Essential Oil of Acorus tatarinowii Schott Ameliorates Aβ-Induced Toxicity in Caenorhabditis elegans through an Autophagy Pathway

Background Acorus tatarinowii Schott [Shi Chang Pu in Chinese (SCP)] is a traditional Chinese medicine frequently used in the clinical treatment of dementia, amnesia, epilepsy, and other mental disorders. Previous studies have shown the potential efficacy of SCP against Alzheimer's disease (AD). Nevertheless, the active constituents and the modes of action of SCP in AD treatment have not been fully elucidated. Purpose The aim of this study was to investigate the protective effects of SCP on abnormal proteins and clarify its molecular mechanisms in the treatment of AD by using a Caenorhabditis elegans (C. elegans) model. Methods This study experimentally assessed the effect of SCP-Oil in CL4176 strains expressing human Aβ in muscle cells and CL2355 strains expressing human Aβ in pan-neurons. Western blotting, qRT-PCR, and fluorescence detection were performed to determine the oxidative stress and signaling pathways affected by SCP-Oil in nematodes. Results SCP-Oil could significantly reduce the deposition of misfolded Aβ and polyQ proteins and improved serotonin sensitivity and olfactory learning skill in worms. The analysis of pharmacological action mechanism of SCP-Oil showed that its maintaining protein homeostasis is dependent on the autophagy pathway regulated partly by hsf-1 and sir-2.1 genes. Conclusion Our results provide new insights to develop treatment strategy for AD by targeting autophagy, and SCP-Oil could be an alternative drug for anti-AD.


Introduction
Alzheimer's disease (AD) is an age-related neurodegenerative disorder clinically featuring loss of memory, cognitive, and behavior functions. To date, only donepezil, galantamine, memantine, and rivastigmine have been approved by the United States Food and Drug Administration for the treatment of mild to moderate AD, but these drugs cannot interrupt or halt disease progression [1]. Therefore, more efficacious therapeutic drugs are required for the management of AD. The pathogenesis of Alzheimer's disease is associated with abnormal proteins including Aβ and tau aggregation in the brain. Generally, there is no deposition of Aβ peptide in the healthy brain, but the increased level of the Aβ peptide aggregation is manifested in the AD brain. Growing studies indicated that autophagy contributes to the degradation of abnormal proteins [2]. It has been reported that many medicinal herbs contain promising autophagy regulators and have great therapeutic potential for AD treatment [3].
Acorus tatarinowii Schott [A. tatarinowii, Shi Chang Pu in Chinese (SCP)] is a renowned traditional Chinese medicine that was first recorded in the Shennong Materia Medica. It is commonly used in the clinical treatment of dementia, amnesia, epilepsy, and other mental disorders [4]. Earlier studies showed that the essential oil from SCP prevented hydrogen peroxide-induced cell injury in PC12 cells (Yan et al., 2020), and β-asarone, a major component of SCP, showed protection against oxidative stress and neuronal damage induced by amyloid-β in rats [5,6]. However, the underlying pharmacological action mechanism of SCP-Oil remains unclear.
In this study, we first used Caenorhabditis elegans (C. elegans) as an in vivo model to elucidate the action mechanism of SCP-Oil. C. elegans is widely used in laboratory research as it is rapidly propagated and has a short life cycle, a simple structure, and extensive homology with mammals [7]. Moreover, C. elegans can be readily induced by gene editing to express human Aβ in their muscle cells and neurons, so it is considered to be a powerful model for screening ADrelated drugs and clarifying their mechanisms [8].
Here, we reported the protection effects of SCP-Oil in a transgenic Aβ C. elegans model. Our results showed that it can reduce ROS accumulation and show protection effects against abnormal Aβ and polyQ proteins by targeting autophagy degradation pathway. Our findings furtherly prove the potential of SCP-Oil to be used in AD treatment. 2.3. Strain Maintenance and Treatment. All strains were maintained at 16°C except for BC12921 and AM140 which were maintained at 20°C. The worms were cultured on solid nematode growth medium (NGM) [9] plates containing a lawn of Escherichia Coli (E. coli) OP50 and fed with the drugs from the time they were age-synchronized to adult stage.

Materials and Methods
2.4. High-Performance Liquid Chromatography (HPLC) of SCP-Oil. A ZORBAX Eclipse XDB C-18 column (Agilent) was used for high-performance liquid chromatography (HPLC) analysis. The operating conditions were as follows: either 10 mg/mL extract or 1.0 mg/mL standard; 1.0 mL/min flow rate; 10 μL injection volume; detection wavelength set at 254 nm; and room temperature (25°C). The eluents were as follows: A (ultrapure water) and B (100% (v/v) methanol). The gradient was as follows: 0.01 min 35% B, 30 min 75% B, 45 min 85% B, 55 min 95% B, and 65 min 95% B. Prior to their injection in the HPLC, all samples were filtered through a 45-μM membrane (Agilent). The analysis result was seen in Supplementary Fig. S2. 2.5. Safety Assessment Assay. Synchronized L1 larvae were cultured in a 96-well plate with OP50, and 1.08 mM FUdR was added by L3. Then, young adult larvae were treated with various concentrations of SCP-Oil. The live worms were observed and recorded daily until day 4. Nematodes that were stiff and unresponsive to strong light or agitation were assumed to be dead.
2.6. Paralysis Assay in Nematodes. Synchronized L1 larvae (>30 worms per treatment) were transferred to 35 mm culture plates containing OP50 and drugs, cultured at 16°C for 36 h, and upshifted to 23°C for transgene induction, scoring the paralyzed worms per hour after cultured at 23°C for another 24 h. Worms were considered paralyzed when they fail to move about, did not respond to platinum wire stimulus, or presented with an anterior halo [10]. The assay was repeated at least three times, and the PT 50 (time duration in which half worms were paralyzed) was calculated [11].

Chemotaxis
Learning Assay. Equal volumes (on demand) of 1 M sodium acetate and 1 M sodium azide were blended and used as an attractant. The control odorant was a mixture of 1 M sodium azide and sterile water. Synchronized CL2355 and control strain CL2122 larvae were cultured on either an untreated or drug-loaded NGM plate at 16°C until L3 (~36 h) and upshifted to 23°C for another 36 h. The worms were collected, and the OP50 with M9 Buffer (3 g KH 2 PO 4 , 6 g Na 2 HPO 4 , 5 g NaCl, 1 mL 1 M MgSO 4 , and ddH 2 O to make up 1 L) were cleared out. 40_60 worms were placed in the center of a clear 10-cm NGM plate, 10 μL attractant was quickly dropped onto one side of the plate, and 10 μL control odorant was dropped onto the other side [12]. After 1 h, the number of worms near each spot was recorded and the chemotaxis index was calculated as follows: chemotaxis index ð CIÞ = ðnumber of worms on attractant side − number of worms on the control sideÞ/total number of worms.
2.9. Fluorescence Assay. Synchronized BC12921 worms were cultured in 96-well plates containing OP50. Drugs were administered at L4, and the worms on day 4 of adulthood were collected. The OP50 and drugs were replaced with M9, and the worms were transferred to a clear 35 mm NGM plate. The worms were then transferred to a black 96-well plate containing 200 μL M9 buffer (10 worms/well,

SCP-Oil Retards Aβ-Induced Paralysis in C. elegans.
Extracellular β-amyloid (Aβ) deposition is the main AD pathogenesis, which is neurotoxic and myotoxic [14]. To determine whether SCP-Oil protects against Aβ-induced toxicity in vivo, we assessed the efficacy of SCP-Oil on delaying Aβ-induced paralysis in transgenic CL4176 worms expressing temperature-induced human Aβ protein.
We treated the worms with 0-1.0 mg/mL SCP-Oil in 96-well plates at 16°C for 4 d. The results showed that SCP-Oil < 1:0 mg/mL had no effect on nematodes' lifespan; it was suggested that SCP-Oil < 1:0 mg/mL was not toxic to the worms (Figure 1(a)). We then performed a paralysis assay on CL4176 worms using 0, 0.001, 0.01, 0.1, 0.4, and 1.0 mg/mL SCP-Oil. Our results showed that SCP-Oil delayed paralysis in CL4176 worms in a dose-dependent manner (Figure 1(b) and Table S1). Further, the PT 50 for worms treated with 1 mg/mL SCP-Oil and the untreated worms were 5:70 ± 0:20 h and 3:25 ± 0:05 h, respectively; 1 mg/mL SCP-Oil significantly extended the PT 50 by up to 75.39% relative to the untreated control (Table S1). Therefore, 1 mg/mL SCP-Oil was used in the subsequent assays. Overall, the above results suggested that none of the tested SCP-Oil concentrations was toxic to the worms and all could potentially protect them against Aβ-induced damage.

SCP-Oil Enhances Olfactory Learning and Serotonin Sensitivity in Nematodes with Neuronal Aβ Expression.
CL2355 is a transgenic strain in which Aβ was expressed in their neuronal cells; it was showed deficits in chemotaxis and associative learning skills [15]. To investigate the protective effects of SCP-Oil on the neurological functions, we determined olfactory adaptation-related learning in this strain. The chemotaxis assay indicated that 1 mg/mL SCP-Oil significantly increased the number of worms on the attractant side of the plate (Figure 2(a)). The chemotaxis indexes for CL2122 control strain and the untreated CL2355 worms were 0:35 ± 0:01 and −0:13 ± 0:02, respectively (Table S2), which suggested that worms with panneuronal human Aβ expression did exhibit severe cognitive deficit. However, the SCP-Oil treatment significantly increased the value of CI to 0:12 ± 0:01 (Figure 2(a) and Table S2). So, SCP-Oil obviously exhibited a protective effect on the neurological functions of worms. Neurotransmitter serotonin plays an important role in locomotion, cognition, and learning-related behavioral plasticity in C. elegans [16]. Here, we incubated the worms in 5 mg/mL exogenous serotonin for 24 h and recorded the number of active individuals. Figure 2(b) shows that 27:40 ± 1:70% of the worms were still alive after treated with 1 mg/mL SCP-Oil, but only 13:34 ± 1:50% of the control worms had survived (Table S3). Moreover, exogenous serotonin had a negligible effect on the CL2122 control (50:56 ± 1:11%; Figure 2(b) and Table S3).
Hence, 1 mg/mL SCP-Oil can obviously improve learning behavior and augment serotonergic excitability against Aβ-induced deficits in the neurological functions.

SCP-Oil Decreases
Aβ Aggregation in C. elegans. Given that SCP-Oil could effectively delay paralysis of CL4176 worms, we evaluated the effect of SCP-Oil on Aβ transcription and protein levels. Relative to the untreated, the Aβ transcription level was decreased by 0.56-fold in nematodes treated with 1 mg/mL SCP-Oil (Figure 3(a)). The amyloid protein levels indicated that 1 mg/mL SCP-Oil can significantly decrease Aβ aggregation in worms; quantitative data analysis showed that Aβ oligomers and monomers were 3 Oxidative Medicine and Cellular Longevity remarkably reduced by~39.10% and 40.07%, respectively, in nematodes treated with 1 mg/mL SCP-Oil (Figures 3(b)-3(d)). Therefore, SCP-Oil resisted Aβ-induced damage in C. elegans by downregulating Aβ transcription and decreasing amyloid protein expression.

SCP-Oil Reduces polyQ Accumulation in C. elegans.
Abnormal polyglutamine (polyQ) aggregation disrupts cellu-lar proteostasis, triggers cytopathy, and induces neurodegenerative diseases, aggregation of polyQ protein elevating amyloidogenic processing of amyloid precursor protein by upregulating β-site amyloid precursor protein-cleaving enzyme 1 [17]. To determine whether SCP-Oil mitigates the accumulation of other abnormal proteins in vivo, we measured polyQ accumulation in AM140 strain. Fluorescence images disclosed there was less polyQ aggregation in  Oxidative Medicine and Cellular Longevity worms treated with 1 mg/mL SCP-Oil than there was in control worms (Figure 4(a)). Quantitative data analysis revealed that the polyQ level was 24.85% lower in the treated group than it was in the control (Figure 4(b) and Table S4). The results suggested that 1 mg/mL SCP-Oil could significantly alleviate the proteotoxic stress induced by polyQ aggregation.

SCP-Oil Decreases Aβ-Induced ROS Accumulation in C.
elegans. Aβ aggregation causes mitochondrial dysfunction which, in turn, leads to the release of reactive oxygen species (ROS). Excessive ROS potentiates Aβ toxicity and promotes neuroinflammation [18]. We measured the ROS levels in AD worms with an H 2 DCFDA kit and found that ROS levels were relatively lower in the CL802 control ( Figure 5(a)). Unlike CL4176, CL802 does not express human Aβ in its muscle cells. In contrast, the cellular ROS level was extremely high in untreated CL4176 ( Figure 5(a)). After being treated with 1 mg/mL SCP-Oil, the intracellular ROS concentration in CL4176 was significantly reduced by 13.55% compared with the ROS level of the control group ( Figure 5(b) and Table S5). Thus, SCP-Oil could mitigate ROS accumulation and ameliorate cellular damage caused by Aβ.

Protection Effects Provided by SCP-Oil Is Autophagy-Dependent.
In order to clarify the underlying signal pathway involved in the protection effects of SCP-Oil in C. elegans, we selected some key transcription factors involved in aging, stress resistance, and protein homeostasis, including daf-2 and daf-16 that play an important role in regulating lifespan and stress resistance [19]; skn-1, hsf-1, and sir-2.1 that participate in stress resistance and protein homeostasis [20,21]; and bec-1, vps-34, unc-51, lgg-1, and other genes that are vital for the autophagy pathway in C. elegans [22]. Our results showed that SCP-Oil could not increase the expression of daf-2 or daf-16, but hsf-1 and sir-2.1 were obviously upregulated, 1.72-and 1.85-fold higher than the con-trol group, respectively (Figure 6(a)), that means protection effects of SCP-Oil partly depend on hsf-1 and sir-2.1 genes.
P62/SQSTM1 is a ubiquitin-and LC3-binding protein and is degraded by autophagy. In vivo, P62/SQSTM1 accumulation is commonly accompanied by reduced autophagy [23]. The observed upregulation of autophagy-associated genes suggested that SCP-Oil could enhance autophagy activity in AD worms. We measured the P62 protein level in BC12921 stain expressing SQST-1::GFP protein. A fluorescence intensity assay demonstrated that P62 protein expression was >60% lower in the SCP-Oil-treated worms than it was in the control (Figure 6(c)). This finding indicated that autophagy was very active in nematodes treated with SCP-Oil. The above results strongly indicated that 1 mg/mL SCP-Oil showing protective effect against Aβ-induced injury was dependent on autophagy pathway.

Discussion
The pathogenic mechanisms of Alzheimer's disease (AD) involve the deposition of abnormally misfolded proteins, amyloid β protein (Aβ), and tau protein. Aβ comprises senile plaques, and tau aggregates form neurofibrillary tangles, both of which are hallmarks of AD. Although it was reported that β-asarone, a major component of SCP, showed protection against neuronal damage induced by amyloid-β in rats [5,6], the underlying molecular mechanism of SCP is still 5 Oxidative Medicine and Cellular Longevity unclear. Here, our experiment results showed that SCP-Oil can remarkably ameliorate Aβ-induced paralysis in worms expressing Aβ protein in their muscle cells compared with DMSO control or vehicle control (Figures 1(b) and 1(c) and S1). An immunoassay disclosed that SCP-Oil decreases Aβ oligomers and monomer protein levels (Figures 3(b)-3(d)), and the Aβ transcription level was significantly decreased in nematodes treated with 1 mg/mL SCP-Oil (Figure 3(a)). The above results furtherly confirmed that SCP-Oil is the main component of SCP and has an inhibitory effect on the toxicity of Aβ protein. Additionally, we found that SCP-Oil improves chemotaxis-related learning and serotonin-associated excitability in worms expressing Aβ in neuron cells (Figure 2), which suggested that SCP-Oil has a protective effect on neuron damage induced by Aβ protein.
Moreover, SCP-Oil can significantly reduce the deposition of misfolded polyQ protein in AM140 strains (Figure 4). These results suggested that SCP-Oil has the effect of maintaining the homeostasis of misfolded proteins and shows the potential to develop a therapeutic for AD.
Oxidative stress has been recognized as a contributing factor in the progression of multiple neurodegenerative diseases including AD [24]. Abnormal proteins in turn could exacerbate ROS production ( Figure 5(a)), thereby contributing to a vicious cycle. We found that SCP-Oil treatment substantially reduced ROS levels in CL4176 strains ( Figure 5(b) and Table S5). Generally, increased production of ROS is associated with loss of mitochondrial function. Sir-2.1, a key gene for controlling mitochondrial function [25], was significantly upregulated (Figure 6(a)). Therefore, SCP-Oil The control group fluorescence intensity was 100% and that for the treatment group was calculated relative to that of the control. Error bars represent the means ± SD. * * P < 0:01; n ≥ 60. Autophagy, the main conserved pathway for the degeneration of aggregated proteins, Aβ, tau, and dysfunctional organelles in the cell, has been discovered to be involved in the pathological changes of AD [3]. Due to the fact that SCP-Oil could significantly reduce the expression of Aβ and polyQ proteins in worms, we inferred that autophagy may make a contribution for this beneficial effect of SCP-Oil. According to the results of autophagy-related genes assay, the mRNA levels of key genes (lgg-1, lgg-2, bec-1, vps-34, and unc-51) were remarkably upregulated ( Figure 6(a)). Moreover, P62 protein, one of the best-known autophagic substrates, was obviously reduced after treatment with SCP-Oil ( Figure 6(c) and Table S6). These findings suggested that the autophagy pathway was involved in the protection against abnormal Aβ and polyQ protein-induced toxicity.

Oxidative Medicine and Cellular Longevity
Autophagy is induced in multiple tissues of Caenorhabditis elegans following HSF-1 overexpression, and downregulation of HSF-1 activity exacerbates misfolded and unfolded protein aggregation [26]. Our present study demonstrated that SCP-Oil significantly upregulated the hsf-1 transcription level (Figure 6(a)). Additionally, sir-2.1 not only participates in oxidative stress but also induces autophagy activity to reduce abnormal protein aggregation and toxicity [27]. Taken together, our results revealed that the effect of SCP-Oil maintaining protein homeostasis is dependent on the autophagy pathway regulated partly by hsf-1 and sir-2.1 genes.

Conclusions
In summary, our results confirmed that SCP-Oil is the main component of SCP showing a protection effect against abnormal proteins in worms. We further clarified the pharmaco-logical action mechanism of SCP-Oil and showed that it reduces Aβ and polyQ deposition through the targeting autophagy pathway. In the recent few years, much progress has been made in finding autophagy regulators from natural products, which provides new insights to develop treatment strategy for AD by targeting autophagy. SCP-Oil could be an alternative drug for anti-AD.

Data Availability
It can be found in Supplementary Materials file. (c) Fluorescence intensity analysis of BC12921 cultured with or without SCP-Oil. All assays were performed at least twice. Data were analyzed by Student's t-test. Error bar indicates the mean ± SD. * * * * P < 0:0001 and * P < 0:05.