Chemicals
Mitragynine, 7-hydroxymitragynine, speciociliatine, and paynantheine were purchased from ChromaDex (California, US). RNA extraction, cDNA synthesis, and real-time PCR kits were provided from Qiagen (Hilden, Germany). HRP-conjugated anti-mouse secondary antibody and mouse primary antibodies against β-arrestin-2, ERK1/2, p-ERK1/2, and β-Actin were obtained from Santa Cruz Biotechnology (California, US). Cell culture media and their supplements were acquired from Gibco (Grand Island, NY, USA). Antioxidation and antigenotoxicity evaluating reagents including DPPH, BHT, linoleic acid, β-carotene, ascorbic acid, agarose as well as all other chemicals and solvents in this study were supplied from Sigma-Aldrich (Sigma-Aldrich, Deisenhofen, Germany). Morphine and all other reagents were obtained from commercial resources in Iran.
Cell culture and cellular growth kinetic
SH-SY5Y (CRL-2266, a neuroblastoma cell line, isolated from a bone marrow biopsy) cell line was generously gifted from Professor Mohammad Saeid Jami (Shahrekord University of Medical Sciences). Cells were cultured in DMEM/F12 medium containing 10 % (v/v) FBS, 100 IU/ml penicillin, 2 mM L-glutamine, and 100 μg/ml streptomycin in a humidified CO2 incubator at 37 °C. Cellular proliferation was monitored during an 8-day incubation. Initially, cells were cultured into 96-well plates at a density of 8000 cells/well. Cells in the wells were counted daily using MTT assay. A standard mathematical model was fitted to the point. The maximum specific growth rate (μmax) was illustrated as the slope of the line depicted on natural logarithms of the growth (dependent variant) versus the time intervals (independent variant). Data was represented as the mean values ± SE of three independent experiments [13].
In-vitro analyses of cytotoxicity
Briefly, SH-SY5Y cells were sub-cultured at a density of 15000 cells/well in 96-well plates and cultivated 24 h at 37 °C. Then, the cells were exposed to serial dilutions (0-300 μM) of morphine, mitragynine, 7-hydroxymitragynine, speciociliatine, or paynantheine. Eventually, cell survival was assessed after a 5-day incubation applying MTT assay. The optical density was read on a plate reader at 570 nm. IC50 values were expressed as the concentration of the agents, reducing cell growth by 50%; also, IC10 values were determined as the concentration of a compound decreased cell growth by 10%. The cytotoxic values were determined by the best regression plot of the percentage viability against any compound concentrations [14].
Antioxidant properties of the alkaloids
The antioxidation properties of the alkaloids have been evaluated either using DPPH or β-carotene/linoleic method. DPPH method is based on the disappearance of the DPPH free radicals and consequently reduction of the absorption at 517 nm. Two milliliter of a fresh DPPH stock was added to various concentrations of each alkaloid (0-400 μM) and placed in the dark for 30 min. Scavenging capacity percentage was determined by [(control absorbance – sample absorbance) × 100 / control absorbance]
equation. Bleaching assay was conducted using an emulsion composed of β-carotene and linoleic acid. Briefly, 1 mg yellow β-carotene pigment, 45 µl of 9-cis-12-cis-linoleic acid (density= 0.902 g/ml), and 200 µl of Tween-20 (density= 1.095 g/ml) were homogenized in 2 ml of chloroform. Then chloroform was rotary evaporated at 40 °C for 30 min and then 100 ml of oxygenated deionized water was mixed with vigorous shaking to form a homogenized stable nanoemulsion. Then, 2.5 ml of the emulsion was added to 350 μl of various alkaloid concentrations (0-400 μM). The mixtures left at 50 °C in the light for 2 h and the optical density was recorded at 470 nm. Bleaching inhibition capacity percentage was calculated from [(sample absorbance at time 0 – sample absorbance after 2 h) × 100 / (control absorbance at time 0 – control absorbance after 2 h)]equation. Controls contained all reagents except the antioxidant factors. The scavenging capacity-50 (SC50) or bleaching inhibitory capacity-50 (BIC50) is an alkaloid concentration required for scavenge 50 % of DPPH radicals or protects half percentage of β-carotene molecules from bleaching, respectively. They were calculated from the calibration curve determined by the regression line from the scavenging capacity or bleaching inhibition percentages versus alkaloid concentrations. Butylated hydroxytoluene (BHT, 0-100 μM) was used as standard antioxidant agents [15-16].
Antigenotoxic activity of alkaloids using COMET assay
The induced DNA damage by the alkaloids was evaluated on SH-SY5Y cells by the COMET assay conducted under alkaline conditions. A serial dilution of each alkaloid (final concentrations of 0 to 600 μM) was supplemented with 120 μM H2O2 solution and stored for 5 min at ambient temperature. Then, 10000 cells were transferred to all dilutions, and the suspension was kept for 30 min at 4 °C. Next, cells were harvested and sandwiched on a slide between two layers of 0.75% w/v low-melting point agarose. The slides were submerged in cold lysis buffer for at least 4 h. The lysis solution was composed of 2.25 M sodium chloride, 90mM ethylenediaminetetraacetic acid, 9mM Tris, and pH was adjusted to 10. The lysis buffer was freshly supplemented with 0.7% w/v sodium hydroxide, 10% v/v dimethyl sulfoxide, 1% v/v Triton X-100 before use. Then, the cells were subjected to a constant electric field in a horizontal electrophoresis chamber (300 mA for 40 min at 4 °C). The Chamber contained freshly prepared alkaline buffer (300 mM Sodium hydroxide; 1 mM ethylenediaminetetraacetic acid; pH ~ 13). The slides were then neutralized using a neutralizing buffer (0.4 M Tris; pH = 7.5). Finally, DNA was stained with 20 μl ethidium bromide (2 μg/ml) and pictured using a fluorescent microscope (BX51; Tokyo, Japan). The DNA damage was analyzed via Open-Comet software and Cells were determined undamaged to maximally damaged, according to tail DNA intensity [Tail DNA×100 ÷ (Head DNA + Tail DNA)], for each alkaloid concentrations. The COMET-inhibitory capacity-50 (CIC50) defined as the concentration of the alkaloid that diminishes the tail DNA percent to 50 % against damage induced by H2O2 and calculated from the calibration curve determined by the best regression line between tail DNA percentage and alkaloid concentrations. BHT (0 to 1000 μM) was used as a standard antigenotoxic agent [17-18].
Quantitative analyses of the transcripts
SH-SY5Y cells were exposed to IC10 concentration of each alkaloid for 24, 48, and 72 h. RNA was extracted from the cells by the RNeasy mini kit, and cDNA was constructed with QuantiTect Reverse Transcription Kit from 1 μg of total RNA according to the company instructions using random hexamer as priming sequences. ARRB2, MAPK1, and MAPK3 transcripts were relatively quantified with the QuantiTect SYBR Green kit on a Rotor‐Gene Q instrument (Qiagen, Hilden, Germany) according to the Pfaffl method. Three primer pairs were designed using Gene Runner Ver. 3.05 software and the sequences were validated using the Primer-BLAST tool available on the NCBI website. Amplification was optimized under the following conditions: a pre-denaturation step at 95 °C for 10 min; 45 cycles for amplification step (15 s denaturation at 95 °C, 20 s annealing temperature at 60°C, and 20 s extension at 72 °C), and a standard melting analysis carried out after the amplification step (1 °C/step between 60-90 °C). The efficiency for each primer pair was determined with a serial dilution of cDNA. The transcript level was normalized to β-actin as the internal reference gene [19-20].
Quantification of protein in the signaling pathway
Western blotting was used for semi-quantification of the targeted proteins. In brief, after treatment time, total proteins were extracted using lysis solution (7 M urea, 2 M thiourea, 10 mM PMSF, 1% w/v DTT, pH 3–10), The protein concentration was determined using the commercial Bradford protein assay kit. Human serum albumin was used as a standard curve for protein quantification. 50 μg of the total protein was electrophoresed on 12 % polyacrylamide SDS-PAGE according to Laemmli method on a discontinuous buffer system. Then, protein bands were blotted onto nitrocellulose membranes via a semi-dry Trans-Blot instrument (Bio-Rad, Richmond, CA) under constant current of 3 mA/cm2 for 60 min. The efficiency of protein transfer was monitored using Ponceau-S staining. Then, the unspecific membrane surface was blocked with 5% BSA for 1 h, and the blot was treated with 1:500 v/v mouse monoclonal IgG antibody against β-actin, β-arrestin-2, ERK1/2, or p-ERK1/2. Secondary mouse IgG kappa binding protein conjugated to horseradish peroxidase (m-IgGκ-HRP, 1:5000 v/v) along with luminol was used for band visualization. The chemiluminescence wave was recorded with a Li-Cor scanner (Lincoln, NE, USA). The density of each protein band was quantified and normalized with β-actin results [19, 21].
Statistical analyses
Three independent runs were carried out in adequate replicates for any analytical experiments. Data analysis was assessed with SPSS-22 statistical software. Stars (Ü), (ÜÜ), and (ÜÜÜ) represent the mean differences between normalized treated and normalized untreated group levels as P<0.05, P<0.01, and P<0.001 using one-way ANOVA, respectively.