted Editorial decision: Revision requested 21 Oct, 2024 Reviews received at journal 12 Oct, 2024 Reviewers agreed at journal 03 Oct, 2024 Reviewers agreed at journal 03 Oct, 2024 Reviewers agreed at journal 03 Oct, 2024 Reviews received at journal 03 Oct, 2024 Reviewers agreed at journal 01 Oct, 2024 Reviewers agreed at journal 01 Oct, 2024 Reviewers agreed at journal 09 Sep, 2024 Reviewers agreed at journal 17 Aug, 2024 Reviewers agreed at journal 06 Aug, 2024 Reviewers invited by journal 01 Aug, 2024 Editor assigned by journal 27 Jun, 2024 Submission checks completed at journal 27 Jun, 2024 First submitted to journal 26 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4641525","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":325228205,"identity":"324404f1-4130-4f8d-a720-bf8759237d54","order_by":0,"name":"Jerome Ndudi Asiwe","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYFACxgYGBh4I6wEDwwHStDAbEKkFAdgkiNLC33+4TeKDjE0+v9jhY9U8NXfk+BmYHz66gUeLxI3ENskZPGmWM2enpd3mOfbMWLKBzdg4B581NxjbbvPwHDYwuJ1jdpuH7XDihgM8bNL4tMifP9h2+w/Pf6CW/G/FPP+I0GJwILHtNgPPAZAtbMy8bURoMbyR2P6zhyfZQHJ2mrHk3L7DxpLNBPwid/74Y4OfPXYG/NLJDz+8+XZYjp+9+eFjvN4HAcYeCM0EjlJmQsrB4AdU6w+iVI+CUTAKRsFIAwC/T06jFpEZgwAAAABJRU5ErkJggg==","orcid":"","institution":"Delta State University","correspondingAuthor":true,"prefix":"","firstName":"Jerome","middleName":"Ndudi","lastName":"Asiwe","suffix":""},{"id":325228206,"identity":"8fa02e14-23e3-4d62-a549-9c615a612cdc","order_by":1,"name":"Godwin D. Yovwi","email":"","orcid":"","institution":"Delta State University","correspondingAuthor":false,"prefix":"","firstName":"Godwin","middleName":"D.","lastName":"Yovwi","suffix":""},{"id":325228207,"identity":"dbf9b16f-faa6-42b3-97aa-a88546902e96","order_by":2,"name":"Mercy Oluwalani Alawode","email":"","orcid":"","institution":"University of Ibadan","correspondingAuthor":false,"prefix":"","firstName":"Mercy","middleName":"Oluwalani","lastName":"Alawode","suffix":""},{"id":325228208,"identity":"df687960-ade8-4f79-9fc5-4059486eeadc","order_by":3,"name":"Theodora Isola","email":"","orcid":"","institution":"Atiba University","correspondingAuthor":false,"prefix":"","firstName":"Theodora","middleName":"","lastName":"Isola","suffix":""},{"id":325228209,"identity":"10406eb7-e52c-4b73-a20a-543653a61cce","order_by":4,"name":"Emuesiri Kohworho Umukoro","email":"","orcid":"","institution":"Delta State University","correspondingAuthor":false,"prefix":"","firstName":"Emuesiri","middleName":"Kohworho","lastName":"Umukoro","suffix":""},{"id":325228210,"identity":"cdcc92b8-625d-4845-91d6-e02903abfae8","order_by":5,"name":"Vincent Ugochukwu Igbokwe","email":"","orcid":"","institution":"Nnamdi Azikiwe University","correspondingAuthor":false,"prefix":"","firstName":"Vincent","middleName":"Ugochukwu","lastName":"Igbokwe","suffix":""},{"id":325228211,"identity":"e74fbaa5-2127-41d7-82ff-0b764a45cac4","order_by":6,"name":"Nicholas Asiwe","email":"","orcid":"","institution":"University of Port-Harcourt","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"","lastName":"Asiwe","suffix":""}],"badges":[],"createdAt":"2024-06-26 09:19:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4641525/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4641525/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00210-024-03650-2","type":"published","date":"2024-11-30T15:58:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60835168,"identity":"bb1b687f-d344-4c18-bea4-efad8c8c7898","added_by":"auto","created_at":"2024-07-22 15:47:54","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":68853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResearch design\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4641525/v1/5b0e3d98af935248323a073b.jpeg"},{"id":60835170,"identity":"f7287d1c-90ef-4685-a102-6d6045772610","added_by":"auto","created_at":"2024-07-22 15:47:54","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":91328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntioxidant impact of lutein on doxorubicin-induced oxidative stress \u003c/strong\u003e(A) Malondialdehyde (MDA), (B) Nitrite, (C) Glutathione (GSH), (D) Catalase, (E) Superoxide dismutase (SOD) and (F) Glutathione \u003cem\u003eS\u003c/em\u003e-transferase (GST). Data is presented in graph as mean±SEM, n=5, \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with control group while \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with DOX group. DOX = Doxorubicin and LUT = Lutein.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4641525/v1/63630fcc87b6e2b16201e1c6.jpeg"},{"id":60835173,"identity":"49fa29e3-aed4-49ae-adc2-c868aefc9152","added_by":"auto","created_at":"2024-07-22 15:47:54","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72597,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLutein prevents hepatocellular membrane disruption \u003c/strong\u003e(A) Aspartate transaminase (AST), (B) Alanine aminotransferase (ALT), (C) Alkaline phosphatase (ALP) and (D) lactate dehydrogenase (LDH). Data is presented in graphs as Mean ± SEM, n=5, \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with control group while \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with DOX group. DOX = Doxorubicin and LUT = Lutein.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4641525/v1/0c40dec2886827635bc66326.jpeg"},{"id":60835171,"identity":"2a4e6140-786a-4bbd-a7c8-db6d8ff2b86f","added_by":"auto","created_at":"2024-07-22 15:47:54","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38179,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLutein prevents pro-inflammatory reactions \u003c/strong\u003e(A) Interleukin- 6 (IL-6) and (B) Tumor necrosis factor- alpha (TNF-α). Data is presented in graphs as Mean ± SEM, n=5, \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with control group while \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with DOX group. DOX = Doxorubicin and LUT = Lutein.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4641525/v1/185a4216c03875df36823afd.jpeg"},{"id":60835172,"identity":"e49b3c42-b325-4bbc-b73d-72c88f5238fd","added_by":"auto","created_at":"2024-07-22 15:47:54","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":41191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLutein prevents against doxorubicin associated cell death \u003c/strong\u003e(A) B-cell lymphoma factor-2 (Bcl-2) and (D) Caspase-3. Data is presented in graphs as Mean ± SEM, n=5, \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with control group while \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with DOX group. DOX = Doxorubicin and LUT = Lutein.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4641525/v1/4f4ba705016f93e7eda69a8f.jpeg"},{"id":60835843,"identity":"47a5337d-e74f-42bc-b710-ea1309993c91","added_by":"auto","created_at":"2024-07-22 15:55:54","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":42435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLutein modulates autophagy mediators \u003c/strong\u003e(A) Beclin-1 and (D) Mammalian target of rapamycin (mTOR). Data is presented in graphs as Mean ± SEM, n=5, \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with control group while \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with DOX group. DOX = Doxorubicin and LUT = Lutein.\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4641525/v1/741b58a4f008a1884d5eeacd.jpeg"},{"id":60835176,"identity":"f89969af-08be-43eb-9995-c23c7909192f","added_by":"auto","created_at":"2024-07-22 15:47:54","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":30839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLutein prevents liver atrophy\u003c/strong\u003e Data is presented in graphs as Mean ± SEM, n=5, \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with control group while \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with DOX group. DOX = Doxorubicin and LUT = Lutein.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4641525/v1/7901336333a0fe65b7c62ef3.jpeg"},{"id":60835174,"identity":"6240e3eb-b299-4dac-91f9-972f5bac79e0","added_by":"auto","created_at":"2024-07-22 15:47:54","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":322522,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLutein prevents liver fibrosis. \u003c/strong\u003eThe representative immunohistographs\u003cstrong\u003e \u003c/strong\u003e(X400 magnification) (A) control group (B) DOX group (C) LUT group and (D) LUT+DOX group. Data is presented in graphs as Mean ± SEM, n=5, \u003csup\u003e\u003cem\u003ea\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with control group while \u003csup\u003e\u003cem\u003eb\u003c/em\u003e\u003c/sup\u003e\u003cem\u003ep\u0026lt;0.05\u003c/em\u003e is significant when compared with DOX group. DOX = Doxorubicin and LUT = Lutein.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4641525/v1/386ca845ac90890b2e28de92.jpeg"},{"id":60835844,"identity":"d5b4d684-ae83-4aff-983e-ec9b73fcb555","added_by":"auto","created_at":"2024-07-22 15:55:54","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":245967,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of lutein pretreatment on liver histology \u003c/strong\u003e(A) Control (B) Doxorubicin (DOX) (C) Lutein (LUT) (D) lutein before doxorubicin\u003cem\u003e \u003c/em\u003e(LUT+DOX). The tissues were stained with H\u0026amp;E and x400 magnification was used to capture the slides with light microscope. Arrow indicates significant lesion.\u003c/p\u003e","description":"","filename":"image9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4641525/v1/512ccaafc3db96c150b385c4.jpeg"},{"id":70390006,"identity":"dd469dd3-fb7d-47f2-bd13-5ee037e0f8c3","added_by":"auto","created_at":"2024-12-02 17:29:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1650879,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4641525/v1/2975d50c-fcc6-4cae-9b59-3472141927d4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lutein protection against doxorubicin-induced liver damage in male Wistar rat is associated with inhibition of oxido-inflammatory stress and modulation of Beclin-1/mTOR activities","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDoxorubicin (DOX), an anthracycline class of antibiotic is frequently used to treat a range of cancers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Concerns over the cumulative dose-dependent hepatic damage during cancer therapy have long existed among oncologists. Nevertheless, the extensive toxicity exhibited has limited the safe usage range of this life-saving medication [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The negative effects of DOX that have been described includes neurotoxicity, cardiotoxicity and reproductive toxicity [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Reactive oxygen species (ROS), which include hydroxyl radicals and superoxide anions as well as lipid peroxide can damage tissues, which may be the source of toxicity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Cheah et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] have postulated that the mechanism of DOX-induced oxidative stress involves the creation of an anthracycline-iron (Fe\u003csup\u003e2+\u003c/sup\u003e) free radical complex. Reactive oxygen species (ROS) levels may increase in stressful circumstances which could lead to oxidative stress, a disorder that can cause major damage to cell architecture. Also, according to Akeel and Jaleel [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], ROS can harm lipids, proteins, carbohydrates and nucleotides, which are essential building blocks of membranes, enzymes, and DNA, among other biological components.\u003c/p\u003e \u003cp\u003eThe liver, a vital metabolic organ, is vulnerable to oxidative damage as a result of ongoing exposure to cytotoxic chemicals [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among the liver enzymes that are suggestive of hepatic injury are lactate dehydrogenase, alanine transaminase, aspartate transaminase, and alkaline phosphatase [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Immune cell activation, which results in the generation of pro-inflammatory cytokines, is nevertheless one of the numerous patho-biological processes that ultimately lead to the death of the liver cell, also known as necrosis. Moreso, Panwar et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], have reported that beclin-1 and mammalian target of rapamycin (mTOR) have a major impact on liver cell homeostasis. It has been discovered that Beclin-1, an essential autophagy regulator that acts as an initiator, is involved in these processes. A potential avenue for intervention in liver damage induction is the disruption of Beclin-1-mediated autophagy dysregulation, which has been linked to several diseases [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Simultaneously, the mTOR pathway, a crucial regulator of cell growth and metabolism, interacts intricately with autophagy. Dysregulated mTOR signaling has been associated with tumor growth and proliferation, resulting in the multiplication, dissemination and infiltration of tumor cells into newly created healthy tissues [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It is interesting to note that special enzyme functions are used to phagocytize the dead cell while maintaining and repairing tissue normalcy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To the best of our knowledge, no study has been published in the literature that shows lutein's impact in this capacity. Nonetheless, a number of studies have hypothesized that a new therapeutic target is, the screening of pharmacological substances that have the ability to modify these molecular actors in order to guarantee appropriate cell physiology.\u003c/p\u003e \u003cp\u003eLutein (C40H56O2), also known as tetraterpenoids, is found in eggs and dark green leafy vegetables like kale and spinach [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Lutein acts as a cytoprotector against several diseases, mostly those brought on by oxidative stress, and has antioxidant qualities. Pap et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], have demonstrated that lutein's protective role involves inhibition of hydrogen peroxide-induced apoptosis, hence facilitating photoreceptor survival and development. Pereira et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], found that lutein suppressed the development of atherosclerosis by lowering MDA levels and inflammatory processes. Furthermore, Kim et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] discovered that the NF-κB pathway was inhibited and lutein's anti-inflammatory properties were boosted in lipopolysaccharide (LPS)-induced inflammation in macrophages [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. While studies on lutein's protective effect against doxorubicin-induced liver damage have been scarce in literature. The current study is designed to investigate the preventive mechanism of lutein against doxorubicin-induced liver damage.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eNaman Pharma drugs Mumbai-2 (India) provided the doxorubicin (BDRL2302YE) and Puritan pride Inc., provided the lutein supplement (B51902-00A). J.I. Baker (USA) supplied reduced glutathione (GSH) and trichloracetic acid (TCA). Randox diagnostics, provided the kits for the assays for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea, and creatinine. The sodium hydroxide was supplied by the German business Merck. All of the materials and reagents that were left were of pure grade.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eThe Purchasing and Care of Animals\u003c/h2\u003e \u003cp\u003eIn total, twenty male Wistar rats were procured in this study from Faculty of Basic Medical Science, Delta State University's animal house, Abraka. They weighed between 130 and 150 grams when they were 12 or 12 weeks old. Standard laboratory conditions, comprising 12 hours of light and 12 hours of darkness, 45\u0026ndash;55 percent humidity and 25\u0026deg;C temperature, were maintained while the rats were housed. They also had unrestricted access to food and clean tap water and the animal ethics committee of the Faculty of Basic Medical Science at Delta State University, Abraka, Nigeria authorized the protocols that were used when treating the animals with approval number (RBC/FBMS/DELSU/24/303).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eResearch design\u003c/h2\u003e \u003cp\u003eUsing a randomized controlled experimental design, the study evaluated lutein's putative defense mechanism against doxorubicin-induced liver damage. Twenty male Wistar rats were randomly assigned to four groups of five animals each after a seven-day acclimatization period (n\u0026thinsp;=\u0026thinsp;5). For a period of 28 days, Group 1 received intraperitoneal injections of distilled water at a rate of 10 milliliters per kilogram of body weight. For three days straight, Group 2 received intraperitoneal injections of Doxorubicin (15 mg/kg body weight). Intraperitoneal injections of lutein (40 mg/kg body weight) were given to Group 3 for a duration of 28 days, whereas Group 4 received injections of lutein (40 mg/kg body weight) every day for 25 days, followed by injections of doxorubicin (15 mg/kg body weight) three days in a row (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While the lutein dosage used in this study was taken from past research by Oyovwi et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the doxorubicin dosage was slightly altered from the study by Ahmed et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. At the completion of the investigation, the animals were weighed and had their cervical dislocations to end their lives. Sensitive scales were used to weigh the liver after it was harvest. The liver was homogenized in a cold centrifuge and the supernatant was then decanted in order to get ready for the biochemical test. More liver tissues were kept for histological analysis in 10% phosphate buffered formalin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAssay for liver enzymes\u003c/h2\u003e \u003cp\u003eThe Buhl and Jackson [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] method was utilized to quantify the lactate dehydrogenase (LDH) in the serum samples, and the Reitman and Frankel [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] method was employed to evaluate the levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eLipid peroxidation\u003c/h2\u003e \u003cp\u003eTo determine the malondialdehyde (MDA) levels in the liver, a lipid peroxidation product, or TBA reactants, were calculated using the approach previously described by Ohkawa et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Following an hour-long boil in the presence of TBA reagent, tissue samples were subjected to spectrophotometric measurements at 532 nm. Next, a chromogen in the color pink was created. The data were represented as TBARS formed/mg of protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasuring nitric oxide\u003c/h2\u003e \u003cp\u003eThe main byproduct of nitric oxide oxidation is nitrite (NOx). Griess reagent was added to the nitrite in order to quantify the amount of NO that was present in the sample. Asiwe et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] state that a spectrophotometer operating at 540 nm wavelength was used to measure color variations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGlutathione (GSH) concentration\u003c/h2\u003e \u003cp\u003eThe GSH content was determined using McCord and Fridovich's [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] approach, with a few minor changes. A total of 0.2 ml of dithiobisnitrobenzoic acid (0.4 mg/ml in 1% sodium citrate) and 0.3 M of Na2HPO4\u0026middot;2H2O were added to the tissue sample. The absorbance was measured after the incubation period at 412 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eActivities of antioxidant enzymes\u003c/h2\u003e \u003cp\u003eGoth [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], reported that the SOD activity was determined using reduced nitroblue tetrazolium (NBT) as the basis of the test. Beers and Sizer [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] outlined the methodology for CAT (catalase) activity, which was determined using tissue homogenate dissolved in 50 mM phosphate buffer (pH 7.0) with 30 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e as the reaction mixture. The activity was calculated by measuring the reduction in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e absorbance at 240 nm. For Glutathione-S-Transferase (GST) activity, McCord and Fridovich [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] reported that dinitrophenyl thioether is produced when reduced glutathione reacts with 1-chloro-2, 4-dinitro benzene (CDNB) and is detected at 340 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eCaspase-3 and B-cell lymphoma factor-2 assessment\u003c/h2\u003e \u003cp\u003eThe levels of B-cell lymphoma factor-2 (Bcl-2) and caspase-3 in the liver was measured using the ELISA method according to the manufacturer's instructions. All of the wells were combined and then each contained 100\u0026micro;L of the standard or sample. The incubation period lasted 90 minutes at 37\u0026deg;C. A 100\u0026micro;L working solution of biotinylated detection Ab was added to each well, and it was incubated for 60 minutes at 37\u0026deg;C. The solutions were taken off the plate after three washing cycles. After adding 100\u0026micro;L of HRP conjugate working solution, an incubation time of 30 minutes at 37\u0026deg;C was necessary. On top of that, the solution underwent five washing. Following a 15-minute incubation at 37\u0026deg;C with 90\u0026micro;L of substrate reagent, 50\u0026micro;L of stop solution was added, and the sample was read with microplate reader at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTumor necrosis factor alpha and interleukin-6 assay\u003c/h2\u003e \u003cp\u003eAn ELISA kit (Biolegend, 2018) was used to measure the amounts of Interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) in the supernatant of rat liver tissue according to the manufacturer's instructions. Before being used, all samples, reagents and reference solutions were brought to room temperature. A biotinylated antibody-coated microplate was filled with samples, standards, controls and a streptavidin solution. Subsequently, the microplate was incubated at room temperature for 45 minutes. Each well was filled to capacity with 100\u0026micro;L of TMB One-Step substrate, which was allowed to sit at room temperature for 30 minutes before the addition of the Stop solution (50\u0026micro;l). A log-log logistic curve-fit was utilized to ascertain the unknown sample concentrations in parts per million. A Spectramax M-5 (Molecular Devices, Sunnyvale, CA) multifunctional plate reader equipped with Softmax Pro v 5.4 (SMP 5.4) was used to get a reading at 450 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAssay of Beclin-1 and mammalian target of rapamycin (mTOR) activity\u003c/h2\u003e \u003cp\u003e Beclin-1 together with mTOR activities were determined by employing ELISA techniques with strict adherence to the manufacturer\u0026rsquo;s guideline. Briefly, 50 \u0026micro;L of each sample or standard was added to the appropriate wells. Subsequently, 50 \u0026micro;L of the appropriate Antibody Cocktail were added to each well. After capping the wells, the plates were shaken for an hour at room temperature at 400 rpm. Extra liquid was wiped from the plate by swishing it against clean paper towels after the final wash with wash buffer. Additionally, 100 \u0026micro;L of TMB Development Solution was injected to each well. For ten minutes, the light was off and the plates were shaken at 400 rpm. The signal becomes yellow in color and increases in intensity by around three times when Stop Solution is applied. After stopping the reaction with stop buffer, the end point was measured at 450 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHistopathology\u003c/h2\u003e \u003cp\u003eThe liver samples were embedded in paraffin, dried in alcohol, and then placed in 10% formalin buffered with phosphate saline. Hematoxylin and eosin were used to stain the slide after it had been split into five micrometer sections and deparaffinized. The dyed slides were seen under a light microscope and magnified 400 times before being photographed. The resulting histomicrograph's histoarchitecture was then examined for changes. After the slices were treated with Masson trichrome to check for fibrosis, pictures were taken of them at 40 and 200 times their original size. The cell nucleus was labeled in black, the cytoplasm in red, and the fibrous tissue in blue. Images at 200x magnification were taken while the sections were being inspected under a light microscope. Image-Pro Plus (Media Cybernetics, Rockville, MD, USA) was used to measure the collagen content of the liver's various regions, and the average collagen content was computed for all the regions following random selection of five different sight fields was made.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical test\u003c/h2\u003e \u003cp\u003eGraphPad Prism 8.3 (GraphPad software, San Diego, CA, USA) was used to conduct the statistical analysis. The data is presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. To compare groups, one-way analysis of variance (ANOVA) and the Post hoc Tukey test were employed. The standard for statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAntioxidant impact of lutein on doxorubicin-induced oxidative stress\u003c/h2\u003e \u003cp\u003eThe impact of lutein on doxorubicin-associated oxidative stress was evaluated in this study and as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B, doxorubicin significantly causes an increase in liver MDA [F(3, 12)\u0026thinsp;=\u0026thinsp;69.36, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9455] and nitrite [F(3, 12)\u0026thinsp;=\u0026thinsp;119.5, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9676] levels when compared with control. Also, liver GSH [F(3, 12)\u0026thinsp;=\u0026thinsp;46.50, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9208], catalase [F(3, 12)\u0026thinsp;=\u0026thinsp;22.29, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.8478], SOD [F(3, 12)\u0026thinsp;=\u0026thinsp;89.91, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9574] and GST [F(3, 12)\u0026thinsp;=\u0026thinsp;21.36, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.8423] were significantly reduced following doxorubicin exposure when compared with control. However, lutein pretreatment significantly alleviated oxidated stress by lowering MDA and Nitrite levels as well as elevating GSH, SOD, GST and catalase levels when compared with DOX-exposed group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eLutein prevents hepatocellular membrane disruption\u003c/h2\u003e \u003cp\u003eDoxorubicin exposure have been reported to cause membrane damage leading to leakage of cytosolic components into the blood stream. As presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D, doxorubicin significantly caused increase in AST [F(3, 12)\u0026thinsp;=\u0026thinsp;93.14, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9588], ALT [F(3, 12)\u0026thinsp;=\u0026thinsp;121.6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9682], ALP [F(3, 12)\u0026thinsp;=\u0026thinsp;55.52, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9328] and LDH [F(3, 12)\u0026thinsp;=\u0026thinsp;321.6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9877] when compared with control group. However, pretreatment with lutein significantly reduced AST, ALT, ALP and LDH when compared DOX-exposed rats.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eLutein prevents pro-inflammatory reactions\u003c/h2\u003e \u003cp\u003eOxidative damage has been reported to cause inflammatory responses. Meanwhile, doxorubicin in this study, cause a significant increase in IL-6 [F(3, 12)\u0026thinsp;=\u0026thinsp;41.95, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9129] and TNF-α [F(3, 12)\u0026thinsp;=\u0026thinsp;8.954, p\u0026thinsp;=\u0026thinsp;0.0022, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.6912] when compared with control. However, pretreatment with lutein reduced significantly the release of these pro-inflammatory cytokines when compared with DOX-exposed animals as presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eLutein prevents against doxorubicin associated cell death\u003c/h2\u003e \u003cp\u003eIn this study, we investigated the protective impact of lutein on doxorubicin-induced apoptosis and our result showed that animals exposed to doxorubicin caused significant decrease in Bcl-2 [F(3, 12)\u0026thinsp;=\u0026thinsp;120.8, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9680] level as well as increased in caspase-3 [F(3, 12)\u0026thinsp;=\u0026thinsp;166.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9765] level when compared with control as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B. However, apoptosis was inhibited by lutein pretreatment as indicated by significant increase in Bcl-2 and a reduction in caspase-3 activities when compared with DOX-treated animals.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eLutein modulates autophagy mediators\u003c/h2\u003e \u003cp\u003eLiver beclin-1 [F(3, 12)\u0026thinsp;=\u0026thinsp;32.44, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.8902] activities was significantly increased by doxorubicin while suppressing mTOR [F(3, 12)\u0026thinsp;=\u0026thinsp;49.36, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9250] activation when compared with control. However, pretreatment with lutein modulated these autophagic process by significantly inhibiting beclin-1 and increased mTOR activities when compared with doxorubicin exposed animals as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eLutein prevents liver atrophy and fibrosis\u003c/h2\u003e \u003cp\u003eAs presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, doxorubicin exposure caused a significant necrosis leading to reduced liver weight [F(3, 12)\u0026thinsp;=\u0026thinsp;33.85, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.8943]. Following lutein pretreatment, the liver weight was significantly restored comparatively to the doxorubicin group. Also, there was an increased area of fibrosis [F(3, 12)\u0026thinsp;=\u0026thinsp;128.6, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9698] in DOX-exposed group. However, pretreatment with lutein reduced the fibrotic area significantly as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEffect of lutein pretreatment on liver histology\u003c/h2\u003e \u003cp\u003eAs presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, eosinophilic infiltration, inflammation and liver cell shrinkage and necrosis was observed in doxorubicin exposed group. However, no significant lesion was observed in lutein pretreatment group which showed restoration of normal histoarchitecture.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eA wide range of clinical applications are reported for doxorubicin (DOX), yet both people and research animals experience substantial tissue damage when exposed to DOX, according to Sheibani et al. [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Hanumegowda and Davis, [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and Eguchi et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], have reported that the cytotoxicity of DOX is associated with the generation of acrolein during drug metabolism which resulted to lipid peroxidation, excess reactive oxygen species (ROS) production, reduced antioxidant defense system and a severe inflammatory response via neutrophil attraction. Dietary antioxidants have recently drawn more attention for their function in protecting the liver from DOX-induced toxicity due to the minimal side effects of utilization [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Strong antioxidant qualities and cytoprotective efficiency against a variety of diseases induced by free radicals are attributes of lutein, according to Maiuolo et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Thus, in order to evaluate lutein's potential protective mechanism against doxorubicin-induced liver damage in male Wistar rats, this experiment was conducted.\u003c/p\u003e \u003cp\u003eThe structure and functionality of biological membranes can be harmed by the chemical process known as lipid peroxidation. It is caused by lipids being attacked by free radicals [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The present investigation demonstrated increased levels of hepatic malondialdehyde, a lipid peroxidation marker. This implies that doxorubicin can generate reactive oxygen species (ROS) in the liver that attack lipids affixed to the hepatic cell membrane, leading to peroxidative damage. In keeping with earlier research, animals given doxorubicin also showed noticeably higher hepatocellular nitrite levels. Belenchev et al. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] state that the production of extremely cytotoxic peroxynitrite, which is the result of the interaction between superoxide anions and nitric oxide, mediates nitrergic damage. Asiwe \u003cem\u003eet al.\u003c/em\u003e, [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], hypothesized that increased nitrite levels in the liver brought on by doxorubicin therapy may have caused the release of glutamate and pro-inflammatory cytokines. Thus, an environment that encouraged hepatocellular injury might have been produced. Interestingly, lutein pretreatment significantly decreased liver nitrite and MDA levels, indicating cytoprotection against lipid peroxidative damage. One of the key mechanisms underlying the cytotoxic effects of DOX is the overproduction of reactive oxygen species (ROS), which in turn causes enhanced lipid peroxidation and the consequent inhibition of the antioxidant defense system. This report's findings, which are consistent with earlier studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], indicated that DOX administration increased oxidative stress. This was shown in the liver tissues by a significant decrease in the levels of GST, SOD, catalase activities as well as GSH level. The overproduction of ROS mediated by doxorubicin may inactivate catalase, GST, and SOD, resulting in a decrease in their activity. Conversely, GSH depletion was previously thought to be caused by the direct conjugation of DOX and its metabolites (acrolein) with free or protein-bound SH groups, which interfered with the antioxidant functions [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. As mentioned earlier, GSH deficiency weakens the body's defenses against free radical-induced cellular damage, which can result in necrotic cell death [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The pretreatment with lutein showed remarkable anti-oxidative action, correcting the poor antioxidant status in liver tissues by significantly raising the GSH level, SOD, catalase and GST activities. Notwithstanding, due to its chemical composition, lutein has a considerable scavenging activity, which is noteworthy [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Additionally, it has been shown that lutein can repair the cell's deficient thiol status by promoting the de novo synthesis of GSH [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Still, the increase in GSH after lutein injection might be part of the defense against DOX-induced liver injury.\u003c/p\u003e \u003cp\u003eThe activities of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine transaminase (ALT) were significantly elevated after doxorubicin treatment. Due to its propensity to increase cell membrane permeability and release these enzymes into the bloodstream, hepatocellular necrosis may be the cause of these consequences. Similar results were found in the serum of rats exposed to lead acetate by Asiwe et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and Asiwe et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. According to supplementary research, the principal factor responsible for liver damage in rats receiving doxorubicin is an increase in serum levels of LDH, AST, ALT, and ALP [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The capacity of lutein to preserve membranes may be the reason why pretreatment with lutein greatly decreased the high levels of AST, ALT, ALP, and LDH. As a biological defensive mechanism against substances that cause disease, Maheshwari et al. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], have connected immune cell activation generated by oxidative damage to inflammatory responses. In this study, it was discovered that exposure to doxorubicin raised levels of pro-inflammatory cytokines (TNF-α and IL-6). Nevertheless, lutein pretreatment significantly reduced the levels of TNF-α and IL-6 in the liver. This is consistent with a previous study that found that lutein can prevent NF-kB activation, a protein that causes the production of inflammatory cytokines. Numerous studies have connected oxidative stress and inflammatory reactions to mitochondria-mediated cell death [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The primary theory is that excessive ROS production leading to DNA uncoupling releases cytochrome-C into the mitochondria, which starts the signaling cascade that ends in cell death. According to this study, doxorubicin treatment significantly reduced Bcl-2 and increased caspase-3 activity, which in turn activated the apoptotic process. But as previous studies have shown, lutein administration significantly decreased caspase-3 activity while increasing liver Bcl-2.\u003c/p\u003e \u003cp\u003eRecent research has demonstrated that dysregulated cellular processes, specifically autophagy and the mTOR pathway, are responsible for the hepatocellular damage caused by doxorubicin. Autophagy, a cellular breakdown mechanism that exposes intracellular entities to lysosomal degradation, is essentially necessary for maintaining cellular homeostasis [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Many liver disorders have been linked to dysregulation of Beclin-1-mediated autophagy, an important regulator of autophagy that acts as an initiator [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Also, Ballesteros-\u0026Aacute;lvarez and Andersen [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], have observed intricate relationships between autophagy and the mTOR pathway, which is a crucial regulator of cell growth and metabolism. In this investigation, doxorubicin exposure significantly increased beclin-1 levels and lowered mTOR levels in the liver, indicating a dysregulated autophagic mechanism [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The pretreatment of lutein inhibited Beclin-1 and activated mTOR, protecting hepatocellular components and restoring normal hepatocellular activity.\u003c/p\u003e \u003cp\u003eUpon histological inspection, the effects of doxorubicin exposure were observed, including substantial eosinophilic infiltration, inflammation, and liver cell shrinkage and necrosis. Additionally, doxorubicin therapy caused interstitial collagen fibrils to deposit and a significant infiltration of mono nuclear cells into the liver tissues which was consistent with the reports of El-Horany \u003cem\u003eet al.\u003c/em\u003e, [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Furthermore, doxorubicin therapy dramatically reduced liver weight, indicating liver tissue mass necrosis and atrophy. However, following lutein pretreatment, the fibrotic area significantly reduced and the liver's histoarchitecture appeared normal. Furthermore, there was a discernible improvement in the liver's weight, indicating that liver cells were regenerating and tissue mass was being restored. Other studies have demonstrated improvements in oxidative stress and restoration of the antioxidant defense in tissues, which may account for the improved liver tissue structure brought about by lutein therapy [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFinally, when doxorubicin was given to animals in this study, it was observed to be associated with oxidative stress, inflammation and apoptosis. Due to hepatocellular membrane disruption, hepatic cytosolic enzymes were able to leak out and cause significant liver damage. Moreover, the disturbance of hepatocellular homeostasis caused by doxorubicin resulted in shrinkage and aberrant histoarchitecture. Lutein pretreatment drastically corrected these alterations via modulation of Beclin-1/mTOR activities and decreased oxido-inflammatory stress. Potential limitations of the research include the lack of evidence supporting apoptotic gene profiling and the requirement for inflammatory substances such as nuclear factor kappa-B to facilitate the assessment of lutein's impact on damage caused by chemotherapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest:\u003c/h2\u003e \u003cp\u003eThe authors have no known conflict of interest.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthical approval:\u003c/strong\u003e \u003cp\u003e The study was approved by the research ethics committee of Faculty of Basic Medical Sciences, Delta State University, Abraka with approval number; RBC/FBMS/DELSU/24/303.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe study did not receive funding from any funding body.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization JNA, and VUI; data curation, writing original draft preparation GDY, JNA, MOA; review and editing TI JNA, NA; supervision JNA VUI; funding acquisition MOA, EKU, TI, GDY and VUI. All authors have read and agreed to the publishing of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement:\u003c/h2\u003e \u003cp\u003eThe author acknowledges Dr A.M Ajayi and Dr O.G Adebayo with sincere gratitude for the technical assistance rendered during the animal experimentation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRawat PS, Jaiswal A, Khurana A, Bhatti JS, Navik U (2021) Doxorubicin-induced cardiotoxicity: An update on the molecular mechanism and novel therapeutic strategies for effective management. Biomed Pharmacother 139:111708\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUmapathi A, Kumawat M, Daima HK (2022) Engineered nanomaterials for biomedical applications and their toxicity: a review. 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Aging Cell, 20(8), e13431\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Horany HES, Atef MM, Abdel Ghafar MT, Fouda MH, Nasef NA, Hegab II, Helal DS, Elseady W, Hafez YM, Hagag RY, Seleem MA (2023) Empagliflozin ameliorates bleomycin-induced pulmonary fibrosis in rats by modulating Sesn2/AMPK/Nrf2 signaling and targeting ferroptosis and autophagy. Int J Mol Sci 24(11):9481\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSurai PF, Kochish II, Kidd MT (2021) Redox homeostasis in poultry: Regulatory roles of NF-κB. Antioxidants 10(2):186\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGopal SS, Sukhdeo SV, Vallikannan B, Ponesakki G (2023) Lutein ameliorates high-fat diet‐induced obesity, fatty liver, and glucose intolerance in C57BL/6J mice. Phytother Res 37(1):329\u0026ndash;341\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Antioxidants, Apoptosis, Autophagy, Doxorubicin, Inflammation, Lutein","lastPublishedDoi":"10.21203/rs.3.rs-4641525/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4641525/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA wide range of clinical applications are reported for doxorubicin (DOX), yet both people and research animals experience substantial tissue damage. However, the protective mechanism of lutein, a natural carotenoid against doxorubicin associated liver toxicity has not been fully studied. Therefore, the aim of this study is to investigate the protective mechanism of lutein in doxorubicin-induced liver damage. Twenty male Wistar rats were randomly assigned to four groups and treated as follows: Group 1 was administered 10 ml/kg body weight of distilled water intraperitoneally for a duration of 28 days. Group 2 was administered Doxorubicin (15 mg/kg body weight) intraperitoneally for three days in a row. Group 3 was administered intraperitoneal injections of Lutein (40 mg/kg body weight) daily for 28 days, and Group 4 was administered intraperitoneal injections of Lutein (40 mg/kg body weight) daily for 25 days and three days in a row of injections of Doxorubicin (15 mg/kg body weight). Our results showed that lutein reduced levels of AST, ALT, ALP, LDH, MDA, nitrite, beclin-1, caspase-3, IL-6 as well as TNF-α against the increase caused by doxorubicin. GSH, SOD, GST, catalase, mTOR as well as Bcl-2 were markedly increased by lutein against the harmful effect of doxorubicin. Moreso, lutein restored normal histoarchitecture as well as reduced fibrosis. In conclusion, Lutein protection against doxorubicin-induced liver damage in male Wistar rat is associated with inhibition of oxidative stress, pro-inflammatory reactions and modulation of Beclin-1/mTOR activities\u003c/p\u003e","manuscriptTitle":"Lutein protection against doxorubicin-induced liver damage in male Wistar rat is associated with inhibition of oxido-inflammatory stress and modulation of Beclin-1/mTOR activities","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 15:47:49","doi":"10.21203/rs.3.rs-4641525/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-21T07:33:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-12T11:18:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157946641976618132057048956974333675551","date":"2024-10-03T16:30:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16693065866414228249304216257742399407","date":"2024-10-03T13:55:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136082449024024567328940280187440128011","date":"2024-10-03T13:18:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-03T09:55:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271241044555395321016168527127400832011","date":"2024-10-01T10:55:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138916096708830987155116566178950634525","date":"2024-10-01T09:16:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"153630033775417967672213736634161637823","date":"2024-09-09T08:03:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"22952608278965576392850169730127282029","date":"2024-08-17T04:04:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36157009032931264885676709059618386500","date":"2024-08-06T06:45:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-01T06:36:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-27T05:34:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-27T05:33:15+00:00","index":"","fulltext":""},{"type":"submitted","content":"Naunyn-Schmiedeberg's Archives of Pharmacology","date":"2024-06-26T09:16:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"naunyn-schmiedebergs-archives-of-pharmacology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nsap","sideBox":"Learn more about [Naunyn-Schmiedeberg's Archives of Pharmacology](https://www.springer.com/journal/210)","snPcode":"210","submissionUrl":"https://submission.nature.com/new-submission/210/3","title":"Naunyn-Schmiedeberg's Archives of Pharmacology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a3749329-b678-4ec5-b583-666e028ba283","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-02T17:25:43+00:00","versionOfRecord":{"articleIdentity":"rs-4641525","link":"https://doi.org/10.1007/s00210-024-03650-2","journal":{"identity":"naunyn-schmiedebergs-archives-of-pharmacology","isVorOnly":false,"title":"Naunyn-Schmiedeberg's Archives of Pharmacology"},"publishedOn":"2024-11-30 15:58:07","publishedOnDateReadable":"November 30th, 2024"},"versionCreatedAt":"2024-07-22 15:47:49","video":"","vorDoi":"10.1007/s00210-024-03650-2","vorDoiUrl":"https://doi.org/10.1007/s00210-024-03650-2","workflowStages":[]},"version":"v1","identity":"rs-4641525","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4641525","identity":"rs-4641525","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}