Effects of Squalene/Squalane on Dopamine Levels, Antioxidant

Journal of Oleo Science Copyright ©2013 by Japan Oil Chemists’ Society J. Oleo Sci. 62, (1) 21-28 (2013) Effects of squalene/squalane on dopamine levels, antioxidant enzyme activity, and fatty acid composition in the striatum of Parkinson’s disease mouse model Hideaki Kabuto1＊ , Tomoko T. Yamanushi1, Najma Janjua1, Fusako Takayama2 and Mitsumasa Mankura2 1 Department of Health Sciences, Kagawa Prefectural University of Health Sciences, 281-1, Mure-cho, Takamatsu 761-0123, Japan. 2 ‌Faculty of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Kitaku, Okayama 700-8530, Japan. Abstract: Active oxygen has been implicated in the pathogenesis of Parkinson’s disease (PD); therefore, antioxidants have attracted attention as a potential way to prevent this disease. Squalene, a natural triterpene and an intermediate in the biosynthesis of cholesterol, is known to have active oxygen scavenging activities. Squalane, synthesized by complete hydrogenation of squalene, does not have active oxygen scavenging activities. We examined the effects of oral administration of squalene or squalane on a PD mouse model, which was developed by intracerebroventricular injection of 6-hydroxydopamine (6-OHDA). Squalene administration 7 days before and 7 days after one 6-OHDA injection prevented a reduction in striatal dopamine (DA) levels, while the same administration of squalane enhanced the levels. Neither squalene nor squalane administration for 7 days changed the levels of catalase, glutathione peroxidase, or superoxide dismutase activities in the striatum. Squalane increased thiobarbituric acid reactive substances, a marker of lipid peroxidation, in the striatum. Both squalane and squalene increased the ratio of linoleic acid/linolenic acid in the striatum. These results suggest that the administration of squalene or squalane induces similar changes in the composition of fatty acids and has no effect on the activities of active oxygen scavenging enzymes in the striatum. However, squalane increases oxidative damage in the striatum and exacerbates the toxicity of 6-OHDA, while squalene prevents it. The effects of squalene or squalane treatment in this model suggest their possible uses and risks in the treatment of PD. Key words: Parkinson’s disease, squalene, squalane, fatty acid composition, oxidative stress 1 INTRODUCTION These active oxygen species are neurotoxic because of Parkinson’s diseas（e PD）is characterized by the progres- their strong oxidizing potential. We have already reported sive degeneration of dopaminergic neurons of the nigros- that the phosphoric diester of alpha-tocopherol and ascor- triatal system and dopamin（e DA）depletion in the striatum. bic acid, which has active oxygen scavenging activity, can While the pathogenesis of PD is not clear, damage to the prevent 6-OHDA-induced neurotoxicity6）. We also reported dopaminergic neurons by oxygen-derived free radicals is that some alkaloids, such as zingerone and eugenol, which considered to be an important contributing mechanism1）. are known antioxidants, provide protection against 6-OH- 6-Hydroxydopamin（e 6-OHDA）is used to produce an excel- DA-induced depletion7, 8）. lent animal model of PD2）, and is considered to be an en- DA is a neurotransmitter. Once released to synaptic dogenous toxin because it is found in the urine of PD pa- cleft, it binds to and activates postsynaptic neuron, and is tients3）. The toxicity of 6-OHDA is thought to be related to inactivated by reuptake into the presynaptic cell, then en- its ability to produce free radicals and to cause oxidative zymatic breakdown by monoamine oxidase into 3,4-dihy- stress4, 5）. 6-OHDA is susceptible to auto-oxidation, result- droxyphenylacetic acid（DOPAC）. Finally, catechol-O- ing in the formation of 6-OHDA quinine and hydrogen per- methyl transferase reduces DOPAC to homovanillic acid oxide, as well as superoxide and hydroxyl radicals4, 5）. （HVA）. ＊Correspondence to: Hideaki Kabuto, Department of Health Sciences, Kagawa Prefectural University of Health Sciences, 281-1 Mure-cho, Takamatsu 761-0123, JAPAN E-mail: [email protected] Accepted August 1, 2012 (recieved for review May 25, 2012) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs 21 H. Kabuto, T. Yamanushi, N. Janjua et al. Squalen（e SQLE）is an isoprenoid compound structurally acids levels in mouse striatum similar to beta-carotene, coenzyme Q10, vitamin A, vitamin Mice were orally administered with SQLE or SQLA at a E, and vitamin K, which are physiologically active sub- dose of 1 g/kg body weight. The control was given vehicle stances9）. It has six double bonds and is a highly effective （0.1％ Tween 20/water solution）. Thirty minutes after the oxygen scavenging agent10）, and protects human skin sur- final administration of a 7-day course of SQLE, SQLA or faces from lipid peroxidation as a quencher of singlet vehicle（once daily）, mice were sacrificed by deep sodium oxygen11）. Furthermore, it is reported to have antitumor pentobarbital anesthesia. The brain was removed and the properties and immune enhancing activities12, 13）. Squalane striatum was separated immediately on an ice plate, and （SQLA）is found naturally in sebaceous secretions14）. It is a stored at －80℃ until analysis. saturated analogue of SQLE, from which it can also be pro- duced by complete hydrogenation and possessing no active 2.4 DA and its metabolites（DOPAC and HVA）levels oxygen scavenging effects. Like SQLE, SQLA is widely em- Striatal tissues were homogenized with 300 μL of 200 ployed as a skin lubricant, ingredient of suppositories, mM ice-cold perchloric acid containing 10 mM disodium carrier of lipid-soluble drugs and adjuvants9, 15）. EDTA. After centrifugation（12000×g for 10 min at 4℃）, In this study, we examined the effects of SQLE and the supernatant was filtered（0.45 μm pore size）and then SQLA on 6-OHDA-induced DA depletion in the mouse stri- injected directly into an HPLC column（Shimadzu, Kyoto, atum, and on lipid peroxidation-inhibiting activity using an Japan）with an electrochemical detecto（r ECD; Eicom, in vivo model. The purpose of this study is to investigate Kyoto, Japan）. The appendant potential of the ECD how SQLE/SQLA has the preventive effect against Parkin- （carbon electrode vs. Ag/AgCl reference electrode）was set son’s disease pathogenesis and its mechanisms. at 700 mV. The analytical column was TSKgel ODS-80TM （4.6 mm I.D.×250 mm; Tosoh, Tokyo, Japan）, and the mobile phase consisted of 0.1 M citrate-sodium acetate buffe（r pH 3.9）containing methano（l 18％, v/v）, disodium 2 MATERIALS AND METHODS EDTA（4 mg/L）, and sodium octanesulfonat（e 0.8 mM）. 2.1 Animals Male ICR mice（Charles River Japan, Yokohama, Japan） 2.5 SOD activity weighing 30 to 35 g were used in all experiments. They Cytosolic SOD activity was assayed using a SOD assay were provided with water and a standard laboratory diet ki（t SOD Test Wako; Wako Pure Chemical, Osaka, Japan）, containing 24％ protein（MF; Oriental Yeast, Tokyo, Japan） based on the method described by Oberley and Spitz 16）. ad libitum, and housed at 25℃ with a 12-h light, 12-h dark Briefly, tissues were homogenized in 20 volumes of ice-cold cycle（lights on 7 am to 7 pm）. New mice were used for phosphate buffe（r 10 mM, pH 8.0）and centrifuged at 12000 each assay. The experimental protocol was approved by ×g for 10 min at 4℃. Xanthine（0.4 mM）, nitroblue tetra- the Ethics Review Committee for Animal Experimentation zolium（0.24 mM）, and xanthine oxidase（0.049 U/mL）dis- of Kagawa Prefectural College of Health Sciences. solved in phosphate butte（r 0.1 M, pH 8.0）were added to the supernatants, and the mixture was incubated for 20 2.2 Effect of treatment with SQLE/SQLA on 6-OHDA tox- min at 37℃. The reaction was stopped by adding sodium icity dodecyl sulfat（e 69 mM）. Absorbance was measured at 560 SQLE or SQLA was administered orally at a dose of 1.0 nm. SOD from bovine erythrocyte（s Sigma Life Science, St. or 0.1 g/kg body weight for 14 days. After 7-day administra- Louis, MO, USA）was used as the standard. tion（once daily）, 6-OHDA was injected intracerebroven- tricularly（i.c.v.）at a dose of 60 μg in 2 μL of 0.05％ L- 2.6 Catalase activity ascorbic acid/saline 30 min after SQLE or SQLA Catalase activity was assayed according to the method administration. SQLE/SQLA administration was continued described by Aebi17）. In brief, the striatum was homoge- for 7 days after the 6-OHDA injection. The control was nized in 20 volumes of ice-cold RIPA buffe［r 0.1 M phos- given vehicle（0.1％ Tween 20/water solution for SQLE/ phate buffe（r pH 7.4）containing 5 mM EDTA, 0.01％ digito- SQLA administration and 0.05％ L-ascorbic acid/saline for nin, and 0.25％ sodium cholate］and centrifuged at 12000 6-OHDA injection）in the same manner. Twenty-four hours ×g for 30 min at 4℃. Phosphate buffe（r 50 mM, pH 7.0） after the final administration, mice were sacrificed under containing EDTA（5 mM）and H2O（2 10 mM）pre-incubated sodium pentobarbital anesthesia and the striatum was for 10 min at 37℃ was added to the supernatant, and the removed on an ice plate and stored at －80℃ until analysis. decomposition of H2O2 was assayed directly by measuring the decrease in absorbance at 240 nm for 3 min. Catalase 2.3 Effect of treatment with SQLE/SQLA on activity of from bovine live（r Wako Pure Chemical, Osaka, Japan）was striatal active oxygen scavenging enzymes, thiobar- used as a standard.

Load more