The Neuroprotective Effect of Byu d Mar 25 in LPS-Induced Alzheimer's Disease Mice Model

Inflammatory factors play an important role in the pathogenesis of Alzheimer's disease (AD). Byu d Mar 25 (BM25) has been suggested to have protective effects in the central nervous system. However, the effect of BM25 on AD has not been determined. This study aims to investigate the neuroprotective effect of BM25 in AD. A total of 40 AD model mice were randomly assigned to the following five groups (n = 8 per group): the AD + NS group, the AD + donepezil group, and three AD + BM25 groups treated with either 58.39 mg/kg (AD + BM25-L), 116.77 mg/kg (AD + BM25-M), or 233.54 mg/kg BM25 (AD + BM25-H). The Morris water maze test was performed to assess alterations in spatial learning and memory deficits. Nissl staining was performed to detect Nissl bodies and neuronal damage. The expression of IL-1β and TNF-α was evaluated by ELISA. The protein expression of P-P38, P38, P-IκBα, caspase 1, COX2, and iNOS was determined by western blotting. The expression of Aβ, p-Tau, and CD11b was measured by immunohistochemistry. The mRNA expression levels of IL-1β, TNF-α, COX2, and iNOS were measured by qRT-PCR. Spatial memory significantly improved in the AD + BM25-M and AD + BM25-H groups compared with the AD + NS group (p < 0.05). The expression of Aβ and p-Tau significantly decreased in the AD + BM25-M and AD + BM25-H groups (p < 0.05). The neuron density and hierarchy and number of pyramidal neurons significantly increased in the AD + BM25-M and AD + BM25-H groups (p < 0.05). In addition, the expression levels of CD11b, IL-1β, TNF-α, COX2, iNOS, caspase 1, p-IκBα, and p-P38 significantly decreased in the AD + BM25-M and AD + BM25-H groups (p < 0.05). In conclusion, our findings suggest that BM25 may exert anti-inflammatory and neuroprotective effects in AD model mice by suppressing the activity of microglia and inhibiting the phosphorylation of IκBα and p38 MAPK.


Introduction
Alzheimer's disease (AD) is a common neurodegenerative disease in the elderly population that causes declines in learning and memory [1][2][3][4]. e incidence of AD in people over the age of 65 is approximately 5% [5]. However, the pathogenesis of sporadic AD is still not fully understood. Neuroinflammation has been suggested to play an important role in the development of AD [6,7]. At present, the role of glial cell activation, especially microglial cells, in neuroinflammation has been widely confirmed [8,9].
Byu d Mar 25 (BM25) was developed by the Tibetan Medicine Master Dima Danzeng Peng Cuo in the 18th century and is still used today for multiple neurological disorders [10]. BM25 is composed of 25 rare herbs, such as saffron, calamus, and musk. It has the functions of opening the orifices and relieving pain. A clinical study has shown that BM25 has positive effects on neuropathic pain, epilepsy, stroke, and multiple peripheral neuropathies and neurological disorders [11]. In addition, BM25 has been shown to attenuate neuronal and astrocyte injury by inhibiting the neuronal denaturation and astrocyte overactivation induced by D-galactose [12]. ese findings may indicate a potential therapeutic role for BM25 in AD. e pharmacological mechanism of BM25 at the molecular level has been less studied. Du et al. found that BM25 reduced the expression levels of nitric oxide (NO) and nitric oxide synthase (NOS) in the plasma of migraine rat models [11]. Liu et al. reported that BM25 inhibited the phosphorylation of NF-kB P65 in human neuroblastoma cells (SH-SY5Y) [13].
e results of network pharmacological analysis suggested that the anti-AD mechanism of BM25 might be related to the regulation of the MAPK, insulin, and mTOR signal transduction pathways; intervention in inflammation and immunity; apoptosis and autophagy; and intervention in Aβ expression and clearance in brain tissue [14].
However, no systematic research on the effect and mechanism of BM25 in AD has been conducted. In the present study, we aimed to illustrate the effect and mechanism of BM25 in an LPS-induced AD mouse model.

Animals and Drug Administration.
is experiment followed the ethical standards of the Declaration of Helsinki as well as national and international guidelines. e research procedures were approved by the Ethics Committee of Tibet University, China (EC20190018). e LPS-induced AD mouse model was established according to our previous study [15]. A total of 40 LPS-induced AD mice were randomly divided into the following five groups: (1) AD + donepezil group (donepezil (1 mg/ml), 0.1 ml/ 10 g) (n � 8) (2) AD + NS group (normal saline (NS) (0.9%), 0.1 ml/ 10 g) (n � 8) (3) AD + BM25-L group (low dose, L) (58.39 mg/kg, 0.1 ml/10 g) (n � 8) (4) AD + BM25-M group (medium dose, M) (116.77 mg/ kg, 0.1 ml/10 g) (n � 8) (5) AD + BM25-H group (high dose, H) (233.54 mg/kg, 0.1 ml/10 g) (n � 8) e dosing and duration of BM25 followed the studies conducted by Du et al. [16] and Li et al. [17]. Drug treatments were performed by lateral ventricular stereotactic injection and lasted for four weeks. e Morris water maze test was performed on the last day of treatment to assess the alterations in spatial learning and memory deficits. Nissl staining was performed to detect Nissl bodies and neuronal damage. e expression of IL-1β and TNF-α was evaluated by ELISA. e expression of P-P38, P38, P-IκBα, Caspase1, COX2, and iNOS proteins was determined by western blotting. e expression of Aβ, p-Tau, and CD11b was measured by immunohistochemistry. e mRNA expression levels of IL-1β, TNF-α, COX2, and iNOS were measured by qRT-PCR.

Morris Water Maze
Test. Spatial learning and memory deficits in the five groups were evaluated by the Morris water maze on the last day of treatment. e test protocols followed a previously published study by Vorhees et al. [18]. An ANY-maze Video Tracking System (Stoelting Co., USA) was used to track and record animal movement during the trials. e swim path, escape latency, and frequency of crossing the target platform were recorded and analyzed.

Tissue Collection.
e mice were anesthetized with pentasorbital sodium (0.2%, 0.1 ml/10 g) by intraperitoneal injection. e brain tissue samples (n � 8) from each group were stored in 10% neutral formalin, and other specimens (n � 8) were stored at −80°C until further analysis.

Nissl Staining.
Nissl bodies in the cytoplasm of surviving neurons were detected by Nissl staining (Beyotime Institute of Biotechnology, China). e number of positive cells per unit area (mm 2 ) at the same site in the hippocampus was detected by using Image-Pro Plus 5.1 software (Media Cybernetics, Inc., Bethesda).

Enzyme-Linked Immunosorbent Assay (ELISA).
e expression levels of IL-1β and TNF-α in brain tissues were measured by ELISA with ELISA kits that were purchased from Sigma (Tokyo, Japan). e levels of IL-1β and TNF-α were detected by a microplate spectrophotometer (Multiskan MK, Finland). e measurement data are expressed as the mean ± standard deviation (SD).

qRT-PCR.
TRIzol Reagent (Invitrogen, Grand Island, NY, USA) was used to isolate total RNA from each brain tissue sample. e RNA quantity and integrity were measured by an ultraviolet spectrophotometer (UV-9000) (Shanghai Precision Instrument Co., Ltd.). Total RNA samples were purified with DNase, and cDNA was syn- 2.9. Statistical Analysis. All the data are presented as the mean ± standard deviation (mean ± SD). e significance of difference was analyzed by SPSS 22.0 followed by a t-test. A value of p < 0.05 was considered statistically significant. where the hidden platform was previously placed, than the mice in the AD + NS group, which revealed better spatial memory ability in the AD mice treated with BM25 (p < 0.05). In contrast, the neuron density and hierarchy decreased, the number of pyramidal neurons decreased, the neuron arrangement was disordered, the cell spacing increased, and neurons were significantly lost in the AD + NS group ( Figure 4). Figure 5, the relative expression of the caspase 1 protein was significantly lower in the AD + BM25-H (0.081 ± 0.024) and AD + BM25-M groups (0.140 ± 0.014) than in the AD + NS group (0.400 ± 0.102) (p < 0.05).

BM25 Inhibited the Activity of Microglia and Decreased the Expression Levels of IL-1β, TNF-α, COX2, and iNOS
3.3.1. Activity of Microglia. As shown in Figure 6, the microglial cells in the hippocampus of the AD + BM25-H and AD + BM25-M groups were small, rod-shaped, and thin and had few branches. e microglial cells in the AD + NS group were branched, and the cell bodies became larger and rounder with more branches. e number of activated microglial cells (CD11b-positive cells) in the AD + NS group was significantly increased compared with those in the AD + BM25-H (1216.63 ± 217.91) and AD + BM25-M groups (1404.20 ± 120.01) (p < 0.05).

Discussion
e etiology of AD is complex. Neuroinflammation is one of the main factors involved in the occurrence and development of AD and one of the important therapeutic targets for AD [19]. At present, the role of glial cells, especially microglia, in neuroinflammation has become a hotspot of research. Studies have shown that the inflammatory response induced by microglial activation is one of the pathogeneses of AD [20,21]. Microglia can be activated and then produce a large number of proinflammatory factors, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) [22]. Research has shown that IL-1β can be induced by the beta amyloid precursor (beta amyloid, Aβ) and cause Aβ deposition and Tau protein phosphorylation by reducing the expression of genes related to Aβ clearance [23]. Additionally, Aβ can bind to a specific receptor, activate microglia, and promote the release a large number of inflammatory factors and toxic substances [24]. Furthermore, activated microglial cells can express large quantities of iNOS and produce excess NO, which damages neurons by inhibiting cytochrome oxidase in the mitochondria of neurons [25]. Cox-2 is an inducible isoenzyme that is expressed in small amounts in microglia at rest. Under the action of proinflammatory molecules such as LPS, intracellular COX-2 mRNA levels increase, microglia are activated, and inflammatory mediators such as TNF-α and IL-6 are released [26].
BM25 is mainly used to invigorate the circulation of blood and to remove blood stasis. Chen et al. showed that calamus reduced the expression of the aquaporin-4 gene in glial cells [27]. Shi et al. reported that musk extract had a significant protective effect against the inflammatory damage of nerve cells caused by LPS, possibly by reducing the secretion of IL-6 by glial cells [28]. Our results confirmed that BM25 can significantly reduce the production of TNF-α, IL-1β, iNOS, COX-2, Aβ, and p-Tau and improve spatial memory, suggesting that BM25 may improve     Evidence-Based Complementary and Alternative Medicine proinflammatory cytokines and other genes [30]. e IκB family includes IκBα, IκBβ, and IκBε. IκBα is the most important inhibitor of NF-κB. When stimulated by an external signal, the IκB kinase (IKK) complex is activated. Activated IKK phosphorylates IKKα and IKKβ, which subsequently bind to ubiquitin ligases. IκBα is ubiquitinated and degraded by the proteasome, leading to NF-κB activation [31,32]. us, the phosphorylation of IκB is essential for NF-κB activation. Currently, NF-κB target genes include cytokines and inflammatory mediators (such as TNF-α, IL-6, IL-1, and iNOS). Excessive activation of NF-κB leads to the production of a large number of inflammatory cytokines, which aggravates the inflammatory response. In the present study, the phosphorylation of IκBα was detected by western blotting, and the results showed that BM25 significantly reduced the phosphorylation of IκBα, which indicated that BM25 may act as an anti-inflammatory agent by suppressing the phosphorylation of IκBα and inhibiting the expression of cytokines and inflammatory mediators (such as TNF-α, IL-1β, iNOS, and COX-2). In addition to the NF-κB signaling pathway, MAPK signaling plays an important role in the expression of inflammation-related factors after microglial activation. Youssef et al. showed that LPS can quickly activate p38, ERK, and JNK signaling in microglia [33]. Other molecules, such as ATP, thrombin, and TNF-α, also activate the MAPK signaling pathway, causing microglial activation [34]. Mitogen-activated protein kinases (MAPKs) are a type of Evidence-Based Complementary and Alternative Medicine 9 serine/threonine protein kinase in cells [35]. Normally, MAPK exists in cells in a nonphosphorylated form. Stimulation via recognition of LPS by TLR4 receptors on the surface of microglia can induce the phosphorylation of MAPK and activate the expression of related cytokines and inflammatory mediator genes [36]. Researchers have suggested that, among the three MAPK subfamilies (p38 MAPK, JNK, and ERK), p38 MAPK is most closely related to the inflammatory response [37]. Studies have shown that LPS promotes the phosphorylation of P38 MAPK in a dosedependent and time-dependent manner, thereby promoting the expression of inflammatory mediators such as TNF-α, IL-1β, and iNOS. Inhibiting the activation of P38 MAPK can inhibit the production of inflammatory mediators and protect neurons [38][39][40]. In the present study, the phosphorylation of P38 MAPK was detected by western blotting, and the results showed that BM25 significantly reduced the phosphorylation of P38 MAPK. erefore, the results of this study suggest that BM25 may inhibit the release of inflammatory mediators by inhibiting the P38 MAPK pathway. However, this study only investigated the effects and preliminary molecular mechanism of inflammatory factor release in an LPS-induced AD mouse model treated with BM25. Cell culture experiments are still lacking at present. erefore, it will be necessary to carry out cell experiments to better explain the anti-inflammatory and neuroprotective effects of BM25 in neuronal cells.

Conclusion
BM25 can significantly improve spatial memory, reduce neuronal apoptosis and death, and inhibit the production of Aβ, p-Tau, IL-1β, iNOS, COX-2, and TNF-α in an LPSinduced AD mouse model. Furthermore, BM25 can exert anti-inflammatory and neuroprotective effects by inhibiting the phosphorylation of IκBα in the NF-κB signaling pathway and p38 MAPK in the MAPK signaling pathway.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.