Optimized-SopungSunkiwon, a Herbal Formula, Attenuates Aβ Oligomer-Induced Neurotoxicity in Alzheimer's Disease Models

Alzheimer's disease (AD), the most common form of dementia, is an age-related neurodegenerative disease that is characterized by memory dysfunction, neuronal cell damage, and neuroinflammation. It is believed that AD-related pathology is mostly due to the overproduction of Aβ, especially the oligomeric form (AβO), in the brain. Evidence of the effects of multifunctional medicinal herbs in the treatment of AD has been steadily increasing. Optimized-SopungSunkiwon (OSS), a multiherbal formulation that is composed of six medicinal herbs derived from SopungSunkiwon, is a traditional medicine that is prescribed for neurodegenerative disorders in elderly patients. We previously reported that OSS showed an antiamnesic and memory enhancing effect in mice, but it is unknown whether OSS has a protective effect against AβO neurotoxicity. In this study, we investigated the effects of OSS in AD models induced by AβO in vitro and in vivo. We found that OSS protected neuronal cells and inhibited the generation of nitric oxide and reactive oxygen species against AβO toxicity in vitro. These results were confirmed by in vivo data that oral administration of OSS for 14 days attenuated memory impairments and neuronal cell death by modulating gliosis, glutathione depletion, and synaptic damage in the mouse hippocampus induced by AβO.


Introduction
Alzheimer's disease (AD) is characterized by progressive memory and learning disorders coupled with severe neuronal degeneration [1]. Although the exact mechanisms of AD pathogenesis remain to be established, it is widely known that amyloid-(A ) deposits play a key role in the disease [2]. Among the different forms of A , the oligomeric form (A O) is thought to be primarily related to the pathogenesis of AD because of its neurotoxicity, which impairs functional synaptic plasticity and induces memory loss by inhibiting hippocampal long-term potentiation (LTP) [3][4][5]. A O has also been implicated in triggering neuronal cell death by activating glial cells and generating reactive oxygen species (ROS) in AD brains [6][7][8]. These characteristics of A O indicate the potential that A O-induced experimental models to show various pathological features of AD may be useful.
The paradigm of drug discovery for neurodegenerative diseases is currently diverging from a single-target to a multitarget approach, because the effects of single-target drugs are too limited to allow for effective treatment of complex neurodegenerative diseases such as AD [9]. Recent studies have provided considerable evidence showing that the multimodal effects of several herbal extracts or herbal formulations are highly effective in the treatment of AD [10,11]. For example, EGb761, a standardized extract of Ginkgo biloba leaves, inhibits A -induced ROS accumulation, neuronal damage, and formation of A fibrils [12][13][14]. B401, a herbal formulation that is famous in traditional Chinese 2 Evidence-Based Complementary and Alternative Medicine medicine and is widely used to treat brain diseases, attenuates glutamate-induced neuronal cell death in SH-SY5Y cells and cognitive dysfunction in triple transgenic AD mice by reducing AD-related pathological proteins including A and tau [15]. Therefore, traditional herbal medicines, which have multitarget and multipotent effects, are emerging as potential treatments options for AD.
Optimized-SopungSunkiwon (OSS) is traditionally prescribed to treat senile constipation and it has been reported that it also works effectively in hyperglycemia, hyperlipidemia, and diabetic nephropathy [16,17]. OSS consists of the following six medicinal herbs: Bombyx mori L., Plantago asiatica L., Rheum palmatum L., Poria cocos Wolf, Gardenia jasminoides Ellis, and Cuscuta chinensis Lam. A previous study showed that Bombycis excrementum, the herb that is present in the largest proportion in the composition of OSS, protects hippocampal neurons and ameliorates memory impairment in mice in which AD-like pathological features are induced by intrahippocampal injection of A O 1-42 [18]. Moreover, we previously confirmed that OSS treatment results in memory enhancing activity as well as recovery from scopolamineinduced memory loss via the facilitation of acetylcholine release and regulation of synaptic proteins in mice [19]. However, the effect of OSS against A neurotoxicity is yet to be investigated.
In this study, we examined whether OSS displays neuroprotective effects against cognitive deficits, neuronal cell death, neuroinflammation, and synaptic loss in A O 1-42induced AD models in vitro and in vivo.

Preparation of OSS Extract.
OSS was prepared as has been previously described [19]. Briefly, OSS was made from a mixture of the following six herbs: Bombycis excrementum, Plantaginis Semen, Rhei Rhizoma, Gardenia Fructus, Poria, and Cuscutae Semen (1.5 : 1.5 : 0.5 : 1 : 1 : 1) obtained from the Kyongdong local market (Seoul, Korea). Each herb mixture (400 g) was extracted three times with sonication in distilled water for 2 h. Following filtration, the solution was evaporated in a vacuum and lyophilized (yield: 1.925%). The powder was kept at 4 ∘ C before use. This extract was previously standardized by analysis of sennoside A, crocin, and geniposide contents [19].

DPPH Radical Scavenging Activity
Assay. Various concentrations of OSS were mixed with 0.20 mM DPPH ethanolic solution (1 : 1). After incubation at dark room temperature for 30 min, the mixture determined at the absorbance of 517 nm using spectrophotometer. Also, the antioxidant activity of OSS was expressed as half maximal inhibiting concentration (IC 50 ) which is defined as the concentration of OSS required to scavenge 50% of DPPH radicals. IC 50 values were estimated by a nonlinear regression. DPPH radical scavenging activity (%) = {control -(sample -blank)}/control × 100.

ABTS Cation
Scavenging Activity Assay. 7.40 mM ABTS solution was added to 2.60 mM potassium phosphate 1 day before starting the experiment in the dark. Various concentrations of OSS were mixed with 7.40 mM ABTS solution and 2.60 mM potassium phosphate. After incubation at room temperature for 5 min, the mixture determined at the absorbance of 732 nm using spectrophotometer. Also, the antioxidant activity of OSS was expressed as IC 50 , which were estimated by a nonlinear regression. ABTS cation scavenging activity (%) = (control − sample)/control × 100.
2.6. Cell Culture and Treatment. Rat pheochromocytoma PC12 cells were maintained in RPMI, supplemented with 5% heat-inactivated FBS, 10% HS, and 1% P/S in an atmosphere of 5% CO 2 at 37 ∘ C. Mouse BV-2 microglial cells were maintained in DMEM, supplemented with 10% heat-inactivated FBS and 1% P/S in the same conditions. All experiments were carried out 12 h after PC12 and BV-2 cells were seeded in 96well plates at a density of 2.0 × 10 5 cells/ml. After the cells were about 70% confluent, various concentrations (0.1-100 g/ml) Evidence-Based Complementary and Alternative Medicine 3 of OSS in FBS free media were added to the cells for 24 h at 37 ∘ C, with or without 1 M A O 1-42 . An equal volume of vehicles was administered to the control and toxin groups, for each.

Measurement of Cell
Viability. PC12 cells were seeded on 96-well plates and were treated with OSS at doses of 0.1-100 g/ml for 24 h or pretreated with OSS for 1 h. They were then stimulated with 1 M A O 1-42 for 23 additional hours (pretreatment) or 1 M A O 1-42 was added for 1 h before treatment with OSS for 23 additional hours (posttreatment). After the treatment, supernatants were removed, and 1 mg/ml of tetrazolium dye (MTT) was added to the cells for 3 h. MTT medium was carefully removed from the wells, and the MTT formazan dye was eluted using dimethyl sulfoxide (DMSO). Absorbance was measured at a wavelength of 570 nm using a spectrophotometer (Versamax microplate reader, Molecular Device; Sunnyvale, CA, USA). Data were expressed as percentages of the values obtained for the controls.

Measurement of Extracellular NO.
The accumulated level of NO in culture supernatants was measured using a colorimetric reaction with Griess reagent using a slightly modified variant of the methods that have previously been described [20]. The supernatants (100 l) were transferred to a separate plate and added to 100 l of Griess reagent in the dark for 10 min at room temperature. Absorbance at 550 nm was measured. For each experiment, freshly prepared sodium nitrite that had been serially diluted was used as a standard, in parallel with culture supernatants.
2.9. Measurement of Intracellular ROS. Intracellular ROS generation was measured with DCFH-DA fluorescence dye, using a slightly modified version of previously described methods [20]. DCFH-DA enters cells passively and is converted into nonfluorescent DCFH, which reacts with ROS to form the fluorescent product dichlorofluorescin (DCF). Cells were seeded onto coverslips in 24-well plates and treated with OSS at 0.1, 1, and 10 g/mL for 1 h. Then, they were stimulated with 1 M A O 1-42 and incubated for an additional 30 min. The cells were incubated with 25 M DCFH-DA for 30 min. The fluorescence intensity was determined at 485 nm excitation and 535 nm emission, using a fluorescence microplate reader (SpectraMax Gemini EM; Molecular Device, Sunnyvale, CA, USA). Representative images were obtained using a fluorescence microscope (Olympus Microscope System BX51; Olympus, Tokyo, Japan).

Measurement of Total
Glutathione. The levels of total glutathione (GSH) were measured using the Total Glutathione Quantification kit (Dojindo Molecular Tech., Tokyo, Japan) according to the instruction manual and previously described method [21]. Briefly, hippocampal tissues were lysed and treated with 5% 5-sulfosalicylic acid. A coenzyme working solution, buffer solution, and enzyme working solution were added to each well at 37 ∘ C for 5 min. Then, a GSH standard solution, sample solution, and substrate working solution were added for 10 min each. Absorbance was measured using a spectrophotometer at a wavelength of 405 nm, and concentrations of GSH were determined in the sample solution using a GSH standard curve.

Animals and Surgery
Procedure. Male ICR mice (8 weeks, 27-30 g) were purchased from Daehan Biolink Co. Ltd. (Eumseong, Korea). Animals were housed in cages of 5 or 6, had free access to water and food, and were maintained under a constant temperature (23 ± 1 ∘ C), humidity (60 ± 10%), and a 12 h light/dark cycle. Animal treatment and maintenance were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication number 85-23, revised 1985) and the Animal Care and Use Guidelines of Kyung Hee University, Seoul, Korea. Stereotaxic injections of A O 1-42 into mouse hippocampi were performed as previously described [18,22]. In brief, mice were anesthetized and mounted in a stereotaxic apparatus (myNeuroLab, St. Louis, MO, USA). Each mouse was unilaterally injected (at a rate of 0.5 l/min) with 3 l of A O 1-42 (10 M) into the granule cell layer (GCL) of the hippocampus (coordinates with respect to bregma in mm: AP −2.0, ML 1.5, DV 2.0), according to a stereotaxic atlas of the mouse brain [23]. Shamoperated mice were injected with the same volume of saline alone. The accuracy of stereotaxic injection to the targeted region was monitored in all animals by examination of the needle tract within brain sections. In all groups, saline and OSS solutions were administered intraorally. OSS dissolved in saline was administered once per day for 14 days (5 days before surgery and for 9 days after surgery).

2.13.
Step-through Passive Avoidance Test. The step-through passive avoidance test (PAT) was performed according to a method described previously [18]. A learning and memory test was performed using a two-compartment step-through passive avoidance test apparatus. The box was divided into bright and dark compartments (21 × 21 × 21 cm 3 each) by a guillotine door. The bright compartment contained a 50 W electric lamp, and the floor of the dark compartment was composed of 2 mm stainless steel rods spaced 1 cm apart. Mice were treated with either OSS or vehicle 1 h before the acquisition trial and were initially placed in the bright compartment for the acquisition trial. The door between the two compartments was opened 10 s later. When the hind legs of the mice entered the dark chamber, the guillotine door was closed and an electrical foot shock (0.6 mA) was delivered through the grid floor for 3 s. The mice were again placed in the bright chamber for the retention trial, which was conducted 24 h after the acquisition trial. The time taken for a mouse to enter the dark chamber after the door was 4 Evidence-Based Complementary and Alternative Medicine opened was defined as the latency time. This was recorded for latencies of up to 300 s.

Novel Object Recognition Test.
The novel object recognition test (NORT) was performed according to a method described previously [18]. The experiments were carried out in a grey open field box (45 × 45 × 50 cm 3 ). Prior to the test, mice were habituated to the test box for 5 min without the presence of objects. After the habituation period, mice were placed into the test box containing two identical objects and were allowed to explore for 3 min. The objects used in this study were wooden blocks of the same size but different shape. The time spent by the animal exploring each object was measured (defined as the training session). Twenty-four hours after the training session, mice were allowed to explore the objects in the test box for 3 min, during which the familiar object used in the previous training session was placed with a novel object. The time that the animals spent exploring the novel and the familiar objects was recorded (defined as the test session). Animals were considered to be exploring an object when they were facing, sniffing, or biting it. The test box and objects were cleaned with 70% ethanol between sessions. Results were expressed as percentages of novel object recognition time (time percentage = exploring time for novel object/[exploring time for novel object + exploring time for familiar object] × 100).

Brain Tissue Preparation.
At 24 h after the memory examination, hippocampal tissue was dissected from the brains of 3 mice from each group in order to measure total glutathione levels. The remaining mice were transcardially perfused with 0.05 M phosphate-buffered saline (PBS) and then fixed with cold 4% PFA in 0.1 M phosphate buffer for cresyl violet staining and immunohistochemistry (n = 5 per group). The perfused brains were removed (whole) and postfixed overnight at 4 ∘ C in 0.1 M phosphate buffer containing 4% PFA. The brains were then immersed in a solution containing 30% sucrose in 0.05 M PBS for cryoprotection. Coronal sections (30 m) were serially cut using a freezing microtome (Leica, Nussloch, Germany) and stored in cryoprotectant (25% ethylene glycol, 25% glycerol, 0.05 M phosphate buffer) at 4 ∘ C until use in immunohistochemistry.

Cresyl Violet Staining and Immunohistochemistry.
For histological assessment of cell loss, free floating sections of mice brains were processed for cresyl violet staining and immunohistochemistry as described in the section above, following a method that had previously been used [18]. For cresyl violet staining, the sections were stained with 0.5% cresyl violet, after which they were mounted onto gelatin-coated slides, dehydrated through graded alcohols (70%, 80%, 90%, and 100%), placed in xylene, and coverslipped using histomount medium. For immunohistochemistry, brain sections were briefly rinsed in PBS and treated with 1% hydrogen peroxide for 15 min. The sections were incubated with a rabbit anti-GFAP antibody (1 : 3000 dilution), a rat anti-Mac-1 (1 : 1000 dilution), a mouse anti-SYN (1 : 200 dilution), or a rabbit anti-PSD-95 antibody (1 : 500 dilution) overnight at 4 ∘ C in the presence of 0.3% triton x-100 and NGS or NRS.
After rinsing in PBS, the sections were then incubated with biotinylated anti-rabbit IgG, anti-rat IgG, or anti-goat IgG (1 : 200 dilution) for 90 min and with ABC (1 : 100 dilution) for 1 h at room temperature. Peroxidase activity was visualized by incubating sections with DAB in 0.05 M tris-buffered saline (pH 7.6). After several rinses with PBS, sections were mounted onto gelatin-coated slides, dehydrated, and coverslipped with histomount medium. The optical densities of cresyl violet, GFAP, Mac-1, SYN, and PSD-95-positive cells in the dentate gyrus (DG) or CA3 region of the hippocampus were analyzed using ImageJ software (Bethesda, MD, USA). The images were taken at a 400x magnification using an optical light microscope (Olympus Microscope System BX51; Olympus, Tokyo, Japan) equipped with a 20x objective lens. Data are presented as percentages of the sham group values obtained.
2.17. Statistical Analysis. All statistical parameters were calculated using GraphPad Prism 5.0 software. Values are expressed as the mean ± standard error of the mean (SEM). Results were analyzed by one-way analysis of variance (ANOVA) analysis followed by the Newman-Keuls multiple comparison post hoc test. Differences with a p value lower than 0.05 were considered statistically significant.

Effect of OSS against
1-42 -Induced Neurotoxicity In Vitro. It has been reported that A O 1-42 induces PC12 cell death by inducing apoptosis [24]. In this study, we investigated whether OSS provides protection against A O 1-42induced cell death in vitro. Treatment with OSS only at 0.1-100 g/ml for 24 h showed no significant difference in cell viability compared to the control group (Figure 1(a)). Pretreatment with OSS at 10 and 100 g/ml significantly inhibited the reduction of cell viability (82.40 ± 3.02% and 88.40 ± 3.60%, resp.) compared with that of the 1 M A O 1-42 only treatment group (68.20 ± 2.16%; Figure 1(b)). Posttreatment with OSS at 10 g/ml also significantly ameliorated cell viability (67.73 ± 2.59%) compared with that of A O 1-42 only treatment group (55.10 ± 0.96%; Figure 1(c)).

Effect of OSS
1-42 -Induced NO Generation In Vitro. NO plays a key role in a variety of inflammatory statuses, being released in response to pathological stimuli [25]. Excessive concentrations of NO also lead to the formation of oxidative stress cascades, thereby contributing to a neurotoxic cascade such as A -mediated neurodegeneration [26]. To examine the anti-inflammatory effects of OSS against A O 1-42 , we evaluated whether OSS inhibits NO production in activated microglia cells induced by A O 1-42 . Incubation with 1 M A O 1-42 increased NO production up to about 10 M. Compared to the group treated with A O 1-42 only, the group that underwent pretreatment with OSS at 1 and 10 g/ml significantly inhibited NO generation (6.18 ± 0.46 M and 5.98 ± 0.64 M, resp.) (Figure 2(a)). Posttreatment with OSS showed that NO generation was inhibited compared to the production levels observed in the A O 1-42 only treatment group. However, the observed difference was not significant (Figure 2(b)).

Antioxidant Effects of OSS In Vitro and In Vivo.
To evaluate the antioxidant potential of OSS, we performed the DPPH free radical and ABTS cation scavenging assay. We found that OSS showed higher scavenging activity than an extract of Scutellaria baicalensis Georgi (SBE), used as a positive control in both the DPPH and ABTS assays ( Table 1). This trend is in accordance with the inhibitory effects of OSS against A O 1-42 -induced ROS generation.
In this study, pretreatment with OSS at 10 g/ml significantly inhibited ROS generation (144.53 ± 11.44%) when compared to the values obtained with the A O 1-42 only treatment group (168.77 ± 14.53%; Figure 3(a)). Posttreatment with OSS at 10 g/ml also led to significantly lower ROS generation values (109.61 ± 8.25%) after A O 1-42 insult compared to those obtained from the A O 1-42 only treatment group (139.74 ± 7.20%; Figure 3(b)). Moreover, we investigated the effects of OSS on the induction of GSH as an antioxidant in the mouse hippocampus. The levels of GSH, the most prevalent antioxidant in the brain, consistently decrease with increasing oxidative stress in AD [27,28].     Injection in Mice. To investigate whether OSS ameliorated memory impairment in mice receiving an intrahippocampal injection of A O 1-42 , NORT and PAT were performed in this study. In the NORT, the A O 1-42injected mice spent similar amounts of time (50.90 ± 2.29%) exploring the novel object and the familiar object during the test session. In contrast, sham-operated mice spent more time exploring the novel object (70.28 ± 2.30%). Treatment with OSS at 50, 100, and 200 mg/kg/day significantly improved A O 1-42 -induced cognitive deficits in this test (61.76 ± 0.81%, 63.75 ± 1.62%, and 58.34 ± 0.60%, resp.; Figure 4(a)). No significant differences in novel object recognition time were found between any of the tested groups during the training session.

Effect of OSS on Memory Impairment Induced by Intrahippocampal
In PAT, the mean latency time of the A O 1-42 -injected vehicle-treated group (85.13 ± 6.04 s) was significantly shorter than that of the sham-operated group (229.42 ± 9.72 s). OSS administered at 50, 100, and 200 mg/kg/day significantly reversed the observed effect of the A O 1-42 -injected vehicletreatment in this test (140.27 ± 11.16 s, 186.08 ± 14.08 s, and 140.51 ± 7.48 s, resp.; Figure 4(b)). No differences in latency time were observed between any of the tested groups during the acquisition trial.

Effect of OSS on
1-42 -Triggered Neuronal Atrophy in the Mouse Hippocampus. Brain atrophy caused by neuronal death is a pathological hallmark of AD in humans and hippocampal atrophy, in particular, is closely related to memory dysfunction [29,30]. To further understand the mechanisms underlying the recovery of memory function, the inhibition of A O 1-42 -triggered hippocampal neuronal death by OSS was investigated using cresyl violet staining.
The A O 1-42 -induced reductions in neuronal density in the granule cell layer (GCL) of the DG (89.23 ± 1.47%) and CA3 (60.25 ± 2.45%) hippocampal regions were significant compared to those of the sham-operated group. This loss was significantly inhibited by OSS treatment at 50, 100, and 200 mg/kg/day in the CA3 region of the mouse hippocampus ( Figure 5).

Effects of OSS on
1-42 -Induced Astrocyte and Microglia Activation in the Mouse Hippocampus. It is known that the activation of astrocyte and microglia under neuroinflammatory conditions plays an important role in the destruction of neurons and leads to synaptic dysfunction, thereby resulting in memory deficits [31]. The intensity of GFAP, a specific marker for astrocytes, in the hilus region of the DG was significantly increased in the A O 1-42 -injected group (190.37 ± 5.10%) as compared with the sham-operated group. This intensity was significantly reduced after OSS treatment at 100 and 200 mg/kg/day (160.89 ± 4.62% and 160.27 ± 6.30%, resp.; Figure 6(a)).
The intensity of mac-1, a specific marker for microglia, in the hilus region of the DG was also nearly doubled in the A O 1-42 -injected group (188.56 ± 9.92%) compared with the sham-operated group. In contrast, mac-1-positive intensity of A O 1-42 -injected mice treated with OSS at 100 and 200 mg/kg/day was significantly decreased (159.25 ± 2.76% and 156.56 ± 4.44%, resp.; Figure 6(b)). Taken together, OSS treatment effectively inhibits hyperactivation of astrocyte and microglia triggered by A O 1-42 toxicity.  in the Mouse Hippocampus. Growing evidence shows that A O induce depletion of hippocampal synaptic proteins such as SYN and PSD-95, resulting in memory dysfunction in  AD [32,33]. As shown in Figure 7, the immunoreactivity of both SYN and PSD-95 in the hippocampal CA3 region was markedly decreased in the A O 1-42 -injected group (75.21 ± 2.66% and 75.95 ± 1.27%, resp.) compared with shamoperated group. This trend was significantly reversed by OSS treatment at 50, 100, and 200 mg/kg/day for both SYN (85.10 ± 2.27%, 88.10 ± 1.96%, and 92.65 ± 2.70%, resp.) and PSD-95 (87.18 ± 2.12%, 92.32 ± 0.93%, and 92.74 ± 1.38%, resp.). These data suggest that OSS restores A O 1-42 -induced synaptic disruption, which is linked to the amelioration of memory impairment.

Discussion
A O, the most toxic form of A , is considered to play a central role in AD pathogenesis rather than A monomers or fibrils [34,35]. He et al. demonstrated that memory impairment and hippocampal CA1 neuronal damage were more remarkable in A O 1-42 -infused rats than in those where features of AD pathology were induced by A 1-42 fibrils due to the observation that A O 1-42 more evidently exhibited proinflammatory factor stimulation than A 1-42 fibrils [36]. Our present data shows that systemic treatment with OSS ameliorates memory dysfunction by blocking A O 1-42 -induced hippocampal cell damage, hippocampal GSH depletion, glial hyperactivation, and synaptic disruption in a mouse model of AD. It was also confirmed that OSS directly inhibited A O 1-42 -induced cell degeneration as well as overproduction of NO and ROS in vitro.
Oxidative stress is an important pathological factor of AD [37]. Several studies indicate that A 1-42 peptide is at the center of oxidative damage as it is an indicator of ROS generation in AD brains [38]. Additionally, increased A -mediated ROS generation can damage the endogenous  antioxidant GSH and enzymes such as superoxide dismutase, GSH peroxidase, and catalase, thus inducing A deposits to form in the brain [39]. A deposits stimulate activation of nearby microglia and astrocytes, generating an inflammatory response through the release of proinflammatory mediators [40]. It has been suggested that activated glia are involved in neuronal degeneration because they produce potent toxic molecules including NO and cytokines [41,42]. The present study demonstrates that OSS treatment inhibits ROS generation in PC12 cells and restores GSH contents depleted by A O 1-42 in hippocampal tissue. Intracellular ROS concentration and endogenous oxidant system normalized by OSS also has an influence on the deactivation of glial cells in the hippocampus as well as on the reduction of NO production in BV-2 microglia cells.
The hippocampal synapse network originates from axons of the CA3 pyramidal region, which connect to almost all regions of the hippocampus [43,44]. Furthermore, CA3 synapses modulate homeostatic plasticity connected to hippocampal neurons [45]. Thus, a marked decrease of synaptic density in the hippocampal CA3 region is highly relevant to synaptic disruption, which is closely linked to memory decline in the pathogenesis of AD [46]. Soluble A O impair hippocampal LTP and can also induce memory dysfunction [3,32]. In this study, it has been demonstrated that OSS treatment rescues synaptic damage in the hippocampal CA3 region based on the results obtained using SYN and PSD-95 markers, which are specific pre-and postsynaptic proteins, respectively [47,48]. In this context, it can be hypothesized that the restoration of memory function after OSS treatment is mediated by facilitated hippocampal synapses.
Other approaches investigating potential therapeutics for AD indicate that the design of multitarget drugs is increasingly necessary because most single-target candidates have been unsuccessful in the treatment of AD given that it is a complex and multifaceted pathogenesis [49,50]. This paradigm of drug discovery for AD is in accordance  with the multifunctional actions of medicinal herbs. The neuroprotective effects of OSS observed in this study can be due to each individual herb of OSS. Water extract from silkworm feces (Bombycis excrementum), for example, was shown to protect hippocampal neurons and memory impairment induced by A O 1-42 in our previous report [18]. Rhaponticin and rhapontigenin isolated from rhubarb roots (Rhei Rhizoma) significantly inhibit A 1-42 -induced apoptotic mechanisms by regulating Bax/Bcl-2 proapoptotic genes in human neuroblastoma cells [51]. Additionally, the protective effects of Poria water extract against A 1-42mediated cell death in PC12 cells were also reported [52]. Furthermore, geniposide, one of the active compounds of Gardeniae fructus, has been shown to exhibit multifunctional neuroprotective effects by blocking receptors for advanced end product-mediated signaling in APP/PS1 transgenic mice and BV-2 microglia cells [53,54]. These constituents of OSS may have contributed to its neuroprotective effects against A O 1-42 neurotoxicity.

Conclusion
In summary, OSS treatment alleviates A O 1-42 -induced damage of memory function and hippocampal neurons. This effect is likely to be mediated by the inhibition of oxidative stress, neuroinflammation, and decline in hippocampal synaptic density. Further detailed investigation is required to reveal the underlying mechanisms that might explain how OSS treatment regulates neuroinflammation and hippocampal neuronal and synaptic damage. Taken together, our data suggest that OSS may be a potential multitargeted candidate for AD treatment.