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Effect of vegetable oils on the experimental infection of mice with Trypanosoma congolense
Experimental Parasitology 210 (2020) 107845
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Effect of vegetable oils on the experimental infection of mice with Trypanosoma congolense
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Aiko Kume, Keisuke Suganuma, Rika Umemiya-Shirafuji, Hiroshi Suzuki∗ National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Trypanosoma congolense Vegetable oil Mice Fatty acid Solvent
Vegetable oils are frequently used as solvents for lipophilic materials; accordingly, the effects of their components should be considered in animal experiments. In this study, the effects of various vegetable oils on the course of Trypanosoma congolense infection were examined in mice. C57BL/6J mice were orally administered four kinds of oils (i.e., coconut oil, olive oil, high oleic safflower oil, and high linoleic safflower oil) with different fatty acid compositions and infected with T. congolense IL-3000. Oil-treated mice infected with T. congolense showed significantly higher survival rates and lower parasitemia than those of control mice. Notably, coconut oil, which mainly consists of saturated fatty acids, delayed the development of parasitemia at the early stage of infection. These results indicated that vegetable oil intake could affect T. congolense infection in mice. These findings have important practical implications; for example, they suggest the potential effectiveness of vegetable oils as a part of the regular animal diet for controlling tropical diseases and indicate that vegetable oils are not suitable solvents for studies of the efficacy of lipophilic agents against T. congolense.
1. Introduction Trypanosomes are kinetoplastid protozoans that cause fatal diseases in livestock and humans. These parasites proliferate extracellularly in blood and tissue fluids of their mammalian hosts and are cyclically transmitted by the tsetse fly (Glossina spp.). Humans have sensitivity to two species of trypanosomes, Trypanosoma brucei rhodesiense and T. b. gambiense (Barrett et al., 2003; Giordani et al., 2016). Animal African trypanosomiasis (AAT), called Nagana, is caused by T. congolense, T. vivax, and, to a lesser extent, T. brucei spp. and is widespread in subSaharan Africa (Giordani et al., 2016). AAT in livestock leads to acute and/or chronic cases of wasting syndrome, causing high morbidity, mortality, and infertility in the absence of suitable treatment (Leach and Roberts, 1981; Connor, 1991; Giordani et al., 2016). Control strategies for AAT rely primarily on vector control using insecticides or traps and on parasite control using trypanocides (Holmes, 2013; Giordani et al., 2016). Despite the effectiveness of vector control methods for AAT, such programs are expensive and frequent re-infestation occurs (Holmes, 2013). Currently, only 6 trypanocidal compounds are used as therapeutic and/or prophylactic drugs against AAT (i.e., diminazene aceturate, homidium salts, isometamidium chloride, quinapyramine sulphate, suramin sodium, and melarsomine dihydrochloride), and many of these have been used since
around the first half of the 20th century (Giordani et al., 2016). In addition, there are five drugs for Human African Trypanosomiasis (HAT) (i.e., eflornithine, melarsoprol, nifurtimox, pentamidine and suramin) (Thomas et al., 2018), and eflornithine is the only new treatment registered for HAT in the past six decades (Priotto et al., 2009). However, these drugs have unsatisfactory therapeutic effects and often have severe side effects. Their extensive utilization has led to the emergence of drug-resistant parasites. Moreover, many of these drugs are chemically related, which has exacerbated the situation by resulting in cross-resistance (Peregrine, 1994; Giordani et al., 2016). The beneficial effects of foods or oils on the bioavailability of hydrophobic drugs have been described previously (Humberstone and Charman, 1997; Pouton, 2000). Thus, vegetable oils are frequently used as solvents for lipophilic materials in animal experiments and have advantages with respect to safety and drug stability (Pouton and Porter, 2008). However, for the interpretation of experimental data, it is essential to understand the effects of solvent components. Vegetable oils consist of a mixture of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs), and the fatty acid (FA) compositions specifically depend on the plant sources (Orsavova et al., 2015). For example, coconut oil and olive oil are mainly composed of lauric and myristic acids (SFAs) and oleic acid (MUFA), respectively (Orsavova et al., 2015). Classic safflower oil
∗ Corresponding author. Research Unit for Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada, Obihiro, Hokkaido, 080-8555, Japan. E-mail address: [email protected] (H. Suzuki).
https://doi.org/10.1016/j.exppara.2020.107845 Received 23 February 2019; Received in revised form 17 October 2019; Accepted 24 January 2020 Available online 28 January 2020 0014-4894/ © 2020 Elsevier Inc. All rights reserved.
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normally contains 10–15% oleic acid (MUFA) and over 70% linoleic acid (PUFA), and another safflower oil made from a natural mutant contains up to 70% oleic acid (MUFA) (Cao et al., 2013; Orsavova et al., 2015). FAs composed of lipids are classified into three types according to the absence or presence of double bonds in the structural formula. SFAs have no double bond, MUFAs have one, and PUFAs have two or more double bonds (Orsavova et al., 2015). In the present study, the effects of various vegetable oils on the course of T. congolense infection were examined in mice. C57BL/6J mice were orally administered 4 kinds of oils, coconut oil, olive oil, high oleic safflower oil, and high linoleic safflower oil, with different FA compositions, followed by infection with T. congolense IL-3000. 2. Materials and methods 2.1. Mice C57BL/6J mice were bred and maintained in specific pathogen-free conditions at the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Japan. Male mice aged 8–12 weeks were used. The temperature (24 ± 1 °C) and humidity (50 ± 10%) were regulated and lighting was controlled (lights on from 7:00 to 19:00). Mice had free access to water and a commercial regular diet (3.4 kcal/g, containing 4.88% crude fat, CA-1; CLEA Japan, Tokyo, Japan). All animal experiments were conducted in accordance with the standards of the Care and Management of Experimental Animals of Obihiro University of Agriculture and Veterinary Medicine, Japan.
Fig. 1. Effect of oil intake on the survival rate of T. congolense-infected mice. Mice were treated with PBS, olive oil, high oleic safflower oil (Safflower-HO), or high linoleic safflower oil (Safflower-HL) from 3 weeks before infection to 1 week after infection. Compared with the PBS group, all oil treatment groups showed significantly higher survival rates. *p < 0.05.
infection with T. congolense IL-3000. There were no statistically significant differences in the survival rate among the experimental groups. The median survival times of T. congolense IL-3000-infected mice treated with PBS, coconut oil, olive oil, high oleic safflower oil, and high linoleic safflower oil were 8.0, 26.0, 25.0, 22.0, and 12.0 dpi, respectively. The median survival times for the coconut oil, olive oil, and high oleic safflower oil groups were longer than that for the high linoleic safflower oil group. All oil intake groups showed significantly lower parasitemia than that of the PBS group on 4 and 6 dpi (Fig. 2). In addition, oil intake, especially coconut oil, tended to delay the detection of parasites in peripheral blood compared with PBS treatment.
2.2. Effect of oil intake on T. congolense IL-3000 infection in mice C57BL/6J male mice were orally administered 0.2 ml of coconut oil (Cocowell, Osaka, Japan), olive oil (Nisshin Oillio Group, Tokyo, Japan), high oleic safflower oil (Ajunomoto, Tokyo, Japan), and high linoleic safflower oil (Holbein Works, Osaka, Japan) or PBS by a feeding needle once a day for 3 weeks and were then inoculated with 3 × 104 cells of T. congolense IL-3000 (International Livestock Research Institute, Nairobi, Kenya) by intraperitoneal injection. Oil treatment was continued for 1 week after infection. Parasitemia was monitored after the infection once every 2 days from 2 to 12 days post infection (dpi) and once every 3–6 days from 15 to 32 dpi. Blood samples for parasitemia estimates were collected from the tails of mice. To account for stress caused by handling, PBS was administered as a control. 2.3. Effect of oil intake on body weight and diet intake C57BL/6J male mice were treated with 4 kinds of oils or PBS (0.2 ml/head) by a feeding needle once a day for 2 weeks. During the experimental period, mouse body weight was measured every 2 days and the weight of food residue was measured every day. Dietary caloric intake was estimated by the difference in weight between food provided and food remaining for each experimental group. The daily caloric intake from oils (0.91 g/ml) was calculated as 1.62 kcal/head. 2.4. Statistical analysis Parasitemia and body weights were analyzed by Dunnett's tests. The survival rate was analyzed using the log-rank (Mantel-Cox) and GehanBreslow-Wilcoxon tests implemented in GraphPad Prism 5. For all analyses, a p-value of less than 0.05 was considered statistically significant.
Fig. 2. Effect of oil intake on parasitemia in T. congolense-infected mice. Mice were treated with PBS, olive oil, high oleic safflower oil (Safflower-HO), or high linoleic safflower oil (Safflower-HL) from 3 weeks before infection to 1 week after infection. The lower graph is an enlarged image of the upper plot from day 0 to day 10. Compared with the PBS group, the coconut oil, olive oil, and high oleic safflower oil treatment groups showed significantly lower parasitemia at 3, 4, 5, and 6 dpi. High linoleic safflower oil-treated mice showed significantly lower parasitemia than that of PBS-treated mice on 4, 5, and 6 dpi. *p < 0.05.
3. Results As shown in Fig. 1, all oil intake groups exhibited significantly longer survival than that of the PBS intake group (p < 0.05) after 2
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Fig. 3. Effect of oil intake on the dietary energy intake (A) and total energy intake (B) of mice. Mice were treated with PBS, olive oil, high oleic safflower oil (Safflower-HO), or high linoleic safflower oil (Safflower-HL) for 2 weeks. Dietary caloric intake was calculated based on the weight of food residue for each experimental group (5 mice/group).
Additionally, oxidative stress, immune responses, and alterations in the FA composition of host lipids might inhibit the development of clinical symptoms and/or parasite growth. Dietary caloric intake is also predicted to affect the course of infection. In sheep experimentally infected with T. congolense, a low-energy diet results in a longer prepatent period but more severe anemia following patency and greater growth retardation than those for a highenergy diet (Katunguka-Rwakishaya et al., 1999). In this study, the dietary caloric intake of oil-treated uninfected mice tended to be lower than that of PBS-treated mice (Fig. 3A), while the total caloric intake, including diet and oils, was similar in all groups (Fig. 3B). Therefore, total caloric intake might be maintained at a constant level based on dietary requirements. Furthermore, the four vegetable oils contained the same energy, despite different plant sources. However, the extent of the protective effect against T. congolense infection was not equivalent among these oils. Therefore, an improvement in the nutritional status of the host by caloric intake might not influence the rescue of mice infected with T. congolense. In this study, the percentage of total caloric intake derived from fats, including oils, was high. High fat diets (HFDs) are associated with obesity and elevated basal and postprandial blood sugar values, particularly when the fat content exceeds 30% of the energy (Buettner et al., 2007), and the effects depend on the taxon, fat type, and feeding duration (Buettner et al., 2007). A/J and C57BL/6 mice are resistant and sensitive to diet-induced steatohepatitis and obesity, respectively (Poussin et al., 2011; Kakimoto and Kowaltowski, 2016). The liver proinflammatory state is greater in C57BL/6 mice than in A/J mice, and oxygen consumption in mitochondria from A/J mice is higher than that in C57BL/6 mice when they are fed a HFD containing 58% energy from fat derived mainly from coconut oil (Hall et al., 2010; Poussin et al., 2011; Kakimoto and Kowaltowski, 2016). Rodents, unlike humans, are considered obese when their body weights or body fat contents are elevated, and are not usually evaluated by the measurement of body mass indexes (Hariri and Thibault, 2010; Kakimoto and Kowaltowski, 2016). Feeding a HFD containing fat mainly from coconut oil for 10 days does not affect the body weight of C57BL/6 mice. Their liver mitochondria energized by succinate or palmitoylcarnitine exhibit greater oxygen consumption than that of mice fed with normal chow (Poussin et al., 2011; Kakimoto and Kowaltowski, 2016). In mice, the upregulation of the pathways involved in reactive oxygen species (ROS)
There was no statistical difference between mice treated with each oil on parasitemia. In the uninfected groups, the dietary caloric intake, calculated by the weight of food residues, tended to be lower in oil-treated mice than in PBS-treated mice (Fig. 3A). Total caloric intake, including diet and oils, was similar in all groups (Fig. 3B). In addition, there were no significant differences in the rate of change of body weight between uninfected oil-treated groups and the uninfected PBS-treated group (Fig. 4). 4. Discussion The results of this study indicated that vegetable oil intake affects the course of T. congolense infection in mice (Figs. 1 and 2). In addition, the effects of oil intake varied among oils. Several factors were suspected to lead to the rescue of mice infected with T. congolense. Energy intake might simply improve the nutritional status of hosts.
Fig. 4. Effect of oil intake on the rate of change in mouse body weight. Mice were treated with PBS, olive oil, high oleic safflower oil (Safflower-HO), or high linoleic safflower oil (Safflower-HL) for 2 weeks. Rates of change were estimated as the body weight on a particular day divided by the body weight at day 0. There were no significant differences among groups. 3
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lipoproteins by a lipoprotein scavenger receptor. In addition, the plasma membrane of T. brucei BSF has a high degree of fluidity and the membrane fluidity of parasites is suspected to be a vital factor for BSF survival (Harrington et al., 2012). Dietary FA intake influences the relative FA composition of lipoproteins and biological membranes, including mitochondrial membranes (Ney et al., 1989; Quiles et al., 2001; Varela-López et al., 2015). Rats fed with 12% (w/w) olive or high oleic safflower oil show higher very low density lipoprotein (VLDL) concentrations and distinctly larger triglyceride-enriched VLDL particles than those of rats fed high linoleic safflower oil (Ney et al., 1989). The characteristics and functions of biological membranes, such as sensitivity to oxidative stress and membrane fluidity, are strongly influenced by the FA composition of the membrane (Varela-López et al., 2015). Hence, oil intake might affect parasites via alterations of the FA composition of host lipids utilized by parasites. Drug delivery systems of a lipid-based formulation for oral administration generally consist of a drug dissolved in blends of two or more excipients, which may be triglyceride oils, partial glycerides, surfactants, or co-surfactants. Oils in the systems are blends of food glycerides derived from vegetable oils (Pouton, 2000; Pouton and Porter, 2008). The lipid-based formulation is designed for improved oral absorption. The strategy to maintain supersaturated drug concentration in vivo is achieved through dissolving the hydrophobic drug in lipid-based formulation, emulsifying the oil phase in water, and maintaining the drug in the solubilised state in the gastrointestinal tract until absorption has occurred (Efendy Goon et al., 2019). Several successful oral pharmaceutical products have recently been marketed as lipid systems, including cyclosporin A and the two protease inhibitors ritonavir and saquinavir (Pouton, 2000). Since the current anti animal anti-trypanosomal drugs are administered by intramuscular, intravenous and/or subcutaneous injection, not oral administration (Giordani et al., 2016), oils could not be used as solvent for these drugs. However, the chance for new drug development might be expanded by lipid-based drug delivery systems. Currently, self-nanoemulsifying drug delivery systems have been researched for the development of highly effective and efficient new drug delivery systems. And other forms of lipid-based formulations such as solid lipid particles, nanostructured lipid carriers, solid dispersions and nanocapsules have also emerged and providing promising results (Efendy Goon et al., 2019). Most often, vegetable oils are incorporated into nanoemulsions, not just for nanoencapsulating several therapeutic classes of drugs inside an oil core (Bajerski et al., 2016). This approach enhances the action of drugs, and enables the use of much lower concentrations of them without problems related to toxicity, resistance and side effects (Bajerski et al., 2016). The nanoencapsulation allows vegetable oils to use on the alternative routes of administration including skin, intravenous, oral and inhalatory forms (Bajerski et al., 2016). In addition, the choice of the vegetable oils also allows to modulate the control of the drug release from lipid-core nanocapsules (Rigo et al., 2014). The research conducted with soybean oil, sunflower oils and rice bran oil showed that the type of vegetable oil did not affect the physicochemical characteristic of the lipid-core nanocapsules but affected their drug release control (Rigo et al., 2014). Our results indicated that oils may be useful for lipid-based anti-trypanosome drug delivery systems, not only for the improvement of drug biokinetics, such as absorption, distribution, metabolism, and excretion but also for the inhibition of pathogenic mechanisms. Since oil administration by oral route was started before T. congolense infection in this study, the preventive effect of vegetable oils against Trypanosomiasis was shown. It means that the oils could be used as a part of regular animal diet for prevention method. On the other hand, it is unknown whether oils affect to T. congolense infection when oil treatment was started after infection. Hence, future studies are needed to investigate the therapeutic effects of vegetable oils on animals that are infested with trypanosomes. In addition, the further investigation of the anti-trypanosomal effect of oils extracted from crops traditionally used as animal diet such as cottonseed cake is required to
production and oxidative stress are coordinately induced by HFD intake in both the liver and adipose tissues prior to the onset of metabolic dysfunction, such as insulin resistance, by a discrete mechanism (Matsuzawa-Nagata et al., 2008). In this study, there were no significant differences in the rate of change of body weight between all uninfected oil-treated groups and the uninfected PBS-treated group (Fig. 4). However, energy intake in oil-treated mice was approximately 27% from fat and 16% from oils. It is possible that oil-treated mice developed metabolic dysfunctions, but metabolic alterations were not evaluated in this study. In a previous study, C57BL/6 male mice were fed HFDs with different concentrations of SFAs (6%, 12%, and 24%) and identical total fat contents (40%) for 16 weeks; the 12% SFA group gained more body and adipose tissue weight than the groups fed the control diet containing 12.2–12.5% fat and other SFA concentrations (Enos et al., 2013). In addition, 12% SFA resulted in the greatest degree of dysregulation in oxidative stress, endoplasmic reticulum stress, and inflammation (Enos et al., 2014; Kakimoto and Kowaltowski, 2016). Accordingly, the concentration of SFA might be related to the survival rate in this experiment (Fig. 1). Since trypanosomes lack glutathione reductase, thioredoxin reductase, catalase, and selenium-containing glutathione peroxidases, they are vulnerable to oxidative stress (Torrie et al., 2009; Banerjee et al., 2017). Intracellular ROS generation is targeted by several antitrypanosome drugs (Fotie et al., 2010; Banerjee et al., 2017). Trypanosomes are also influenced by the oxidative status of host animals. Mice deficient in α-tocopherol, a potent anti-oxidant, exhibit resistance to T. congolense infection owing to oxidative damage to parasite DNA (Herbas et al., 2009). An HFD induces oxidative stress in animals. HFD intake increases the level of chylomicrons in the intestine. These chylomicrons enter the circulation and result in the generation of free fatty acids, which are taken up by the liver (Cole et al., 2011; Tan et al., 2018). This promotes the mitochondrial β-oxidation of free fatty acids and leads to an excess electron flow from cytochrome-c oxidase, which elevates ROS accumulation. Moreover, HFD-induced ROS may stimulate the proinflammatory state and thereby activate NF-κB, which induces NF-κB-dependent proinflammatory agents, such as tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and interferon-γ (IFN-γ) (Ruggiero et al., 2011; Tan et al., 2018). Pre-stimulation by IFN-γ is required to activate macrophage responses to parasites (Drennan et al., 2005; Dos-Santos et al., 2016). Macrophage activation is very important for the immune response to parasites during the early stage of Trypanosoma infection (Stijlemans et al., 2010), which is associated with an increase in Th1 cytokines, such as IFN-γ and TNF-α (Onyilagha et al., 2017). Nitric oxide (NO), ROS, and TNF-α have trypanocidal activity in vitro and activate macrophage production (Drennan et al., 2005; Stijlemans et al., 2010; Dos-Santos et al., 2016). Therefore, ROS production and the associated immune response caused by oil intake might affect T. congolense infection. Cells involved in the metabolic system and immune response show evidence for coordination and coevolution (Ruiz-Núñez et al., 2016). During Trypanosoma infection, Toll-like receptors (TLRs) activate innate immunity for protection against parasites (Bafica et al., 2006; DosSantos et al., 2016). TLR2 and TLR4 recognize glycoisnositol phospholipids and glycosylphosphatidylinositol, which coat the surface of Trypanosoma spp (Drennan et al., 2005; Dos-Santos et al., 2016). TLR2 and TLR4 recognize lipid-based structures and trigger the induction of inflammatory cytokines (Kawai and Akira, 2005; O'Neill, 2006). SFA causes adipose tissue inflammation by the activation of TLR4 and TLR2 (O'Neill, 2006; Ruiz-Núñez et al., 2016). In our study, coconut oil, which consists of mainly SFAs, strongly suppressed the development of parasitemia at the early stage (Fig. 2). This may be explained by TLR activation. T. brucei bloodstream forms (BSFs) reportedly regulate FA synthesis in response to host environmental FAs (Lee et al., 2006). Trypanosomes bind to and take up low-density lipoproteins and high-density 4
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clarify the effectiveness and advantage of oils used as parts of regular animal diet on controlling Trypanosomiasis. The results of the present study indicated that vegetable oils might be beneficial for the usage as animal diet, and the development of combination therapies with lipophilic anti-trypanosome drugs to obtain additive and synergistic effects. However, the vegetable oils were not suitable solvents to investigate the efficacy of lipophilic agents against T. congolense owing to their potential anti-T. congolense effects.
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Declaration of competing interest The authors declare that they have no competing interests. Acknowledgments We would like to thank Editage (www.editage.jp) for English language editing. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.exppara.2020.107845. References Bafica, A., Santiago, H.C., Goldszmid, R., Ropert, C., Gazzinelli, R.T., Sher, A., 2006. Cutting edge: TLR9 and TLR2 signaling together account for MyD88-dependent control of parasitemia in Trypanosoma cruzi infection. J. Immunol. 177, 3515–3519. https://doi.org/10.4049/jimmunol.177.6.3515. Bajerski, L., Michels, L.R., Colomé, L.M., Bender, E.A., Freddo, R.J., Bruxel, F., Haas, S.E., 2016. The use of Brazilian vegetable oils in nanoemulsions: an update on preparation and biological applications. Braz. J. Pharm. Sci. 52, 347–363. https://doi.org/10. 1590/s1984-82502016000300001. Banerjee, M., Parai, D., Dhar, P., Roy, M., Barik, R., Chattopadhyay, S., Mukherjee, S.K., 2017. Andrographolide induces oxidative stress-dependent cell death in unicellular protozoan parasite Trypanosoma brucei. Acta Trop. 176, 58–67. https://doi.org/10. 1016/j.actatropica.2017.07.023. Barrett, M.P., Burchmore, R.J., Stich, A., Lazzari, J.O., Frasch, A.C., Cazzulo, J.J., Krishna, S., 2003. The trypanosomiases. Lancet 362, 1469–1480. https://doi.org/10.1016/ S0140-6736(03)14694-6. Buettner, R., Schölmerich, J., Bollheimer, L.C., 2007. High-fat diets: modeling the metabolic disorders of human obesity in rodents. Obesity 15, 798–808. https://doi.org/ 10.1038/oby.2007.608. Cao, S., Zhu, Q.H., Shen, W., Jiao, X., Zhao, X., Wang, M.B., Liu, L., Singh, S.P., Liu, Q., 2013. Comparative profiling of miRNA expression in developing seeds of high linoleic and high oleic safflower (Carthamus tinctorius L.) plants. Front. Plant Sci. 4, 489. https://doi.org/10.3389/fpls.2013.00489. Cole, M.A., Murray, A.J., Cochlin, L.E., Heather, L.C., McAleese, S., Knight, N.S., Sutton, E., Jamil, A.A., Parassol, N., Clarke, K., 2011. A high fat diet increases mitochondrial fatty acid oxidation and uncoupling to decrease efficiency in rat heart. Basic Res. Cardiol. 106, 447–457. https://doi.org/10.1007/s00395-011-0156-1. Connor, R.J., 1991. The diagnosis, treatment and prevention of animal trypanosomiasis under field conditions. In: Programme for the Control of African Animal Trypanosomiasis and Related Development: Ecological and Technical Aspects. Food and Agriculture Organisation of the United Nations (FAO), Rome, Italy FAO Animal Production and Health Paper No. 100. http://www.fao.org/docrep/004/T0599E/ T0599E01.htm#ch1. Dos-Santos, A.L., Carvalho-Kelly, L.F., Dick, C.F., Meyer-Fernandes, J.R., 2016. Innate immunomodulation to trypanosomatid parasite infections. Exp. Parasitol. 167, 67–75. https://doi.org/10.1016/j.exppara.2016.05.005. Drennan, M.B., Stijlemans, B., Van den Abbeele, J., Quesniaux, V.J., Barkhuizen, M., Brombacher, F., De Baetselier, P., Ryffel, B., Magez, S., 2005. The induction of a type 1 immune response following a Trypanosoma brucei infection is MyD88 dependent. J. Immunol. 175, 2501–2509. https://doi.org/10.4049/jimmunol.175.4.2501. Efendy Goon, D., Sheikh Abdul Kadir, S.H., Latip, N.A., Ab Rahim, S., Mazlan, M., 2019. Palm oil in lipid-based formulations and drug delivery systems. Biomolecules 9, E64. https://doi.org/10.3390/biom9020064. Enos, R.T., Davis, J.M., Velázquez, K.T., McClellan, J.L., Day, S.D., Carnevale, K.A., Murphy, E.A., 2013. Influence of dietary saturated fat content on adiposity, macrophage behavior, inflammation, and metabolism: composition matters. J. Lipid Res. 54, 152–163. https://doi.org/10.1194/jlr.M030700. Enos, R.T., Velázquez, K.T., Murphy, E.A., 2014. Insight into the impact of dietary saturated fat on tissue-specific cellular processes underlying obesity-related diseases. J. Nutr. Biochem. 25, 600–612. https://doi.org/10.1016/j.jnutbio.2014.01.011. Fotie, J., Kaiser, M., Delfín, D.A., Manley, J., Reid, C.S., Paris, J.M., Wenzler, T., Maes, L., Mahasenan, K.V., Li, C., Werbovetz, K.A., 2010. Antitrypanosomal activity of 1,2dihydroquinolin-6-ols and their ester derivatives. J. Med. Chem. 53, 966–982. https://doi.org/10.1021/jm900723w.
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Effect of vegetable oils on the experimental infection of mice with Trypanosoma congolense
T
Aiko Kume, Keisuke Suganuma, Rika Umemiya-Shirafuji, Hiroshi Suzuki∗ National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada-cho, Obihiro, Hokkaido, 080-8555, Japan
A R T I C LE I N FO
A B S T R A C T
Keywords: Trypanosoma congolense Vegetable oil Mice Fatty acid Solvent
Vegetable oils are frequently used as solvents for lipophilic materials; accordingly, the effects of their components should be considered in animal experiments. In this study, the effects of various vegetable oils on the course of Trypanosoma congolense infection were examined in mice. C57BL/6J mice were orally administered four kinds of oils (i.e., coconut oil, olive oil, high oleic safflower oil, and high linoleic safflower oil) with different fatty acid compositions and infected with T. congolense IL-3000. Oil-treated mice infected with T. congolense showed significantly higher survival rates and lower parasitemia than those of control mice. Notably, coconut oil, which mainly consists of saturated fatty acids, delayed the development of parasitemia at the early stage of infection. These results indicated that vegetable oil intake could affect T. congolense infection in mice. These findings have important practical implications; for example, they suggest the potential effectiveness of vegetable oils as a part of the regular animal diet for controlling tropical diseases and indicate that vegetable oils are not suitable solvents for studies of the efficacy of lipophilic agents against T. congolense.
1. Introduction Trypanosomes are kinetoplastid protozoans that cause fatal diseases in livestock and humans. These parasites proliferate extracellularly in blood and tissue fluids of their mammalian hosts and are cyclically transmitted by the tsetse fly (Glossina spp.). Humans have sensitivity to two species of trypanosomes, Trypanosoma brucei rhodesiense and T. b. gambiense (Barrett et al., 2003; Giordani et al., 2016). Animal African trypanosomiasis (AAT), called Nagana, is caused by T. congolense, T. vivax, and, to a lesser extent, T. brucei spp. and is widespread in subSaharan Africa (Giordani et al., 2016). AAT in livestock leads to acute and/or chronic cases of wasting syndrome, causing high morbidity, mortality, and infertility in the absence of suitable treatment (Leach and Roberts, 1981; Connor, 1991; Giordani et al., 2016). Control strategies for AAT rely primarily on vector control using insecticides or traps and on parasite control using trypanocides (Holmes, 2013; Giordani et al., 2016). Despite the effectiveness of vector control methods for AAT, such programs are expensive and frequent re-infestation occurs (Holmes, 2013). Currently, only 6 trypanocidal compounds are used as therapeutic and/or prophylactic drugs against AAT (i.e., diminazene aceturate, homidium salts, isometamidium chloride, quinapyramine sulphate, suramin sodium, and melarsomine dihydrochloride), and many of these have been used since
around the first half of the 20th century (Giordani et al., 2016). In addition, there are five drugs for Human African Trypanosomiasis (HAT) (i.e., eflornithine, melarsoprol, nifurtimox, pentamidine and suramin) (Thomas et al., 2018), and eflornithine is the only new treatment registered for HAT in the past six decades (Priotto et al., 2009). However, these drugs have unsatisfactory therapeutic effects and often have severe side effects. Their extensive utilization has led to the emergence of drug-resistant parasites. Moreover, many of these drugs are chemically related, which has exacerbated the situation by resulting in cross-resistance (Peregrine, 1994; Giordani et al., 2016). The beneficial effects of foods or oils on the bioavailability of hydrophobic drugs have been described previously (Humberstone and Charman, 1997; Pouton, 2000). Thus, vegetable oils are frequently used as solvents for lipophilic materials in animal experiments and have advantages with respect to safety and drug stability (Pouton and Porter, 2008). However, for the interpretation of experimental data, it is essential to understand the effects of solvent components. Vegetable oils consist of a mixture of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs), and the fatty acid (FA) compositions specifically depend on the plant sources (Orsavova et al., 2015). For example, coconut oil and olive oil are mainly composed of lauric and myristic acids (SFAs) and oleic acid (MUFA), respectively (Orsavova et al., 2015). Classic safflower oil
∗ Corresponding author. Research Unit for Functional Genomics, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Nishi 2-13, Inada, Obihiro, Hokkaido, 080-8555, Japan. E-mail address: [email protected] (H. Suzuki).
https://doi.org/10.1016/j.exppara.2020.107845 Received 23 February 2019; Received in revised form 17 October 2019; Accepted 24 January 2020 Available online 28 January 2020 0014-4894/ © 2020 Elsevier Inc. All rights reserved.
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normally contains 10–15% oleic acid (MUFA) and over 70% linoleic acid (PUFA), and another safflower oil made from a natural mutant contains up to 70% oleic acid (MUFA) (Cao et al., 2013; Orsavova et al., 2015). FAs composed of lipids are classified into three types according to the absence or presence of double bonds in the structural formula. SFAs have no double bond, MUFAs have one, and PUFAs have two or more double bonds (Orsavova et al., 2015). In the present study, the effects of various vegetable oils on the course of T. congolense infection were examined in mice. C57BL/6J mice were orally administered 4 kinds of oils, coconut oil, olive oil, high oleic safflower oil, and high linoleic safflower oil, with different FA compositions, followed by infection with T. congolense IL-3000. 2. Materials and methods 2.1. Mice C57BL/6J mice were bred and maintained in specific pathogen-free conditions at the National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Japan. Male mice aged 8–12 weeks were used. The temperature (24 ± 1 °C) and humidity (50 ± 10%) were regulated and lighting was controlled (lights on from 7:00 to 19:00). Mice had free access to water and a commercial regular diet (3.4 kcal/g, containing 4.88% crude fat, CA-1; CLEA Japan, Tokyo, Japan). All animal experiments were conducted in accordance with the standards of the Care and Management of Experimental Animals of Obihiro University of Agriculture and Veterinary Medicine, Japan.
Fig. 1. Effect of oil intake on the survival rate of T. congolense-infected mice. Mice were treated with PBS, olive oil, high oleic safflower oil (Safflower-HO), or high linoleic safflower oil (Safflower-HL) from 3 weeks before infection to 1 week after infection. Compared with the PBS group, all oil treatment groups showed significantly higher survival rates. *p < 0.05.
infection with T. congolense IL-3000. There were no statistically significant differences in the survival rate among the experimental groups. The median survival times of T. congolense IL-3000-infected mice treated with PBS, coconut oil, olive oil, high oleic safflower oil, and high linoleic safflower oil were 8.0, 26.0, 25.0, 22.0, and 12.0 dpi, respectively. The median survival times for the coconut oil, olive oil, and high oleic safflower oil groups were longer than that for the high linoleic safflower oil group. All oil intake groups showed significantly lower parasitemia than that of the PBS group on 4 and 6 dpi (Fig. 2). In addition, oil intake, especially coconut oil, tended to delay the detection of parasites in peripheral blood compared with PBS treatment.
2.2. Effect of oil intake on T. congolense IL-3000 infection in mice C57BL/6J male mice were orally administered 0.2 ml of coconut oil (Cocowell, Osaka, Japan), olive oil (Nisshin Oillio Group, Tokyo, Japan), high oleic safflower oil (Ajunomoto, Tokyo, Japan), and high linoleic safflower oil (Holbein Works, Osaka, Japan) or PBS by a feeding needle once a day for 3 weeks and were then inoculated with 3 × 104 cells of T. congolense IL-3000 (International Livestock Research Institute, Nairobi, Kenya) by intraperitoneal injection. Oil treatment was continued for 1 week after infection. Parasitemia was monitored after the infection once every 2 days from 2 to 12 days post infection (dpi) and once every 3–6 days from 15 to 32 dpi. Blood samples for parasitemia estimates were collected from the tails of mice. To account for stress caused by handling, PBS was administered as a control. 2.3. Effect of oil intake on body weight and diet intake C57BL/6J male mice were treated with 4 kinds of oils or PBS (0.2 ml/head) by a feeding needle once a day for 2 weeks. During the experimental period, mouse body weight was measured every 2 days and the weight of food residue was measured every day. Dietary caloric intake was estimated by the difference in weight between food provided and food remaining for each experimental group. The daily caloric intake from oils (0.91 g/ml) was calculated as 1.62 kcal/head. 2.4. Statistical analysis Parasitemia and body weights were analyzed by Dunnett's tests. The survival rate was analyzed using the log-rank (Mantel-Cox) and GehanBreslow-Wilcoxon tests implemented in GraphPad Prism 5. For all analyses, a p-value of less than 0.05 was considered statistically significant.
Fig. 2. Effect of oil intake on parasitemia in T. congolense-infected mice. Mice were treated with PBS, olive oil, high oleic safflower oil (Safflower-HO), or high linoleic safflower oil (Safflower-HL) from 3 weeks before infection to 1 week after infection. The lower graph is an enlarged image of the upper plot from day 0 to day 10. Compared with the PBS group, the coconut oil, olive oil, and high oleic safflower oil treatment groups showed significantly lower parasitemia at 3, 4, 5, and 6 dpi. High linoleic safflower oil-treated mice showed significantly lower parasitemia than that of PBS-treated mice on 4, 5, and 6 dpi. *p < 0.05.
3. Results As shown in Fig. 1, all oil intake groups exhibited significantly longer survival than that of the PBS intake group (p < 0.05) after 2
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Fig. 3. Effect of oil intake on the dietary energy intake (A) and total energy intake (B) of mice. Mice were treated with PBS, olive oil, high oleic safflower oil (Safflower-HO), or high linoleic safflower oil (Safflower-HL) for 2 weeks. Dietary caloric intake was calculated based on the weight of food residue for each experimental group (5 mice/group).
Additionally, oxidative stress, immune responses, and alterations in the FA composition of host lipids might inhibit the development of clinical symptoms and/or parasite growth. Dietary caloric intake is also predicted to affect the course of infection. In sheep experimentally infected with T. congolense, a low-energy diet results in a longer prepatent period but more severe anemia following patency and greater growth retardation than those for a highenergy diet (Katunguka-Rwakishaya et al., 1999). In this study, the dietary caloric intake of oil-treated uninfected mice tended to be lower than that of PBS-treated mice (Fig. 3A), while the total caloric intake, including diet and oils, was similar in all groups (Fig. 3B). Therefore, total caloric intake might be maintained at a constant level based on dietary requirements. Furthermore, the four vegetable oils contained the same energy, despite different plant sources. However, the extent of the protective effect against T. congolense infection was not equivalent among these oils. Therefore, an improvement in the nutritional status of the host by caloric intake might not influence the rescue of mice infected with T. congolense. In this study, the percentage of total caloric intake derived from fats, including oils, was high. High fat diets (HFDs) are associated with obesity and elevated basal and postprandial blood sugar values, particularly when the fat content exceeds 30% of the energy (Buettner et al., 2007), and the effects depend on the taxon, fat type, and feeding duration (Buettner et al., 2007). A/J and C57BL/6 mice are resistant and sensitive to diet-induced steatohepatitis and obesity, respectively (Poussin et al., 2011; Kakimoto and Kowaltowski, 2016). The liver proinflammatory state is greater in C57BL/6 mice than in A/J mice, and oxygen consumption in mitochondria from A/J mice is higher than that in C57BL/6 mice when they are fed a HFD containing 58% energy from fat derived mainly from coconut oil (Hall et al., 2010; Poussin et al., 2011; Kakimoto and Kowaltowski, 2016). Rodents, unlike humans, are considered obese when their body weights or body fat contents are elevated, and are not usually evaluated by the measurement of body mass indexes (Hariri and Thibault, 2010; Kakimoto and Kowaltowski, 2016). Feeding a HFD containing fat mainly from coconut oil for 10 days does not affect the body weight of C57BL/6 mice. Their liver mitochondria energized by succinate or palmitoylcarnitine exhibit greater oxygen consumption than that of mice fed with normal chow (Poussin et al., 2011; Kakimoto and Kowaltowski, 2016). In mice, the upregulation of the pathways involved in reactive oxygen species (ROS)
There was no statistical difference between mice treated with each oil on parasitemia. In the uninfected groups, the dietary caloric intake, calculated by the weight of food residues, tended to be lower in oil-treated mice than in PBS-treated mice (Fig. 3A). Total caloric intake, including diet and oils, was similar in all groups (Fig. 3B). In addition, there were no significant differences in the rate of change of body weight between uninfected oil-treated groups and the uninfected PBS-treated group (Fig. 4). 4. Discussion The results of this study indicated that vegetable oil intake affects the course of T. congolense infection in mice (Figs. 1 and 2). In addition, the effects of oil intake varied among oils. Several factors were suspected to lead to the rescue of mice infected with T. congolense. Energy intake might simply improve the nutritional status of hosts.
Fig. 4. Effect of oil intake on the rate of change in mouse body weight. Mice were treated with PBS, olive oil, high oleic safflower oil (Safflower-HO), or high linoleic safflower oil (Safflower-HL) for 2 weeks. Rates of change were estimated as the body weight on a particular day divided by the body weight at day 0. There were no significant differences among groups. 3
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lipoproteins by a lipoprotein scavenger receptor. In addition, the plasma membrane of T. brucei BSF has a high degree of fluidity and the membrane fluidity of parasites is suspected to be a vital factor for BSF survival (Harrington et al., 2012). Dietary FA intake influences the relative FA composition of lipoproteins and biological membranes, including mitochondrial membranes (Ney et al., 1989; Quiles et al., 2001; Varela-López et al., 2015). Rats fed with 12% (w/w) olive or high oleic safflower oil show higher very low density lipoprotein (VLDL) concentrations and distinctly larger triglyceride-enriched VLDL particles than those of rats fed high linoleic safflower oil (Ney et al., 1989). The characteristics and functions of biological membranes, such as sensitivity to oxidative stress and membrane fluidity, are strongly influenced by the FA composition of the membrane (Varela-López et al., 2015). Hence, oil intake might affect parasites via alterations of the FA composition of host lipids utilized by parasites. Drug delivery systems of a lipid-based formulation for oral administration generally consist of a drug dissolved in blends of two or more excipients, which may be triglyceride oils, partial glycerides, surfactants, or co-surfactants. Oils in the systems are blends of food glycerides derived from vegetable oils (Pouton, 2000; Pouton and Porter, 2008). The lipid-based formulation is designed for improved oral absorption. The strategy to maintain supersaturated drug concentration in vivo is achieved through dissolving the hydrophobic drug in lipid-based formulation, emulsifying the oil phase in water, and maintaining the drug in the solubilised state in the gastrointestinal tract until absorption has occurred (Efendy Goon et al., 2019). Several successful oral pharmaceutical products have recently been marketed as lipid systems, including cyclosporin A and the two protease inhibitors ritonavir and saquinavir (Pouton, 2000). Since the current anti animal anti-trypanosomal drugs are administered by intramuscular, intravenous and/or subcutaneous injection, not oral administration (Giordani et al., 2016), oils could not be used as solvent for these drugs. However, the chance for new drug development might be expanded by lipid-based drug delivery systems. Currently, self-nanoemulsifying drug delivery systems have been researched for the development of highly effective and efficient new drug delivery systems. And other forms of lipid-based formulations such as solid lipid particles, nanostructured lipid carriers, solid dispersions and nanocapsules have also emerged and providing promising results (Efendy Goon et al., 2019). Most often, vegetable oils are incorporated into nanoemulsions, not just for nanoencapsulating several therapeutic classes of drugs inside an oil core (Bajerski et al., 2016). This approach enhances the action of drugs, and enables the use of much lower concentrations of them without problems related to toxicity, resistance and side effects (Bajerski et al., 2016). The nanoencapsulation allows vegetable oils to use on the alternative routes of administration including skin, intravenous, oral and inhalatory forms (Bajerski et al., 2016). In addition, the choice of the vegetable oils also allows to modulate the control of the drug release from lipid-core nanocapsules (Rigo et al., 2014). The research conducted with soybean oil, sunflower oils and rice bran oil showed that the type of vegetable oil did not affect the physicochemical characteristic of the lipid-core nanocapsules but affected their drug release control (Rigo et al., 2014). Our results indicated that oils may be useful for lipid-based anti-trypanosome drug delivery systems, not only for the improvement of drug biokinetics, such as absorption, distribution, metabolism, and excretion but also for the inhibition of pathogenic mechanisms. Since oil administration by oral route was started before T. congolense infection in this study, the preventive effect of vegetable oils against Trypanosomiasis was shown. It means that the oils could be used as a part of regular animal diet for prevention method. On the other hand, it is unknown whether oils affect to T. congolense infection when oil treatment was started after infection. Hence, future studies are needed to investigate the therapeutic effects of vegetable oils on animals that are infested with trypanosomes. In addition, the further investigation of the anti-trypanosomal effect of oils extracted from crops traditionally used as animal diet such as cottonseed cake is required to
production and oxidative stress are coordinately induced by HFD intake in both the liver and adipose tissues prior to the onset of metabolic dysfunction, such as insulin resistance, by a discrete mechanism (Matsuzawa-Nagata et al., 2008). In this study, there were no significant differences in the rate of change of body weight between all uninfected oil-treated groups and the uninfected PBS-treated group (Fig. 4). However, energy intake in oil-treated mice was approximately 27% from fat and 16% from oils. It is possible that oil-treated mice developed metabolic dysfunctions, but metabolic alterations were not evaluated in this study. In a previous study, C57BL/6 male mice were fed HFDs with different concentrations of SFAs (6%, 12%, and 24%) and identical total fat contents (40%) for 16 weeks; the 12% SFA group gained more body and adipose tissue weight than the groups fed the control diet containing 12.2–12.5% fat and other SFA concentrations (Enos et al., 2013). In addition, 12% SFA resulted in the greatest degree of dysregulation in oxidative stress, endoplasmic reticulum stress, and inflammation (Enos et al., 2014; Kakimoto and Kowaltowski, 2016). Accordingly, the concentration of SFA might be related to the survival rate in this experiment (Fig. 1). Since trypanosomes lack glutathione reductase, thioredoxin reductase, catalase, and selenium-containing glutathione peroxidases, they are vulnerable to oxidative stress (Torrie et al., 2009; Banerjee et al., 2017). Intracellular ROS generation is targeted by several antitrypanosome drugs (Fotie et al., 2010; Banerjee et al., 2017). Trypanosomes are also influenced by the oxidative status of host animals. Mice deficient in α-tocopherol, a potent anti-oxidant, exhibit resistance to T. congolense infection owing to oxidative damage to parasite DNA (Herbas et al., 2009). An HFD induces oxidative stress in animals. HFD intake increases the level of chylomicrons in the intestine. These chylomicrons enter the circulation and result in the generation of free fatty acids, which are taken up by the liver (Cole et al., 2011; Tan et al., 2018). This promotes the mitochondrial β-oxidation of free fatty acids and leads to an excess electron flow from cytochrome-c oxidase, which elevates ROS accumulation. Moreover, HFD-induced ROS may stimulate the proinflammatory state and thereby activate NF-κB, which induces NF-κB-dependent proinflammatory agents, such as tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and interferon-γ (IFN-γ) (Ruggiero et al., 2011; Tan et al., 2018). Pre-stimulation by IFN-γ is required to activate macrophage responses to parasites (Drennan et al., 2005; Dos-Santos et al., 2016). Macrophage activation is very important for the immune response to parasites during the early stage of Trypanosoma infection (Stijlemans et al., 2010), which is associated with an increase in Th1 cytokines, such as IFN-γ and TNF-α (Onyilagha et al., 2017). Nitric oxide (NO), ROS, and TNF-α have trypanocidal activity in vitro and activate macrophage production (Drennan et al., 2005; Stijlemans et al., 2010; Dos-Santos et al., 2016). Therefore, ROS production and the associated immune response caused by oil intake might affect T. congolense infection. Cells involved in the metabolic system and immune response show evidence for coordination and coevolution (Ruiz-Núñez et al., 2016). During Trypanosoma infection, Toll-like receptors (TLRs) activate innate immunity for protection against parasites (Bafica et al., 2006; DosSantos et al., 2016). TLR2 and TLR4 recognize glycoisnositol phospholipids and glycosylphosphatidylinositol, which coat the surface of Trypanosoma spp (Drennan et al., 2005; Dos-Santos et al., 2016). TLR2 and TLR4 recognize lipid-based structures and trigger the induction of inflammatory cytokines (Kawai and Akira, 2005; O'Neill, 2006). SFA causes adipose tissue inflammation by the activation of TLR4 and TLR2 (O'Neill, 2006; Ruiz-Núñez et al., 2016). In our study, coconut oil, which consists of mainly SFAs, strongly suppressed the development of parasitemia at the early stage (Fig. 2). This may be explained by TLR activation. T. brucei bloodstream forms (BSFs) reportedly regulate FA synthesis in response to host environmental FAs (Lee et al., 2006). Trypanosomes bind to and take up low-density lipoproteins and high-density 4
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clarify the effectiveness and advantage of oils used as parts of regular animal diet on controlling Trypanosomiasis. The results of the present study indicated that vegetable oils might be beneficial for the usage as animal diet, and the development of combination therapies with lipophilic anti-trypanosome drugs to obtain additive and synergistic effects. However, the vegetable oils were not suitable solvents to investigate the efficacy of lipophilic agents against T. congolense owing to their potential anti-T. congolense effects.
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