Acetylshikonin, a Novel AChE Inhibitor, Inhibits Apoptosis via Upregulation of Heme Oxygenase-1 Expression in SH-SY5Y Cells

Acetylcholinesterase inhibitors are prominent alternative in current clinical treatment for AD patients. Therefore, there is a continued need to search for novel AChEIs with good clinical efficacy and less side effects. By using our in-house natural product database and AutoDock Vina as a tool in docking study, we have identified twelve phytochemicals (emodin, aloe-emodin, chrysophanol, and rhein in Rhei Radix Et Rhizoma; xanthotoxin, phellopterin, alloisoimperatorin, and imperatorin in Angelicae dahuricae Radix; shikonin, acetylshikonin, isovalerylshikonin, and β,β-dimethylacrylshikonin in Arnebiae Radix) as candidates of AChEIs that were not previously reported in the literature. In addition to AChEI activity, a series of cell-based experiments were conducted for the investigation of their neuroprotective activities. We found that acetylshikonin and its derivatives prevented apoptotic cell death induced by hydrogen peroxide in human and rat neuronal SH-SY5Y and PC12 cells at 10 μM. We showed that acetylshikonin exhibited the most potent antiapoptosis activity through the inhibition of the generation of reactive oxygen species as well as protection of the loss of mitochondria membrane potential. Furthermore, we identified for the first time that the upregulation of heme oxygenase 1 by acetylshikonin is a key step mediating its antiapoptotic activity from oxidative stress in SH-SY5Y cells.


Introduction
Alzheimer's disease (AD) is one of the most devastating neurodegeneration diseases characterized by progressive memory loss and cognitive dysfunction in the aging population. Although beta-amyloid aggregation and fibrillar tau-tangles have been identified as the major pathogenesis markers in AD patients and they are now promising targets for drug development, there is still no available drug against these targets (reviewed in [1][2][3]). Therefore, acetylcholinesterase inhibitors (AChEIs) are alternative option in current clinical treatment, and there is a continued need to search for novel AChEIs with less side effect to treat AD [4].
Synthetic compounds are now a central focus when searching any AChEIs. Many of these AChEIs potently inhibited the enzyme at the nanomolar level [5][6][7]. However, not much information regarding the potency and efficacy of these AChEIs in animal study or clinical trials can be gathered, due to the fact that the potency of AChEIs inhibition may not correlate with their neuroprotection efficacy due to their increases in cellular toxicity. It is supported by the recent study that the role of AChEIs against AD might be far beyond its AChE inhibition that enhances neuronal transmission acetylcholine [8]. Abundant evidence from in vitro and in vivo studies has demonstrated that AChEIs exhibited remarkably neuroprotective effects through attenuation of oxidative stress and enhancement of antioxidant status [9,10]. Therefore, both anti-AChE activity and antioxidative stress should be considered when searching novel AChEIs as drugs to treat AD.
In recent years, in silico virtual drug screening became a preferred approach to screen novel compounds given that the structures of molecular targets (enzymes or receptors) are determined. The high-throughput docking screen can provide possible candidates for further biomedical validation so as to reduce the time and cost of research and development in drug discovery (reviewed in [11]). The availability of the structure of AChEs has provided the opportunity of 2 Evidence-Based Complementary and Alternative Medicine widespread in silico screening of novel AChEIs [12][13][14][15][16][17][18]. The objective of the present study is to compile a comprehensive database from natural herbs in which the key constituents have been chemically characterized. It was inspired by the fact that the natural AChEI, galantamine, the FDA approved drug to treat mild-to-moderate AD, is a natural alkaloid that has only mild AChEI activity but strong neuroprotective efficacy [19]. Using this database, we have successfully identified some groups of phytochemicals that have mild AChEI activity but showed very promising neuroprotection in neuronal cell cultures induced by oxidative damages.

AChE Assay.
Candidate phytochemicals dissolved in DMSO were tested for AChE inhibitory activity by the Ellman assay with minor modifications [20]. Ten L of human recombinant AChE (prepared in-house [20]) and 1 L of drug were added into 190 L of PBS buffer (100 mM, pH 7.4) and incubated in a 96-well plate at 37 ∘ C for 10 min. Then 25 L of 12.5 mM ATCI and 25 L of 10 mM DTNB were premixed and added into each well. After 10 min incubation with the substrate, the optical densities were measured in a 96-well plate reader at 412 nm. The optical density was inversely proportional to the inhibitory activity. By contrast, a blank control without the tested compound was also performed in parallel; the normal hydrolytic rate of the enzyme can be represented by the blank control. Each assay was performed in triplicate.
The percentage inhibitory activities of the various compounds were calculated by comparison with the positive control and the blank control. The formula was shown as follows: percent of inhibitory activity of the compound = (1 − absorbance of sample/absorbance of blank control)/(1 − absorbance of positive control/absorbance of blank control) × 100%. Data analysis was performed with Prism software. Inhibitory effects were expressed as IC 50 value calculated by regression analysis.
2.5. Cell Viability Assay. MTT colorimetric assay was performed to determine the cell viability. Cells were seeded in 96-well plates at a density of 5×10 3 cells/well and treated with test chemicals at desired concentration at 37 ∘ C for 12 hours. Subsequently, cells were stimulated with H 2 O 2 (500 M) for 4 hours. After the exposure period, the cells were incubated with 20 L MTT (5 mg/mL) for 4 h. The cells were eluted with DMSO and quantified with a spectrophotometer (Ultramark Microplate Reader, Bio-Rad) at a wavelength of 590 nm.

Measurement of Mitochondrial Membrane Potential.
The cells were treated with desired concentration of acetylshikonin for 12 hours, and then the cells were stimulated with H 2 O 2 (500 M) for 2 h. Rhodamine 123 (2 M) was added to cells after the treatment for 30 min at 37 ∘ C. Fluorescence intensity of Rhodamine 123 was measured/detected by a fluorescence spectrophotometer (M1000, TECAN, Austria GmbH, Austria) using excitation/emission of 485/530 nm and a fluorescence microscope (Nikon Live Cell Imaging System Ti-E, Japan) and using excitation/emission of 490/530 nm, respectively. Fluorescence intensity of each group was normalized to the control group.

Western Blot Assay.
Proteins in the total cell lysate were separated by 10% SDS polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane (Immobilon-P membrane; Millipore, Bedford, MA, USA). After the blot was blocked in a solution of 5% bovine serum albumin, membrane was incubated overnight with primary antibodies against Bcl-2, Bax, Caspase-3, p53, HO-1, or betaactin followed by incubation with horseradish peroxidaseconjugated secondary antibodies for 1 h. Specific bands were detected with ECL-plus western blotting detection reagent (GE Healthcare Bio-Sciences) and photographed with Fuji-Film LAS-3000 (Fujifilm, Tokyo, Japan).

Statistics.
Statistical significance was determined using the One-Way ANOVA (GraphPad Software, CA, USA). The results are presented as the means ± SEM. The significance was accepted when value was < 0.05.

Potential AChE Inhibitors from Natural Products Were
Identified by Molecular Docking Screen. Using the natural product database and AutoDock vina for screening, we have identified 12 phytochemicals reportedly (emodin, aloe-emodin, chrysophanol, and rhein from anthraquinone fraction in RHEI RADIX ET RHIZOMA; xanthotoxin, phellopterin, alloisoimperatorin, and imperatorin from furanocoumarin fraction in ANGELICAE DAHURICAE RADIX; shikonin, acetylshikonin, isovalerylshikonin, and , -dimethylacrylshikonin from naphthoquinone fraction in ARNEBIAE RADIX) which can act as AChEIs. Huperzine A, the positive control, exhibited the highest docking score in the ranking list. It is noted that Trp86 is the key residue interacting with all AChEIs through -interaction in docking simulation, which is consistent with the key role of Trp86 in the catalytic pocket of AChE (Table 1) [21]. In vitro validation demonstrated that anthraquinones from RHEI RADIX ET RHIZOMA were the strongest AChEIs (   (Figure 1(c)). In PC12 cells, five chemicals rescued H 2 O 2 -induced cell death (Figure 1(d)). Notably, acetylshikonin-treated cells exhibited the highest viability during H 2 O 2 stimulation, indicating that acetylshikonin might be the strongest neuroprotective candidate among these potential AChE inhibitors. Thus, study in the latter part will be focused on neuroprotective effects of acetylshikonin on H 2 O 2 -induced cell apoptosis in both SH-SY5Y and PC12 cells.

Acetylshikonin Modulated H 2 O 2 -Induced Apoptosis-Related Protein Expression in Both SH-SY5Y and PC12 Cells.
To further explore the detailed neuroprotective mechanisms of acetylshikonin on H 2 O 2 -induced apoptosis, the possible related proteins were measured by western blot. As shown in Figures 4(a) and 4(b), H 2 O 2 -stimulation decreased Bcl-2 expression level, while it increased Bax and p53 expression level. In contrast, acetylshikonin concentration dependently led to increased expression of Bcl-2 as well as decreased Bax and p53 expression in H 2 O 2 -induced SH-SY5Y and PC12 cells. Caspase cascade has been identified as the critical executor for apoptosis. In H 2 O 2 -induced cells, the decreased caspase-3 and the increased cleaved caspase-3 were observed, which was rescued by acetylshikonin in a dose-dependent manner.

Upregulation of Heme Oxygenase-1 (HO-1) by Acetylshikonin Played a Key Role of Its Antiapoptotic Activity in
It has been widely accepted that upregulation of Heme oxygenase 1 (HO-1) expression protects cells against the oxidative-stress cellular injury [23]. Western blot results showed that HO-1 expression was increased after acetylshikonin treatment in SH-SY5Y cells. However, acetylshikonin treatment has no impact on the level of HO-1 expression in PC12 cells ( Figure 5(a)). To further confirm the role of HO-1 in antiapoptotic effects of acetylshikonin, we cotreated acetylshikonin with ZnPP (HO-1 inhibitors, 10 M) in H 2 O 2 -induced cells. Cell viability results demonstrated that ZnPP reversed the protective effects of acetylshikonin on H 2 O 2 -induced cell death in SH-SY5Y cells ( Figure 5(b), right panel). However, these reversed effects of ZnPP were not observed in PC12 cells ( Figure 5(b), left panel), and they were consistent with Western blot results. Therefore, HO-1 induction by acetylshikonin was critical against the oxidative-stress induced cell apoptosis in SH-SY5Y cells. In contrast, acetylshikonin was not able to upregulate HO-1 expression in PC12 cells, indicating that antiapoptotic activity of acetylshikonin in oxidative stress condition might be mediated through other antioxidant pathway in PC12 cells.

Discussion
With the rapid advances in personal computing power, virtual drug screening is popular and prominent. While there are numerous databases for synthetic compounds, there are only a few natural product databases that are specifically for in silico docking study. To facilitate virtual docking on natural compounds, we have established our in-house natural products database, which contains approximately 8,000 naturally occurring chemicals so far. Based on docking screening, top chemicals in ranking list were selected for the following analysis. The classic analysis, which has been widely accepted in most synthesis chemical virtual screen, is to rank the binding affinity and then choose the high ranking chemicals for further in vitro validation. Natural products, unlike synthesis chemicals, come from natural sources, such as plants, fungus, animals, and minerals. This characteristic might lead to a possibility that the high ranking chemicals have derivatives in the same species or genus. The collection of this kind of derivatives has been identified as bioactive fraction in complementary medicine. Therefore, we selected these top ranking derivatives for further validation. Here we identified three bioactive fractions as AChEIs, including anthraquinone fraction in RHEI RADIX ET RHIZOMA, furanocoumarin fraction in ANGELICAE DAHURICAE RADIX, and naphthoquinone fraction in ARNEBIAE RADIX. In this way, our database is not only suitable to screen pure compounds but also helpful to identify bioactive fractions.
Ellman assay results showed that anthraquinones from RHEI RADIX ET RHIZOMA were the strongest AChEIs among these potential chemicals. In contrast, naphthoquinone from ARNEBIAE RADIX exhibited most potent attenuation activity against H 2 O 2 -induced apoptosis in both SH-SY5Y and PC12 cells. Particularly, acetylshikonin, one naphthoquinone from ARNEBIAE RADIX, not only significantly inhibited AChE activity but also dramatically rescued oxidative stress-induced apoptosis in both SH-SY5Y and PC12 cells.
In addition to the role on the inhibition of Ach hydrolysis, there is evidence that all the FDA-approved AChEIs (tacrine, donepezil, rivastigmine, and galantamine) are neuroprotective agents. Three AChE inhibitors (tacrine, galanthamine, and donepezil) increased the activities of catalase (CAT) and glutathion peroxidase (GSH-Px) and protected PC12 cells from apoptosis generated by hydrogen peroxide. Donepezil also protected rat septal neurons from the toxicity induced by amyloid, while tacrine significantly attenuated hydrogen peroxide-induced injury and reversed hydrogen peroxideinduced overexpression of bax and p53 in PC12 cells [24][25][26].
Acetylshikonin, the major active components of ARNE-BIAE RADIX, exhibit many biological effects including anticancer [27], antioxidant [28], and antiobesity [29]. Recent study also revealed that acetylshikonin might increase antioxidant enzyme activity and nitric oxide levels in ethanolinduced ulcer rat models [28]. Shikonin, the analogs of acetylshikonin, has been reported to protect PC12 against 6-hydroxydopamine-mediated neurotoxicity [30]. However, reports on the antioxidative stress effects of acetylshikonin on neuronal cells are limited.
Mitochondria have been identified as a key site of cell apoptosis and death. The dysfunction of mitochondria resulted in ROS generation as well as mitochondria membrane potential loss. The cleaved caspase-3 and PARP were upregulated by excessive ROS and mitochondria membrane potential loss, which subsequently triggered cell apoptosis [31,32]. Present study demonstrated that acetylshikonin rescued H 2 O 2 -mediated ROS production, ΔΨm loss, and upregulation of cleaved caspase-3. Furthermore, H 2 O 2 stimulation resulted in upregulation of Bax expression and downregulation of Bcl-2 expression, therefore leading to the decline of Bcl-2/Bax ratio that served as another important indicator of mitochondrial dysfunction [33]. Western blot results confirmed that acetylshikonin increased the Bcl-2/Bax ratio by upregulation of Bcl-2 and downregulation of Bax, indicating that H 2 O 2 -induced mitochondrial dysfunction might be attenuated by acetylshikonin. In addition, p53, another proapoptotic factor [34], is essential for H 2 O 2induced apoptosis in glioma cells. A high level of p53 expression was observed in H 2 O 2 -induced apoptosis; however, this apoptosis was significantly reduced by antisense p53 oligonucleotide [35]. The increased p53 has been inhibited by acetylshikonin. Together, acetylshikonin has been reported to protect mitochondrial function from oxidative stress in both SH-SY5Y and PC12 cells.
Upregulation of HO-1 is the major approach to prevent H 2 O 2 -induced cells from apoptosis and cell death [36]. For further mechanistic exploration, the expression level of HO-1 was detected by western blot. Results showed the upregulation of HO-1 by acetylshikonin was observed in SH-SY5Y cells but not in PC12 cells. In cell proliferation assay, the specific antagonist of HO-1 ZnPP abolished the protective effects of acetylshikonin in SH-SY5Y cells. Consistent with western blot results, ZnPP cannot exert its effect on the neuroprotective activity of acetylshikonin in PC12 cells. Notably, shikonin, the analogs of acetylshikonin, has been reported to induce the Nrf2-ARE system that might upregulate the transcription of HO-1 genes [37]. Further systematic studies are needed to investigate whether upregulation of HO-1 effect by acetylshikonin is mediated by Nrf2-ARE pathway.

Conclusion
Together, we first reported that acetylshikonin, a novel AChEI, exhibited antiapoptotic activity through an HO-1 dependent mechanism in SH-SY5Y cells. Therefore, the findings suggested that acetylshikonin might provide potential benefits for Alzheimer's diseases treatment.