Animals

In vivo study was conducted on experimental animals by using 4.5-6 months adult male Wistar rats, weighing 250-300 g obtained from the animal house. All tests were carried out at 20-25°C (12 hours light and 12 hours dark). Municipal tap water was used as drinking water and compressed food as nutrition (consisting of 23% protein, 3.0% crude fat, 7.0% crude fiber: 8% acid insoluble ash, 1-2.5% calcium, 0.9% phosphorus, 0.5-1% sodium, 12% moisture). All the experimental works were approved by the Ethical Committee of Tabriz University of Medical Science (Tabriz, Iran) and conform to the European Communities Council Directive of 24 November 1986 (86/609/EEC).

AChE activity assessment in hippocampal primary culture homogenate

Primary neuronal cultures of rat hippocampus were prepared according to a previous study with some modifications (Seibenhener and Wooten, 2012). Dissected hippocampus (isolated from neonatal rat) was triturated by trypsin and mechanical disruption. The cells were differentiated in neurobasal medium supplemented with B27 and FGF2 for 7 days. The resulting cells were homogenized in cold buffer phosphate, 28 mM (pH 7.8), containing Triton X100 (Sadeghi et al., 2018). The homogenized samples were then centrifuged at 13,000 g for 20 min and subsequent supernatants were considered for AChE assay. AChE activity was determined based on spectroscopic monitoring of 5-thio-2-nitro-benzoate anion production at 430 nm during the reaction between 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) and thiocholine. In this reaction, acetylcholine iodide as a substrate was converted to thiocholine that reacts with DTNB and produces 5-thio-2-nitrobenzoate. The final concentrations of DTNB and substrate were 0.33 mM and 1.56 mM, respectively, in all assay medium. The activity of the enzyme was expressed as μmole of ACh hydrolyzed per minute per milligram protein in the absence and presence of bile acids (10 μM), individually. The homogenate’s protein concentration was evaluated according to the Bradford method (Bradford, 1976). All assays were done in five replicates.

Molecular docking analysis

In order to precisely assess the affinity of CA and SDC to AChE, Auto Dock software (version 4.2) was used to simulate the binding process of bile acid molecules to AChE enzyme (Morris et al., 2009). For this aim, firstly, the crystal structure of AChE (PDB ID: 1DX6) was taken from the Protein Data Bank (https://www.rcsb.org/). Since different subunits of the enzyme are the same, the subunit A was selected to perform docking analysis precisely. The 3D molecular structures of CA and SDC were obtained from the PubChem website (https://pubchem.ncbi.nlm.nih.gov). Their structural conformations were minimized by Gaussian 03 programs based on a theoretical level of B3LYP with 6–31G basis set. Polar hydrogen atoms were added into AChE as a target and Kollman charges were also calculated. In addition, the rotation of bile acid molecules was defined and rotatable bonds were detected. To identify the ideal binding site of each bile acid on AChE protein, a docking calculation using the Lamarckian genetic algorithm (LGA) search method was performed. All other calculation parameters were default settings. Chimera 1.10.1 program was used to visualize the obtained results (Rizvi et al., 2013).

In vivo experimental design

NBML rats were used as an AD model as follows: rats were anesthetized with ketamine (125 mg/kg, i.p.) and xylazine (10 mg/kg, i.p) and then placed in a Stoelting stereotaxic apparatus. The midline of clean scalp was notched and a burr hole was drilled into the skull. Following that, ibotenic acid was injected at the NBM nucleus with coordination as follows: AP=1.2, ML=±3.2, and DV=7.5 mm from the surface of skull (Paxinos and Watson, 2005). Each side of the NBM was injected with 1 μl of 5 μg/μl ibotenic acid solution with a microinjection pump at the speed of 120 μ lit/h. After 7 days of recovery, the animals were divided randomly into three groups as follows:

Control: rats were submitted to surgery and injected with 1 μl saline (as equivalent shock obtained by surgery and local injection) and were orally administered saline after recovery for 25 days.

Rat model of AD: NBML rats in which both NBM nuclei were bilaterally destroyed by ibotenic acid during surgery. These rats received saline orally after recovery for 25 days.

NBML rats treated with bile acids: this group consisted of 24 rats whose NBM nuclei were bilaterally destroyed by ibotenic acid. After recovery, the AD-like rats were randomly divided into three groups (8 rats in each group) and two groups were treated orally with 30 mg/kg of SDC or CA for 25 days. The third group received 15 mg/kg SDC+15 mg/kg CA to study synergic effects of both bile acids. The dose of bile acids and the experimental time duration were selected according to a previous study (Lo et al., 2013).

To confirm the increase of bile acid levels in the brain tissue of rats after 25 days of treatment (15 mg/kg SDC+15 mg/kg CA), the concentration of CA and SDC were evaluated in brain tissue by using CA and DCA ELISA Kit (Cell BIOLABS and MYBIOSOURCE). According to the obtained results, CA and DCA concentrations in the control group were calculated at 0.1±0.02 nmol/g and 0.06±0.01 nmol/g, respectively, which increased to 0.24±0.08 nmol/g and 0.18±0.03 nmol/g, respectively, in rats that received combined bile acids during the 25 days. Therefore, oral administration of bile acids enhanced these molecules’ concentration more than two-fold in brain tissue.

Western blotting analysis

Western blotting was carried out according to a standard protocol (Sadeghi et al., 2017). After SDS-PAGE, the proteins were transferred onto PVDF membrane under 140 V for 1.5-2 h in transfer buffer. The membrane was probed with the primary [Anti-Glial Fibrillary Acidic Protein (GFAP) antibody (G3893, Sigma-Aldrich), Anti-beta Amyloid 1-42 antibody (ab10148, Abcam), rat specific anti-acetylcholinesterase antibody (ab31276, Abcam), rat specific anti-amyloid precursor protein (APP) antibody (ab2072, Abcam)] and secondary specific antibodies after blocking and washed four times in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05 % Tween 20). An ECL detection kit was used to visualize bands containing specific proteins according to the manual. Anti β-actin (1:1000) (Cell Signaling Technology) was used as a housekeeping protein to control protein concentration.

Histological studies

For histological analysis, the rats from each experimental group were sacrificed under deep ketamine and xylazine anesthesia and before fixation with 4% formaldehyde and were subsequently perfused with 0.3% sodium sulphide in 0.1 M phosphate buffer. The separated brains were stored in 30% sucrose for cryoprotection and sectioned on a freezing microtome at 8 μm. The tissue sections were stained with Thioflavin T, a general marker of amyloid deposits, and studied by a fluorescent microscope. To this purpose, the tissue sections were incubated with freshly filtered 0.05 % Thioflavin T solution in PBS for 8 min at room temperature (Batelli et al., 2008).

Statistical analysis

SPSS10 software was used to analyze the data. Analysis and identification of the differences between experimental groups and control were performed using the t-test and p<0.05 was considered as a significant difference level.

Bile acids bind to the active site gorge and inhibit AChE catalytic activity

AChE belongs to the serine hydrolase family that contains the α/β hydrolase fold as a common structural element (Holmquist, 2000). A hallmark of AChE is its catalytic activity that can be assessed by the Ellman method, a sensitive, rapid and reliable method for detecting very low amounts of AChE catalytic function (Sadeghi et al., 2018). In the present study, primary hippocampal neuron cultures were used as a source of AChE to investigate the possible effects of bile acids (CA, SDC and CA+SDC) on catalytic activity of AChE. The results showed that bile acids could significantly inhibit hydrolyzing activity of the enzyme. The catalytic function of AChE was reduced from 6.37±0.52 to 5.56±0.31 μmol/min.mg (protein) in the presence of CA (10 μM), while SDC reduced AChE activity to 3.64±0.37 μmol/min.mg (protein). Further, it was shown that 10 μM of combined bile acids (5 μM CA+ 5 μM SDC) could reduce the enzyme activity to 2.07±0.23 μmol/min.mg (protein). The maximum rates of AChE inhibition occurred in the presence of SDC+CA and decreased more than three-fold (Table I).

Table I.

The effects of bile acids on hydrolytic activity of AChE and binding parameters of each bile acid on the enzyme. Experimental data was expressed as the mean ± S.E.M. of at least five independent tests.

Molecular docking simulation

Molecular docking simulation revealed that both of the evaluated bile acids were capable of binding to the active site gorge of AChE at different affinities. The binding energy was calculated as -7.76 and -8.94, respectively, for the binding of CA and SDC to the active site gorge. It was shown that the active site of AChE consists of catalytic residues (Ser 203, His 447 and Glu 334), which are located within the gorge, similar to other AChEs in mammals (Johnson and Moore, 2006; García-Ayllón et al., 2011; Liu et al., 2017). Also, some anionic and hydrophobic amino acids surround the entrance of the active site known as the PAS site (Tyr 286, Trp 286, Phe 338, Val 294 and Leu 289) (Johnson and Moore, 2006; García-Ayllón et al., 2011). Fig. 1 shows the binding sites of bile acids on the active site of AChE. It was indicated that CA due to the bulky structure could bind to PAS and is not able to reach to the catalytic site at deep of the gorge so could limit substrate access to the catalytic site. While SDC could pass through the PAS tunnel and reach to CAS which limits substrate binding to the active site. All docking results are listed in Table I.

Fig. 1.

Bile acid molecules in the AChE active site gorge. (A) shows the surface view of the active site cavity representing the crystal structure of AChE with two bile acids bound. (B) shows a cartoon form of one subunit of AChE that binds to bile acids by hydrophobic interactions and hydrogen bonds. CA and SDC are represented as a cyan and brown molecule, respectively. (C) indicates the binding position of each bile acid in the active site gorge in comparison with PAS and CAS sites. (D) shows molecular structures of each bile acid with the C₇ marked in red.

Bile acids treatment attenuated catalytic activity of hippocampal AChE that was increased by NBM lesion

NBM lesion causes impaired spatial navigation according to Fig. S1 (supplementary data) in experimental animals. The pathogenesis of AD has been linked to AChE activation that is also accompanied by β-amyloid deposition (Dong et al., 2004; Johnson and Moore, 2006). Therefore, to investigate whether NBM lesion and oral administration of bile acids could affect AChE activity in the rat hippocampus, the enzyme activity in homogenate tissue was evaluated for all groups. In agreement with our previous study (Sadeghi et al., 2013), the results showed that AChE activity was significantly increased in NBML rats up to 2982.71±152.26 nmol/min.mg protein, while the enzyme activity in the control hippocampus was calculated to be 525.34±34.52 nmol/min.mg protein (Fig. 2). NBML rats that treated with 30 mg/kg of SDC and/or CA for 25 days, showed reduced AChE activity compared with the NBML rats that treated with saline. Fig. 2 shows that the activity of AChE was remarkably reduced (3-fold) in NBM lesioned rats were administered 30 mg/kg of SDC. NBML rats received 30 mg/kg of CA revealed a two-fold reduction in AChE activity in hippocampal tissue. The enzyme activity was, however, remarkably inhibited in the rats that received both of the bile acids (520.13±50.25 nmol/min.mg protein), indicating synergistic effects.

Fig. 2.

AChE activity in the hippocampus tissue. Results showed a significant increase in hydrolyzing activity in NBML rat’s hippocampus that was attenuated with bile acids treatment. Co-administration of CA and SDC was most significant. The data were expressed as mean ± S.E.M. of five independent experiments and an asterisk (*) symbol indicates significant changes in comparison with NBML rats (p<0.05).

Bile acids improved abnormal expression of GFAP, Aβ and AChE

Western blot analysis confirmed the increased expression of GFAP, a well-known biomarker for neuronal damage in ibotenic acid received hippocampus (Lei et al., 2015). In this study, it was revealed that the GFAP content of hippocampal tissue was reduced in the rats treated with bile acids, especially the rats that received CA+SDC daily. Our results also revealed that AChE expression was enhanced in hippocampal tissue of NBML rats, while bile acids significantly reduced its expression. Fig. 3 reveals that the effects of CA and the combination of both bile acids are more significant than SDC treatment. According to the results, the level of Aβ content was six-fold in the NBML rats compared with the control. Interestingly, CA caused a greater decrease in the level of Aβ in hippocampal cells (more than five-fold) than SDC (near to two-fold). Our results also confirmed nonlinear cumulative effects of CA and SDC in the reduction of AChE expression.

Fig. 3.

Immunoblotting studies. Results showed upregulation of AChE, Aβ and GFAP proteins in NBML rats that can be interpreted as hippocampal damage, which improved with oral administration of bile acids. (A) Lane 1 represents control hippocampus, lane 2 shows results in NBML rats. Lane 3 represents SDC-treated NBML rats, lane 4 represents NBML rats treated with CA and lane 5 shows results in NBML rats that received CA+SDC. (B) Immunoblotting results quantified with Image J software as a plot. The data were expressed as mean ± S.E.M. of at least three independent experiments and asterisk (*) symbol indicates significant changes in comparison with NBML rats (p<0.05).

Bile acids improved histopathological abnormalities induced by NBM lesion

In this investigation, histological studies were done as confirmation of biochemical results. Thioflavin staining demonstrated a clear abundance of amyloid plaques in the hippocampal tissue of NBML rats; however, deposits were absent in the control rats. In agreement with a previous study (Lo et al., 2013), thioflavin plaque numbers were reduced in the hippocampus of bile acids-supplemented-NBML rat compared to the NBML rats that treated with saline. Thioflavin plaque numbers significantly decreased in the groups treated with SDC and CA. Fig. 4 also shows cumulative effects of both bile acids in AD rats that received SDC and CA simultaneously and resulted to decreased number and size of plaques.

Fig. 4.

Thioflavin staining of hippocampus tissue. Results showed absence of amyloid plaques in control rats. Hippocampal sections representing NBML rats showed high amounts of deposited amyloids. Amyloid deposit amounts were reduced in rats that received the PAS-interacting bile acid (CA) versus SDC, which could not bind to the PAS.