All samples used in the study were collected in accordance with the guidelines approved by the ethics committee of our University with approval ID: IR.IAU.DAMGHAN.REC.1398.005.
Preparation of Aβ1–42 peptide
Aβ1–42 peptides were monomerized by dissolution in Hexafluoro-2-propanol (HFIP) (Sigma-920-66-1) for preparation the 1 mM concentration of Aβ, we used 2.2 ml of HFIP They were aliquoted into microcentrifuge tubes. The HFIP was evaporated using a Speed Vac, and the peptide was stored at -20 °C until use. In order to fibril formation, large aggregates of Aβ1-42 were directly dissolved in dH2O and incubated at 37 °C for 72 h . All other chemicals were of analytical reagent grade.
Astrocytes culture and treatment
Primary fetal human astrocytes were isolated from the hypothalamus and cerebral cortex, which were previously isolated from hypothalamus and cerebral cortex of two human fetuses on gestational weeks 9–12 (gift from Bon Yakhteh Laboratory in Tehran) were cultured in DMEM with 10% heat-inactivated fetal bovine serum (FBS) were purchased from Sigma chemical Co., (Sigma-F2442, St. Louis, USA), and kanamycin (50 mg/mL the cells were incubated at 37 °C in %5 CO2, %85 - %95 humidity. 200000-250000 cells were cultured in each well . According to the IC50, after 24 h, METH (donated by Tehran University) and Aβ were added to the well. METH was added to DMEM, containing 10% FBS, to reach the final concentration of 12.5 µM. METH remained in the vicinity of the cell for 24 h. For treatment with Aβ, 10 µM of Aβ was kept at 37 °C for 72 h (fibril formation) and then added to DMEM plus F12 without FBS . Cells were exposed to amyloid for 24 h. All experiments have been performed according to the following procedures: group-1 cells with Aβ, group-2 cells with METH, group-3 cells with METH after 24 h of adding Aβ (Ab + METH, treated group), group-4 cells with Aβ after 24 h of adding METH (METH + Ab, prevention group) and group-5 as control.
MTT assay for estimation of cell viability
Astrocytes were seeded in a 96-well plate (10000, 15000 and 20000 Cells per well), fed with 5% FBS in DMEM, and incubated for 24 hours. Then, they were exposed to 0.8, 1.6, 3.1, 6.2, 12.5, 25, 50, and 100 µM concentrations of methamphetamine hydrochloride (donated by Tehran University), which were dissolved in water and used for 24-, 48-, and 72-hour treatment. In addition, MTT (Sigma-Aldrich, USA) (5 mg/mL in PBS) was added. We used DMSO to dissolve the crystals. The measured absorbance was at 570 nm.
Astrocyte cells were cultured in a 6-well plate (200000-250000 cells per well). According to the IC50, METH was added to DMEM containing 10% FBS in order to reach the final concentration of 12.5 mM. Methamphetamine exposure for the cell was performed for 24 h . Regarding the treatment with Aβ, 10 µM of Aβ was kept at 37 °C for 72 h (fibril formation), and added to DMEM plus F12 lacking FBS  and the cells were exposed to amyloid for 24 h. The related groups included the cells with Aβ (Group 1), METH (Group 2), Ab + METH (METH after adding Aβ for 24 h: Treated group; Group 3), METH + Ab (Aβ after adding METH for 24 h: Prevention group; Group 4), and control.
Total RNA was isolated from astrocyte culture by using an RNA extraction kit (Roche 11828665001) according to the manufacturer’s recommendation. The concentration of RNA samples was determined by measuring optical density at 260 nm. The quality of RNA was confirmed by detecting 18S and 28S bands on agarose gel electrophoresis. The RNA samples were incubated with DNase at room temperature for 15 min in order to remove residual DNA contamination.
The total RNA from each sample was used to generate cDNA with oligo (dT) primers according to the manufacturer’s protocol thermo (K1621).
GAPDH was used for housekeeping genes, and primer 3 was applied to design all of the primers. Table 1 indicates the primer sequences.
Quantitative real-time PCR analysis
The GSK3β, Cdk5, PKCα, Bcl-X, and Bax genes related to AD and apoptosis were evaluated by using housekeeping gene (GAPDH). The primers were previously checked by conventional RT-PCR and agarose gel (1.5%) electrophoresis. Real-time quantitative polymerase chain reaction (PCR) was performed by using Ampliqon PCR Master Mix (A314406) and Qiagen Rotor-Gene Q system. All of the experiments were performed in triplicates. Table 3 indicates the real-time PCR primers specific for test and GAPDH gene. The cycling protocol consisted of an initial 5-min denaturation step at 95 °C, followed by 35 cycles of denaturation at 95°C for 1min, annealing at 60 °C for 1min, extension at 72 °C for 1min and a final 5 min extension step at 72 °C. CT relative quantification method was used and the obtained CT values were normalized for endogenous reference .
Western blotting to prepare total protein extraction
The astrocyte cells were digested in NP40 lysis buffer (CMG-NP40). The cocktail protease and phosphatase inhibitor (Roche 11836153001, 04906845001, respectively) were added to the cell lysate for 30 min at 4 °C. Following centrifugation at 14000 g, the supernatant was stored at -70 °C. Then, the protein concentration was determined by using a Bradford Assay Protein Kit (Bio Basic SK3031) according to the manufacturer’s protocol . In addition, the proteins were mixed with a loading buffer (TrisHCl 63 mM, glycerol 30%, SDS 2%, Bromophenol blue 0.05%, pH 6.8), and boiled for 5 min at 95 °C. Further, 10% polyacrylamide gel was used. Accordingly, 80 μg of protein was loaded in each well at that time and the electrophoresis was conducted at 125 mV for 1 h. After gel electrophoresis, all proteins were transported into a PVDF membrane with 320 mA for 2 h at 4 °C (Bio-Rad 1620174), and then blocked with BSA 5% for 1.5 h at the room temperature. Membranes were incubated overnight with monoclonal antibody (p-GSK3β-sc-373800) (1:500), monoclonal antibody (GSKα/β- sc-7291) (1:500) at 4 °C, monoclonal (β-Actin (c4) sc-47778) (1:500) at 4 °C and anti-tau (phospho S396 EPR2731) Abcam (1/10000) at 4 °C. Afterwards, the membranes were washed three times for 5 min in Tris-buffer saline solution with 0.1% tween (TBS/T) incubated with 1:10000 diluted anti-mouse antibody (sigma A9044) and 1:2500 diluted goat Anti-rabbit IgG (Elabscience E_AB_1003), respectively. Then, the membranes were washed three times for 5 min in Tris buffer saline solution with 0.1% Tween, and were detected by chemiluminescence. Bands were scanned and the band intensities were calculated by ImageJ. Finally, the bands were normalized to the intensity of β-actin in each sample.
Apoptosis and necrosis survey
Briefly, pre-treated astrocyte cells were harvested with trypsin and washed with 0.01 M PBS twice. Along with centrifugation at 2000 rpm for 5 min, the cells were re-suspended in 500 μl of binding buffer at the density of 1 × 106 cell/ml, along with adding 5 μl Annexin, V-FITC, and 5 μl PI, ((ANXVF-200T)) respectively. Next, the cells were incubated in the dark at 25 °C for 15 min and analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, and USA).
DNA content cell cycle analysis (propidium iodide)
The pre-treated astrocyte cells were harvested with trypsin and washed with PBS by centrifugation at 2000 rpm for 5 min at 4 °C. The cells were re-suspended in cold PBS including DNase-free Ranse (Sigma) and stained with Propidium Iodide (PI) containing 1% Triton X-100 (v/v) (Sigma). The solution was incubated at 20 °C for 30 min (protected from light) and analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) .
Hoechst staining assay
The treated astrocyte cells were harvested and washed with PBS. The density of cell should be1*104 cells/mL in PBS (1:1 v/v). The cells were incubated for 30 min at the room temperature with Hoechst 33342 (Invitrogen, H3570) (5 mg/mL).
SPSS v16 and GraphPad Prism were used for statistical analysis. Each of the treatment group was compared with Ab group by using independent sample t-test in real-time PCR. ANOVA followed by Dunnett post hoc test was used to evaluate the results of western blotting analysis. P < 0.05 was accepted as the level of significance. All error bars in figures are based on the results of mean ± standard deviation (SD). Each experiment was performed in triplicate.