Rose Essential Oil Delayed Alzheimer's Disease ... - ACS Publications

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Rose Essential Oil Delayed Alzheimer’s Disease-Like Symptoms by SKN‑1 Pathway in C. elegans Shuqian Zhu, Hongyu Li, Juan Dong, Wenqi Yang, Ting Liu, Yu Wang, Xin Wang, Meizhu Wang, and Dejuan Zhi* Gansu High Throughput Screening and Creation Center for Health Products, School of Pharmacy, Lanzhou University, Lanzhou, 730000, P.R. China S Supporting Information *

ABSTRACT: There are no effective medications for delaying the progress of Alzheimer’s disease (AD), the most common neurodegenerative disease in the world. In this study, our results with C. elegans showed that rose essential oil (REO) significantly inhibited AD-like symptoms of worm paralysis and hypersensivity to exogenous 5-HT in a dose-dependent manner. Its main components of β-citronellol and geraniol acted less effectively than the oil itself. REO significantly suppressed Aβ deposits and reduced the Aβ oligomers to alleviate the toxicity induced by Aβ overexpression. Additionally, the inhibitory effects of REO on worm paralysis phenotype were abrogated only after skn-1 RNAi but not daf-16 and hsf-1 RNAi. REO markedly activated the expression of gst-4 gene, which further supported SKN-1 signaling pathway was involved in the therapeutic effect of REO on AD C. elegans. Our results provided direct evidence on REO for treating AD on an organism level and relative theoretical foundation for reshaping medicinal products of REO in the future. KEYWORDS: rose essential oil, Alzheimer’s disease, Caenorhabditis elegans, Aβ, SKN-1 development of AD.14 Many evidence have shown that neuritic plaque was composed of extracellular Aβ peptides which comprise 39−42 amino acids.15,16 The oligomers are the most common and toxic form of Aβ peptides among monomers, oligomers, and fibrils.17 Caenorhabditis elegans provides a convenient and useful tool for evaluating the efficacy of anti-AD drug candidates and elucidating the pharmacological mechanisms of those drug candidates on treating the toxicity of Aβ aggregation.18−22 In this study, we tested the activity of REO to resist the toxicity induced by Aβ1−42 by using transgenic AD C. elegans. Meanwhile, we also tried to reveal the mechanism of REO for countering Aβ toxicity. The results will provide data to support the notion that REO is promising to be used for AD prevention before the onset of this disease.

1. INTRODUCTION Rose species have been used for a long history as food and folk medicines in many cultures, they have been nowadays developed into various functional foods.1 Rosa rugosa Thunb is one of the most important rose species producing valuable essential oil with fantastic fragrance.2 Essential oil is widely known as natural fragrant raw material in perfumes, cosmetics, and food industry.3 Previous studies report that rose essential oil (REO) possesses antioxidant properties, antibacterial, and antifungal activity, anti-inflammatory effects.4−7 Also, it seemed that R. damascena essential oil has an inhibitory effect on acetylcholine esterase (AChE) and butyrylcholine esterase (BChE) in vitro. The result seemed to reveal one of the reasons that REO is beneficial to improve memory impairment.8,9 The previous works suggest that REO has the potential for treating Alzheimer’s disease (AD). However, the underlying pharmacological mechanisms of REO for anti-AD have been far from completely understood. AD is a devastating progressive neurodegenerative disease, and it often occurs among the aging persons in the world.10 Almost all the clinical manifestation of AD patients focus on the deterioration of memory, language, and other cognitive functions, leading to loss of autonomy and psychological ability obstacles.10,11 There are over 46 million people suffering from AD throughout the world, and the prevalence will alarmingly increase to 131.5 million by 2050.12 According to the report in 1997, more than $100 billion have been used to combat against AD or other related dementias per year.13 AD causes the heavy burden and cost in either families or society; however, no efficient medicine is available yet. AD is characteristic of the emergence of extracellular neuritic plaques and neurofibrillary tangles, and amyloid depositions are generally preceded by the neurofibrillary tangles in the © 2017 American Chemical Society

2. MATERIALS AND METHODS 2.1. Materials. REO was provided by Gansu Oriental Tianrun Rose Industry Co., Ltd. (Gansu, China). It was extracted from the fresh flowers of Chinese Kushui rose (Rosa setate × Rosa rugosa) by a standard method of steam-distillation described in Chinese Pharmacopoeia 23 and then quantified by gas chromatography/mass spectrometry (GC/MS) in the Analysis and Testing Center of Lanzhou University. The composition of REO is shown in Table 1. The GC/MS assay was using a Trace DSQ, which is equipped with a DB-5MS capillary column (30 m, 0.25 mm i.d., film thickness 0.25 μm). The initial oven temperature was 40 °C for 3 min and then was programmed to rise to 100 °C at the rate of 8 °C/min, then to 200 °C at the rate of 5 °C/min, finally to 250 °C at the rate of 10 °C/min, and Received: Revised: Accepted: Published: 8855

July 14, 2017 September 4, 2017 September 15, 2017 September 15, 2017 DOI: 10.1021/acs.jafc.7b03224 J. Agric. Food Chem. 2017, 65, 8855−8865

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Journal of Agricultural and Food Chemistry Table 1. Ingredients of Rose Essential Oil Quantified by GC/MS

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DOI: 10.1021/acs.jafc.7b03224 J. Agric. Food Chem. 2017, 65, 8855−8865

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Journal of Agricultural and Food Chemistry Table 1. continued

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kept constant for 10 min at a level of 250 °C. Both the injection and ion source temperatures were set at 250 °C. The carrier gas was helium at 1 mL/min flow rate. REO was diluted at 1:500 in diethyl ether, and 1 μL of REO was injected in split mode with a split ratio of 1/50. The detection was carried out using an electric ionization system with an ionization energy of 70 eV. The mass spectra were acquired over the mass range of 35−650 (m/z). 2.2. C. elegans Strains and Maintenance. The wild type strain N2, and the transgenic nematode strains used in present work were purchased from Caenorhabditis Genetics Center (CGC). The transgenic worm strains are listed as following: CL4176, dvIs27 [myo-3p:: Aβ1−42::let-851 3′UTR) + rol-6 (su1006)]); CL2006, dvIs2 [pCL12 (unc-54/human Aβ peptide1−42 minigene) + pRF4]; CL2355, dvIs50 [pCL45 (snb-1::Aβ1−42::3′ UTR(long)) + mtl-2::GFP]; CL2122, dvIs15 [(pPD30.38) unc-54(vector) + (pCL26) mtl2::GFP]; TJ356, zIs356 [daf-16p::daf-16a/b::GFP + rol-6]; LG333, geIs7 [skn-1b::GFP]; CL2166, dvIs19 [(pAF15)gst-4p::GFP::NLS]. Worms of N2, CL2006, TJ356, LG333, and CL2166 were cultured on nematode growth medium (NGM) plates seeded with E. coli OP50 as food resources in an incubator at 20 °C; the other strains were grown at 15 °C. 2.3. Paralysis Assay. In total, 80−120 egg-synchronized CL4176 worms were transferred onto fresh NGM plates with or without REO at appropriate concentrations. When the animals grew up to L3 larvae, the expression of human Aβ1−42 gene was induced by temperature upshifting from 15 to 25 °C as Dostal et al. described.20 After 34 h, paralyzed individuals were counted at 2-h intervals until all the worms were paralyzed. The nematodes were recognized as paralyzed when they did not move or only waved their heads after repeated touches. PT50 for each group was calculated as the mean duration time when 50% tested worms became paralyzed after temperature upshifting for 34 h. 2.4. Fluorescence Staining of Aβ Deposits Assay. Synchronized wild type N2 and the transgenic nematode strain CL2006 were seeded onto NGM plates. After maintenance at 20 °C for 4 days, the worms were then transferred onto fresh NGM plates with or without REO at appropriate concentrations. Here, N2 were used as negative controls for Aβ deposits. After cultured at 20 °C for 2 days, the worms were collected with M9 buffer, then fixed in 4% paraformaldehyde/ PBS (pH 7.4) at 4 °C for 24 h. After that, worms were permeabilized

in 5% β-mercaptoethanol, 1% Triton X-100, and 125 mM Tris−HCl (pH 7.4) at 37 °C for another 24 h. The individuals were stained with 0.125% thioflavin S in 50% ethanol for 2 min, destained with 50% ethanol two or three times to wash off the residual thioflavin S not adsorbed, and mounted on slides for microscopy.19 Fluorescence images were obtained at the same exposure parameters by a fluorescent microscope (BX53; Olympus, Japan) with a digital camera. The Aβ deposits were quantified after scoring the thioflavin S-positive deposits in the anterior pharyngeal bulb in each nematode. 2.5. Exogenous Serotonin Sensitivity Assay. CL2355 worms were egg-synchronized and placed onto fresh NGM plates seeded with OP50, and they were treated with or without REO at 15 °C for 84 h, then transferred into 25 °C for 36 h. The animals were collected with M9 buffer and we scored the number of the paralyzed worms after exposure to 5 mg/mL of serotonin in a 96-well plate for 5 min. CL2122 was used as a transgenic control strain, and the assay was repeated at least three times.19,24 2.6. RNA Interference (RNAi) Assay. The synchronized CL4176 strain were maintained on RNAi NGM plates from eggs to adults for two generations as reported by Dostal et al.20 Exceptionally, L3 stage larvae were exposed to RNAi bacteria in first generation worms for skn-1 RNAi. The RNAi NGM plates contained 100 μg/mL Amp, 5 μg/mL tetracycline, and 1 mM isopropyl β-D-1-thiogalactopyranoside and were seeded with E. coli HT115 cloned target gene of hsf-1, daf-16, and skn-1 or only empty L4440 vector. After propagated on RNAi NGM plates for two generations, synchronized L1 worms were treated with or without REO for about 70 h at 15 °C and upshifted the temperature to 25 °C for 28 h. The number of paralyzed worms was scored as the method described in Paralysis Assay. 2.7. Subcellular DAF-16 or SKN-1 Nuclear Localization Assay. Synchronous L1 larvae of TJ356 or LG333 worms were treated with or without REO for 72 h, then washed with M9 buffer to remove the bacteria, added in one droplet of 20 mM sodium azide, and mounted on glass slides for microscopy. The subcellular distribution of DAF-16 or SKN-1 were observed under the fluorescence microscope (BX53; Olympus, Japan), and the ratio of the worms with DAF-16 or SKN-1 nuclear localization was counted. The nematodes in positive control for DAF-16 activation were heated at 37 °C for 30 min,21 while the worms in positive control for SKN-1 activation were exposed to 1.6 mM sodium arsenite at 20 °C for 1 h. 8859


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Journal of Agricultural and Food Chemistry 2.8. The gst-4::GFP Expression Assay. Synchronous L1 larvae of transgenic CL2166 nematodes were transferred onto fresh NGM plates with or without REO for 72 h at 20 °C. Afterward, the individuals were collected using M9 buffer and added one droplet of 20 mM sodium azide, finally mounted on glass slides for fluorescent microscopy. Also, the worms of positive control were exposed to 1.6 mM sodium arsenite at 20 °C for 1 h.25 Fluorescence images were obtained at the same exposure parameters by a fluorescent microscope (BX53; Olympus, Japan) with a digital camera. The fluorescence intensities of at least 30 individual worms were measured by ImageJ software, and the mean fluorescence intensities were calculated as the average fluorescence intensities per worm in each treatment. 2.9. Western Blotting. Human Aβ1−42 in the transgenic C. elegans CL4176 was detected by Western blot. Nematodes were harvested in deionized water with protease inhibitor cocktail (Sigma, P2714) added, flash frozen in liquid nitrogen, and stored at −80 °C. Worms were homogenized in RIPA lysis buffer, then sat on ice for 30 min, centrifuged at 14 000g for 5 min. Total protein content in the resultant supernatant was measured by the BCA method. Proteins in each sample were heated at 100 °C in sample loading buffer (62 mM TrisHCl, pH 6.8, 2% SDS, 10% glycerol, 4% β-mercaptoethanol, 0.0005% bromophenol blue) for 5 min. The samples were cooled and centrifuged again at 14 000g for 5 min, then were electrophoresed on the Tris-Tricine gel after loading 80 μg of total proteins in each lane. The gel was transferred onto a 0.22 μm PVDF membrane, and the membrane was blocked with 5% milk in TBS-Tween (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20) at room temperature for 2 h. The blotting was carried out overnight at 4 °C. Aβ proteins were detected by a primary antibody of 6E10 (Biolegend, 803001) at 1:500 dilution. α-Tubulin was used as internal control (Sigma, T6199) at 1:1000 dilution. Goat antimouse HRP was used as a secondary antibody at 1:5000. Finally, immunoreactive bands were visualized by Tanon ECL detection system (Bio-Tanon, China). Densitometry analysis was performed by ImageJ software. The relative band intensities of Aβ monomer or oligomer were calculated as the intensity values of 4 KD bands or 20 KD bands after normalized to the intensity values of α-tubulin, respectively. 2.10. Statistical Analysis. Statistical analyses were performed by SPSS 19.0 software. The data was shown as mean ± SD. Significant differences between groups were analyzed by using one-way analysis of variance (ANOVA). Except for paralysis assays, log rank survival test was carried out to compare the significance level among treatments. The p value of 0.05 or less was considered to be significant statistically.

Figure 1. Rose essential oil delayed AD-like symptoms in transgenic worms with human Aβ overexpressed. (A) curves of not paralyzed fraction of each group, the myo-3p::Aβ1−42::3′UTR (long) worms were treated with 2 μg/mL, 10 μg/mL, 20 μg/mL of REO from eggs, and 0.1% DMSO was regarded as control; (B) PT50s of each group in part A; there is a significant difference among these groups when letters are different (p < 0.05); (C) curves of not paralyzed fraction of each group treated with 2 μg/mL, 10 μg/mL, 20 μg/mL of REO from temperature upshift when the expression of Aβ1−42 was induced; (D) effect of REO on hypersensitivity to exogenous 5-HT in snb1::Aβ1−42 worms, and 0.1% DMSO was regarded as the control. There is a significant difference among these groups when letters are different (p < 0.05); (E) curves of not paralyzed fraction of each group, worms were treated with 20 μg/mL of REO, 10.8 μg/mL of β-citronellol, and 3 μg/mL geraniol, respectively; (F) PT50s of each group in part E, there is significant difference among these groups when letters are different (p < 0.05).

3. RESULTS AND DISCUSSION 3.1. Rose Essential Oil Delayed AD-like Symptoms Induced by Aβ Toxicity in C. elegans. C. elegans CL4176 is a transgenic worm strain, and the muscle-specific expression of human Aβ1−42 can be induced by temperature upshift, and subsequently the accumulation of Aβ deposits in the body muscle cells leads to AD-like symptom of paralysis phenotype.18 In the present work, CL4176 was used to test the effect of REO on AD-like symptom. The results showed that whatever drug treatment started from eggs (Figure 1A,B) or the onset of temperature upshift for induction of Aβ expression (Figure 1C), REO always significantly delayed the progress of worm paralysis in a dose-dependent manner. Behavioral plasticity often involves the alteration of the properties of neurons and synapses, such as locomotion, egglaying, olfactory learning, and mating. Serotonin (5-HT) is an important neuromodulator which can cause these above alterations.26,27 Transgenic C. elegans CL2355 displays hypersensitivity response to exogenous 5-HT due to human Aβ1−42 peptide overexpressed in its nervous system.24 To explore whether REO could exert a protective effect on the nerve system from Aβ toxicity, CL2355 was employed to detect the effect of REO treatment on another AD-like

symptom of 5-HT hypersensitivity caused by Aβ accumulation in nerve cells. The result showed that REO significantly decreased worm paralysis in a dose-dependent manner (Figure 1D), suggesting that REO lowered 5-HT hypersensitivity induced by Aβ toxicity. In contrast, neither exogenous 5-HT nor 5-HT in combination with REO affected the paralysis of CL2122 worms without nerve-specific overexpression of Aβ, which acted as a transgenic control of CL2355 C. elegans (Figure 1D). Considering that REO was mainly composed of 54.01% of βcitronellol and 15.02% of geraniol (Table 1), these two ingredients were further evaluated for their effects on worm AD-like symptoms. Compared to REO, β-citronellol and geraniol on the same dosage in REO delayed worm paralysis 8860


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Figure 2. Rose essential oil decreased Aβ deposits and reduced Aβ oligomers in AD worms. (A) Aβ deposits in unc-54/Aβ1−42 worms treated with 2 μg/mL, 10 μg/mL, and 20 μg/mL of REO, which were stained by ThS, 0.1% DMSO was regarded as the treatment control, and N2 worms were used as ThS staining control. The scale bar is 20 μm. (B) Quantitative analysis of Aβ deposits of each group after ThS staining. There is a significant difference among these groups when letters are different (p < 0.05). (C) Western blot assay were performed using the transgenic worms with myo3p::Aβ1−42::3′UTR (long) as the AD model. Aβ monomer and oligomer band were shown with or without 20 μg/mL REO treatment before and after the skn-1 gene were knocked down by RNAi, and L4440 was used as an empty vector control group; (D) quantitative analysis of each band of treated group. *** indicated that there was a significant difference between the treatment group and the control group at p < 0.001.

dependent manner (Figure 2A,B). Whereas the current evidence shows that Aβ oligomers, rather than senile plaques (SPs) or Aβ fibrils, are the toxic species,29 we performed the Western blotting assay to explore whether REO could lower the toxic Aβ oligomers. In our present work, Aβ-immunoreactive bands were visualized by the Aβ antibody of 6E10. Four KD of Aβ monomer and 20 KD of Aβ oligomer bands were quantified, respectively. The results showed that REO significantly reduced the level of Aβ oligomers, and it was canceled by skn-1 RNAi (Figure 2C, D). To determine whether REO could nonspecifically affect the expression levels of any exogenous proteins, a transgenic worm CL2179 [myo-3p::GFP::3′ UTR (long) + rol-6 (su1006)] with GFP under the control of promoter myo-3 was employed. The result showed that there was no significant difference in GFP fluorescence density among groups with or without drug

to a lesser degree. No therapeutic effects could be observed after treating the animals with β-citronellol and geraniol together (Figure 1E,F). Therefore, only REO was used in all of the following experiments. 3.2. Rose Essential Oil Suppressed Aβ Deposits in Transgenic AD C. elegans. Aβ1−42 protein has been demonstrated to be one of the hallmarks of AD progression and plays an important role in AD pathogenesis. Decreasing Aβ level becomes one of the most promising therapeutic approaches for AD patients.28 In the present work, transgenic C. elegans CL2006, which has constitutive expression of Aβ1−42 in body muscle cells, was used to determine whether REO could affect the Aβ level. The number of Aβ deposits on head regions of the tested worms can be specifically stained by thioflavin S (Th S) and then counted out.19 The result showed that REO could reduce the Aβ1−42 deposits in a dose8861


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Figure 3. Rose essential oil alleviating worm AD-like symptom was mediated by SKN-1 signal pathway. (A) Curves of not paralyzed fraction in myo-3p::Aβ1−42::3′UTR (long) worms after treatment with or without skn-1 RNAi; (B) PT50s of each treated group in part A, and there is a significant difference among these groups when letters are different (p < 0.05); (C) REO induced SKN-1 nuclear localization in skn-1b::GFP worms after treatment with 2 μg/mL and 20 μg/mL of REO. 0.1% DMSO was used as the control, 200 μg/mL of REO treating animals for 1 h was used as the acute group, 1.6 mM sodium arsenite was used as the positive control. The scale bar is 30 μm. (D) REO increased the expression of gst-4 gene in gst-4p::GFP worms treated with REO, 0.1% DMSO was used as the control group, 1.6 mM sodium arsenite was used as the positive control group. The scale bar is 40 μm. (E) Quantitative analysis of SKN-1 nuclear localization of each treated group in part C; there is a significant difference among these groups when letters are different (p < 0.05); (F) quantitative analysis of gst-4p::GFP expression of each treated group in part D; there is a significant difference among these groups when letters are different (p < 0.05).

Figure 4. Rose essential oil inhibiting worm AD-like symptom was independent of DAF-16 and HSF-1 signal pathway. (A) Curves of not paralyzed fraction in the myo-3p::Aβ1−42::3′UTR (long) worms after treatment with or without daf-16 RNAi; (B) PT50s of each treated group in part A, there is significant difference among these groups when letters are different (p < 0.05); (C) nuclear localization of DAF16 in daf-16a/b::GFP worms treated with 2 μg/mL, 20 μg/mL REO, and 37 °C treatment for 30 min was used as the positive control. The scale bar is 40 μm. (D) Quantitative analysis of each treated group in part D; there is a significant difference among these groups when letters are different (p < 0.05). (E) Curves of not paralyzed fraction in the myo-3p::Aβ1−42::3′UTR (long) worms after treatment with or without hsf-1 RNAi; (F) PT50s of each treated group in part E; there is significant difference among these groups when letters are different (p < 0.05).

was required for REO ameliorating AD-like symptoms of the worm paralysis phenotype induced by Aβ toxicity. In C. elegans LG333, intestinal SKN-1 activation was recognized by the GFP reporter translocated from the cytoplasm to the nucleus in response to stress.30 To determine whether REO could activate the SKN-1 pathway, the nuclear localization of SKN-1 was examined in the present work. The result showed that REO significantly increased SKN-1 nuclear distribution in a dose-dependent manner (Figure 3C,E). SKN-1 activation and nuclear translocation plays a critical role in regulating its downstream stress responsive gene expressions.30 As one of the target genes of skn-1, gst-4 glutathione S-transferase is a kind of phase II enzyme in response to oxidative stress.30 To test whether the expression of gst-4 could be induced after REO treatment, CL2166 which is a

treatment, suggesting that REO did not affect the expression of nontoxic protein but it specifically suppressed the toxic ones (Figure S1). 3.3. Rose Essential Oil Delaying AD Symptoms Was Mediated by SKN-1 but Not DAF-16 and HSF-1. SKN-1 pathway in C. elegans is highly homologous to the Nrf2 pathway in mammals. Transcription factor SKN-1 belongs to the Cap’n’collar family, which regulates phase II detoxification response genes to take a protective effect.19,25 To detect whether it was required for REO to resist the toxicity induced by aberrant Aβ generation and aggregation, the expression of skn-1 was knocked down by RNAi.20,21 The result showed that skn-1 RNAi completely counteracted the inhibitory effect of REO on worm paralysis (Figure 3A,B). It suggested that SKN-1 8862


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future work should try to investigate the anti-AD activity of the other components in REO. It is noteworthy that a similar phenomena was observed in previous work; that is, coffee extract significantly alleviates worm AD-like symptoms, but its main component of caffeine has less therapeutic effect.20 Also, coffee improves the cognitive deficits in aged rats, but caffeine alone is less effective.33 REO could inhibit Aβ deposits and activate SKN-1 to upregulate the expressions of downstream stress responsive genes, such as gst-4, thus delaying AD-like symptoms induced by Aβ toxicity in C. elegans (Figure 3A−C,E). SKN-1 is responsible for modulating its downstream genes which can respond to oxidative stress.29,34 REO in our experiment did elevate the expression of gst-4 downstream effector of skn-1 (Figure 3D,F), and this was identified with the results reported by Dostal et al.20 gst-4 codes for glutathione S-transferase catalyzing the electrophiles to conjugate with GSH in phase II deoxification process and then contributes to attenuating oxidative stress in the worm.35 According to our knowledge, Aβ aggregation is closely associated with increased oxidative stress and then leads to neural injury.36,37 Alleviating oxidant stress has been shown to inhibit Aβ aggregation.38 Therefore, the increased expression of gst-4 can contribute to lowering Aβ aggregation. Since REO and its main component of βcitronellol and geraniol at a high concentration of 1 mg/mL only have a mild antioxidant activity in vitro,9 our results supported that REO delaying worm AD-like symptoms of paralysis phenotype was independent of its direct antioxidant activity. Obviously, REO induced antioxidative stress responses in vivo, and our results agreed with previous work reported by Sennol et al.9 Additionally, Western blot results in the present work supported that SKN-1 was required for the reduction of Aβ oligomers (Figure 2C,D). However, we still cannot elucidate that SKN-1 promotes the clearance of Aβ aggregates or inhibits the formation of Aβ aggregates or both. Food restriction has been demonstrated to improve AD symptoms by suppressing Aβ neuropathology in the mouse and squirrel monkeys.39 Previous work shows that REO has antibacterial activity, and its minimum inhibitory concentration against E. coli is 4 mg/mL.10 In our present work, REO functioned at a concentration of 20 μg/mL, and it is much less than 4 mg/mL. Also, the chronic food clearance assay on the 96-well plate showed that there was no difference on the optic density at 600 nm between treatment groups and the control (Figure S2). It indicated that REO neither inhibited the growth of the worm food resource of E. coli OP50 nor did animals food intake behavior. Therefore, direct food restriction was not contributed to REO fighting against AD. However, because sirtuin activators like resveratrol can simulate the effect of caloric restriction,40 we still cannot rule out that REO can mimic the effect of food restriction through relative molecular mechanism. REO ameliorated AD-like symptoms in C. elegans by reducing Aβ deposits and the Aβ level (Figure 2). At present, one of the clinically used anti-AD drugs is memantine, which acts as a kind of N-methyl-D-aspartate receptor antagonist, and it has been proved to decrease the Aβ level and reduce Aβ plaques.41,42 Therefore, memantine has been used as a positive control in our lab, and 100 μM memantine can promote PT50 of the control by 138.7%,43 and 20 μg/mL REO in our present work increased it by 136.1% (Figure 1B). Although the anti-AD activity of REO was almost equal to that of memantine in C.

Figure 5. Schematic diagram of rose essential oil suppressing AD-like symptoms induced by Aβ toxicity in worms.

transgenic worm strain carrying GFP fused to gst-4 promoter were used. The result showed that REO significantly promoted gst-4::GFP expression (Figure 3D,F). Insulin/IGF-1 signaling pathway also plays critical roles in response to stresses, and DAF-16 is the major component of the insulin/IGF-1 signaling pathway.31 In the present work, daf16 RNAi did not affect the inhibitory effect of REO on worm paralysis (Figure 4A,B). TJ356 worms carries a DAF-16:: GFP fusion protein, thus DAF-16 activation can be distinctly visualized by GFP nuclear distribution.21 Again, in the present work, REO did not change the nuclear localization of DAF-16 in the tested TJ356 worms (Figure 4C,D). These results indicated that DAF-16 was not required for REO resisting the toxicity of Aβ species. The transcription factor hsf-1 has been demonstrated to mediate the reduction of proteotoxicity of Aβ1−42.32 Whereas in our work, hsf-1 RNAi did not alter any therapeutic effect of REO on worm paralysis induced by the toxicity of Aβ1−42 (Figure 4E,F), indicating that hsf-1 was also not required for the protective effect of REO. 3.4. Discussion. In the present work, REO significantly alleviated AD-like symptoms of paralysis phenotype induced by Aβ toxicity in C. elegans, suggesting that it could be potentially used to treat AD. This is supported by previous work in that REO from R. damascena has been revealed to promote memory performance, and it is thought to contribute to REO’s remarkable inhibition on the activities of AChE and BChE.9 Phenylethyl alcohol, which is the most active component against AChE and BChE in rose oil from R. damascena, was not detected in REO of Chinese Kushui rose (Rosa setate × Rosa rugosa) (Table 1). In our work, REO at a dose of only 20 μg/ mL could significantly delay AD-like symptoms in worms on an organism level. However, REO cannot inhibit the activity of AChE by 60.86% and BChE by 51.08% in vitro until it reaches a dose of 1000 μg/mL.9 It seemed that REO displayed anti-AD activity mainly through an inhibitory effect on Aβ toxicity but not against the activity of AChE or BChE. Similar to work by Senol et al.,9 REO exerted more effective anti-AD activity than any of its main component alone or their combination (Figure 1). This may be explained by that other bioactive components that exist in REO, and these components, together with geraniol and β-citronellol, exert synergistic or addictive therapeutic effects on AD worms. We still could not exactly explain why geraniol in combination with β-citronellol showed no significantly anti-AD activity any longer; one possibility is that some components in REO are needed to promote the beneficial effect on AD worms. Our 8863


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(8) Senol, F. S.; Orhan, I.; Kurkcuoglu, M.; Khan, M. H.; Altintas, A.; Şener, B.; Başer, K. H. C. An in vitro approach to neuroprotective activity of Rosa damascena Mill, a medieval age traditional medicine used for memory enhancement. Planta Med. 2011, 77, 163. (9) Senol, F. S.; Orhan, I. E.; Kurkcuoglu, M.; Khan, M. T. H.; Altintas, A.; Sener, B.; Baser, K. H. C. A mechanistic investigation on anticholinesterase and antioxidant effects of rose (Rosa damascena Mill.). Food Res. Int. 2013, 53, 502−509. (10) Shohayeb, M.; Abdel-Hameed, E. S. S.; Bazaid, S. A.; Maghrabi, I. Antibacterial and antifungal activity of Rosa damascena MILL. essential oil, different extracts of Rose petals. Global J. Pharmacol. 2014, 8, 01−07. (11) Lambert, J. C.; Ibrahim-Verbaas, C. A.; Harold, D.; Naj, A. C.; Sims, R.; Bellenguez, C.; et al. Meta-analysis of 74,046 individuals identifies 11 new susceptibility loci for Alzheimer’s disease. Nat. Genet. 2013, 45, 1452−1458. (12) Prince, M.; Wimo, A.; Guerchet, M.; Ali, G.; Wu, Y.; Prina, M. World Alzheimer Report 2015. The global impact of dementia. An analysis of prevalence, incidence, cost and trends. Alzheimer’s Disease International: London, 2015. (13) Ernst, R. L.; Hay, J. W. Economic research on Alzheimer disease: a review of the literature. Alzheimer Dis. Assoc. Disord. 1997, 11, 135−145. (14) Santacruz, K.; Lewis, J.; Spires, T.; Paulson, J.; Kotilinek, L.; Ingelsson, M.; et al. Tau suppression in a neurodegenerative mouse model improves memory function. Science 2005, 309, 476. (15) Poling, A.; Morgan-Paisley, K.; Panos, J. J.; Kim, E. M.; O’Hare, E.; James, P.; et al. Oligomers of the amyloid-β protein disrupt working memory: Confirmation with two behavioral procedures. Behav. Brain Res. 2008, 193, 230−234. (16) Cuevasa, M. E.; Haensgena, H.; Sepulveda, F. J.; Zegersa, G.; Roac, J.; Opazo, C.; et al. Soluble Aβ1−40 peptide increases excitatory neurotransmission and induces epileptiform activity in hippocampal neurons. J. Alzheimers Dis. 2011, 23, 673−687. (17) Regitz, C.; Fitzenberger, E.; Mahn, F. L.; Dußling, L. M.; Wenzel, U. Resveratrol reduces amyloid-beta (Aβ1−42)-induced paralysis through targeting proteostasis in an Alzheimer model of Caenorhabditis elegans. Eur. J. Nutr. 2016, 55, 741−747. (18) Link, C. D. Expression of human β-amyloid peptide in transgenic Caenorhabditis elegans. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 9368−9372. (19) Wu, Y.; Wu, Z.; Butko, P.; Christen, Y.; Lambert, M. P.; Klein, W. L.; Link, C. D.; et al. Amyloid-β-induced pathological behaviors are suppressed by Ginkgo biloba extract EGb 761 and ginkgolides in transgenic Caenorhabditis elegans. J. Neurosci. 2006, 26, 13102−13113. (20) Dostal, V.; Roberts, C. M.; Link, C. D. Genetic mechanisms of coffee extract protection in a Caenorhabditis elegans model of βAmyloid peptide toxicity. Genetics 2010, 186, 857−866. (21) Keowkase, R.; Aboukhatwa, M.; Luo, Y. Fluoxetine protects against amyloid-beta toxicity, in part via daf-16 mediated cell signaling pathway, in Caenorhabditis elegans. Neuropharmacology 2010, 59, 358−365. (22) Xin, L.; Yamujala, R.; Wang, Y.; et al. Acetylcholineestaraseinhibiting alkaloids from Lycoris radiata delay paralysis of amyloid beta-expressing transgenic C. elegans CL4176. PLoS One 2013, 8 (5), e63874. (23) The Pharmacopoeia Committee of China. The Chinese Pharmacopoeia, Vol.I; The Chemical Industry Publishing House: Beijing, China, 2005. (24) Arya, U.; Dwivedi, H.; Subramaniam, J. R. Reserpine ameliorates Ab toxicity in the Alzheimer’s disease model in Caenorhabditis elegans. Exp. Gerontol. 2009, 44, 462−466. (25) Rebolledo, D. L.; Aldunate, R.; Kohn, R.; Neira, I.; Minniti, A. N.; Inestrosa, N. C. Copper reduces aβ oligomeric species and ameliorates neuromuscular synaptic defects in a C. elegans model of inclusion body myositis. J. Neurosci. 2011, 31, 10149−10158. (26) Sawin, E. R.; Ranganathan, R.; Horvitz, H. R. C. elegans locomotory rate is modulated by the environment through a

elegans, the therapeutic effect of REO should further be validated in murine or primate AD models. The anti-AD effect of REO of Chinese Kushui rose (Rosa setate × Rosa rugosa) in C. elegans was diagramed as Figure 5. In conclusion, REO significantly improved AD-like symptoms in transgenic C. elegans with human Aβ1−42 overexpressed in the body wall muscular cells and nerve cells. Further, REO reduced Aβ oligomers to treat AD worms through SKN-1 signaling pathway but not the DAF-16 and HSF-1 signaling pathways. Our results provide data and a theoretical foundation for developing medicinal products of REO in the future.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.7b03224. Figure S1, effect of rose essential oil on the level of GFP protein overexpressed in muscle cells of transgenic smg-1 [myo-3p::GFP] worms and Figure S2, rose essential oil did not affect worm food clearance (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-931-8915685. Fax: 86-931-8915685. E-mail: [email protected]. ORCID

Dejuan Zhi: 0000-0002-1787-3735 Funding

This work was supported by the Ministry of Science and Technology New Drug Project of MOST of China (Grant 2015ZX09501-004-003-008); National Natural Science Foundation of China (Grants 31560176, 81403145); and Fundamental Research Funds for the Central Universities of China (Grant lzujbky-2017-206). Nematode strains used in this work were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (Grant P40 OD010440). Notes

The authors declare no competing financial interest.

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REFERENCES

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Rose Essential Oil Delayed Alzheimer's Disease ... - ACS Publications

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