In Silico Analysis of Baicalin’s Binding on Amyloid- β, Presenilin-1 and Tau Proteins: Validation in a Mouse Model of Cerebral Amyloidosis

The production of amyloid-β following activation of presenilin-1 and accumulation of the hyperphosphorylated tau protein contribute to Alzheimer’s disease development. In order to understand Baicalin’s effect on AD treatment, we have used in silico studies to predict its binding on amyloid-β, presenilin-1 and tau protein receptors. Computational molecular docking was performed using Molegro Virtual Docker software. The scores obtained were -112.35, -68 and -53 Kcal/mol respectively for the three receptors, indicating potent inhibitory effect of Baicalin on amyloid-β (highest binding score). Moreover, Baicalin showed high affinity towards 3jq5 (amyloid-β protein receptor) as it showed thirteen hydrogen bonds with nine amino acids. Contrary to what was observed with amyloid-β, a lower binding affinity was observed for tau-protein. In addition, to confirm molecular docking results, in vivo studies using APP/PS1 mice (a model of cerebral amyloidosis) treatment with Baicalin showed that it restores behavioural impairment, inhibits the production of amyloid-β in the cortex, alongside with the inhibition of macrophage infiltration in the brains of these mice. These findings open new ways for deeper understanding of the amyloid hypothesis in AD and consideration of Baicalin as a promising drug for AD treatment.


Introduction
The development of new drugs in the last decade consisted in the association of diseases with the functioning of key proteins which need to be blocked in order to find an effective treatment.
This is the case of Alzheimer's Disease (AD), a chronic disorder associated with cognitive decline, loss of memory and death [1].

from the plant Scutellaria baicalensis. Recent studies showed that
Baicalin inhibits oxidative stress injury in neuronal cells [2]. Baicalin has been established to have potent anti-inflammatory and neuroprotective effects by protecting neurons from microglia-mediated neuroinflammation via suppressing NLRP3 inflammasome and TLR4/NF-KB pathway [3]. The drug was also reported to inhibit Aβ-induced microglial cell activation by regulating the JAK2/ STAT3 pathway i006E AD transgenic mice [4]. As far as Amyloid-β inhibition is concerned, Baicalin was shown to protect SH-SY5Y cells from oxidative injuries induced by Aβ1-42 aggregation [5]. AD patient's brains revealed the close presence of microglia around Aβ plaques, indicating that neuroinflammation is involved in disease progression. With its anti-inflammatory effects [6], Baicalin appears to be a drug worth investigation due to its multiple effects on disease symptoms.
Structure based-drug design is recognized as a promising tool to discover drug inhibitors and has become a famous way of drug discovery. With this new tool, the drug design process has been facilitated and speeded up considerably. Taking advantage of molecular docking tools, we have first investigated the binding potential of Baicalin on the key enzymes involved in AD initiation and progression. Afterwards, identified target receptors have been validated in vivo using a transgenic mice model of the disease (APP/PS1) which is used to mimic human manifestations of AD.

Molecular Docking
Ligand preparation: Baicalin was identified from PubChem chemical database. The structure was drawn using Molview software ( Figure 1) and energy minimization performed using the MM2 force field and saved as a. MOL format. The missing charges and hybridization states of Baicalin structure were assigned with the help of the MVD software.
Protein preparation: The 3D structure of different receptors was retrieved from the Protein Data Bank (PDB) (http://www. rcsb.org). We have chosen respectively: Amyloid-β (PDB ID: 1BA4), presenilin-1 (PDB ID: 5A63) and tau protein (PDB ID:6NK4). The proteins had one or two polypeptides and were co-crystallized with ligands. The targets were visually inspected, and reference ligands identified for each receptor. Receptors were prepared for docking by removal of water molecules, ligands, cofactors and assigning bonds, bond order, hybridization and charges using the MVD software [7].

Docking search algorithm and scoring functions: MVD uses
PLP (Piecewise Linear Potential) algorithm as scoring function for computational screening. In this study, the MolDock simplex evolution search algorithm was used for docking. In practice, the number of receptor cavities was limited to four and using the cavity prediction wizard, the cavity with large volume was selected.
Docking of Baicalin in different receptors was performed and the best poses generated were used based on the docking scores (expressed in Kcal/mol).
Parameters for scoring functions: MVD software uses two scoring functions: the MolDock score and the ReRank score, where MolDock score is an E score (docking scoring function) defined as: E score= E inter+ E intra.
A. E inter: Sum of ligand-protein interaction energy, ligand-water interaction energy and ligand-cofactor interaction energy [8].
B. E intra: Internal energy of the ligand.
ReRank score provides an estimation of the ligand-receptor interaction strength [9].

Animals
For in vivo experimentation, we have used male APP/PS1 mice

Treatment with Baicalin
APP/PS1 transgenic mice were divided into two groups (n=6, three females and three males at the age of 5 months). Mice were treated for 21 days as follows. A) Baicalin daily oral administration, 20mg/kg bw, B) Vehicle-treated used as control in this study.

Nest construction assay
We used the next construction assay with few modifications to evaluate the deficits in social behaviour of APP/PS1 mice and the potential effect of Baicalin following treatment. Mice were individually housed for 24 hours in clean plastic cages with 1cm of wood chip bedding. Individual cages were then supplied with paper towel torn into 5x5cm squared pieces, two hours prior to the onset of the dark phase. On the following day, different cages were inspected for nest construction. Test scores were examined by three independent individuals (who were not aware of the treatment procedure) by observing the paper Powel with the help of a three-point scale system: A. No biting or tears on the paper, B. Moderate biting and/or tears on the paper but no coherent nest C. The vast majority of paper torn into pieces and grouped into a corner of the cage.

Immunohistochemistry and image analysis
Mice treated with Baicalin and control mice were sacrificed after 21-days of treatment. Thereafter, they were perfused intrac-

Statistical analysis
Differences of plaque/cell counts and nest construction data were analysed using the unpaired t-test (Graph Pad Prism 6.0 software). For all statistical analysis, significance levels were set at P<0.05.

Molecular Docking of Baicalin with key Receptors involved in Alzheimer's Disease
After performing docking analysis, the results obtained in Ta

Effect of Baicalin Treatment on Nest Construction Capacity
Transgenic APP/PS1 mice were tested to evaluate the effect of Baicalin treatment on behavioural impairment. Following treatment, we observed an improvement of the nesting capacities of treated mice. As shown in Figure 3, no significant difference was observed between treated and non-treated mice at day 1 (control= 1.6., Baicalin=2.00, n=6). Interestingly, after 21 days of treatment, a significant difference between treated and non-treated mice was observed (control=1.7, Baicalin=2.8, n=6) with an immediate chew-ing and tearing of the paper towel fragments. Contrary to non-treated mice, APP/PS1 transgenic mice chewed the paper towels which were found grouped at one corner of the cage ( Figure 3C).

Effect of Baicalin on Amyloidosis and Neuroinflammation
Mice receiving no treatment did not show any significant dif-   The unpaired t-test was used to calculate the differences of cell counts between treatment group and control, with significance set at p < 0.05.

American Journal of Biomedical Science & Research
Copy@ Brice Ayissi Owona Figure 5: Effect of Baicalin on amyloid-β deposition in mice cortex and hippocampus. APP/PS1 mice were either treated with Baicalin or the vehicle (carboxymethylcellulose). After 21 days of treatment by gavage, brains were dissected and analysed by immunohistochemistry. Plaques of amyloid-β were counted and represented in a graph. The unpaired t-test was used to calculate the differences of plaque counts between treatment group and control, with significance set at p < 0.05. A reduction of amyloid plaques was observed in the cortex of transgenic mice. No significant effect was observed in the hippocampus.

Discussion
Baicalin is a bioactive flavone with reported anti-inflammatory and neuroprotective effects [10]. In order to confirm the results obtained following molecular docking, we tried to study the effect of Baicalin on amyloid-β in vivo. We have used transgenic mice APP/PS1, a mouse model of AD amyloidosis exhibiting remarquable elevation of amyloid-β associated with behavioural deficiencies. Moreover, these mice express the Swedish mutation of APP and the L166P mutation of human presenilin-1. In addition, these mice show plaque formation at 2 months in the neo-cortex and 4 months in the hippocampus [12]. Impairment in nesting construction is another hallmark of AD and this is used to mimic behavioural deficits in vivo. In this study, Baicalin was administered to mice in vivo for a period of 21 days. The results obtained showed that after this treatment time, mice treated with Baicalin showed an increase in nesting score in comparison to control mice, indicating the potential of the drug to improve behavioral impairments in these mice. The same effect obtained with Baicalin was noted with other flavonoid and plant compounds such as Zerumbone, Herperidin and Icariin [13][14][15]. Another hallmark of AD is the deposition and accumulation of amyloid-β in the cortex and hippocampus. The results obtained showed a significant reduction of plaque number in the cortex, but, no significant reduction was observed in the hippocampus of mice. From the results obtained, it appears that the time of treatment with Baicalin may have an effect on its efficiency in plaque reduction.
In a recent study, it was reported that treatment of APP/PS1 mice with Baicalin for 11 days did not show any significant effect in hippocampus and cortex [16]. Increasing the number of days of treatment may therefore considerably improve the treatment, at least by reducing plaque accumulation in the cortex. Accumulation of amyloid-β always results in neuroinflammation with activation of microglia which in turn potentiate inflammation in the brain. Therefore, anti-inflammatory compounds in the brain could be good candidate for AD treatment. Baicalin is a well-known anti-inflammatory drug with in vitro antioxidant and immunomodulatory effects. Treatment of mice with the drug showed a reduction of inflammation in the cortex and hippocampus of treated mice in comparison to the control mice. This was evaluated in this study by measuring the expression of Iba+ cells, a marker of microglia inflammation. In silico inhibition of amyloid-β therefore corroborate in vivo observations, suggesting that the binding of Baicalin with high score and high affinity to its receptor is responsible for the observed improvement in nesting scores, amyloid plaque number reduction in the cortex and the resulting reduction inflammation in mice brains.

Conclusion
This study investigated the capacity of Baicalin to bind amyloid-β, presenilin-1 and tau protein, which are proteins involved in the initiation and development of AD. In silico studies showed that Baicalin binds preferably to amyloid-β with the highest and strongest energy. In vivo, Baicalin showed inhibitory effect on Aβ accumulation in the cortex and restored behavioral impairments in mice. Therefore, further investigation of Baicalin as a potential drug for AD are needed in order to open new ways for drug discovery.