Materials and methods
PBMCs isolation from blood samples
PBMCs were isolated from heparinized blood samples of 45 healthy individuals (for all assays) and 20 COVID-19 patients (for MTT assay only) by Histopaq density gradient centrifugation (Sigma, Missouri, USA) after receiving informed consent from the participants. In details, the obtained samples were diluted with the same volume of PBS. Then, the Ficoll-Paque Plus (Biosra, France) was carefully added to diluted samples in 1:2 ratio respectively. The mixture was then centrifuged at 400 × g for 20 min at 20 °C. The undisturbed PBMC layer was carefully transferred to a new tube and washed twice with three volumes of PBS. This study was approved by the Ethics Committee in Tabriz University of Medical Sciences (Code: IR.TBZMED.REC.1399.1143).
Extraction of plant extracts and treatment with PBMCs
Plant extracts was extracted from 10 g of crushed hyssopus samples using ultrasonic bath at 20 kHz and 100 ml of ethanol (80%) at 45 °C for 20 min. Then, the solvents was evaporated by vacuum evaporator SPS:refid::bib13(13). The Hyssop extract was then treated with PBMCs (1 × 106 cells/well) in complete RPMI-1640 medium (Gibco, Paisley, UK) supplemented 15% heat-inactivated fetal bovin serum (FBS; Gibco, Paisley, UK), 100 mg/ml streptomycin, 100 u/ml penicillin (Gibco) and 2 mM L-glutamine (Gibco, Paisley, UK) for 12, 24 and 48 h at 37 °C in 5% atmospheric CO2 and 95% humidity. Simultaneously, PBMCs were stimulated with suboptimal dose (10 ng/ml) of phorbol myristate acetate (PMA; Gibco, NY, USA).
The viability of PBMCs was measured by the methyl thiazol tetrazolium bromide (MTT) assay. Briefly, 2 × 105 cells/well were seeded in a 96-well plate. The cells were treated with increasing concentrations of Hyssop extract (5–50 µg/ml) for 24 h. Subsequently, 100 ul of MTT (1 mg/ml, Sigma, USA) was added into each well and incubated for 4 h at 37 °C. Then, 100 µl DMSO was added to each well to dissolve the purple formazan crystals and incubated in room temperature for 30 min. Later, the optical density was measured using a spectrophotometer at 540 nm wavelength and compared to untreated cells .
Gene expression assay
The expression levels of genes including TLR 3,7,8,9, as well as NF-κB and Myd88 genes were assessed in PBMCs treated with Hyssop extract by real-time polymerase chain reaction (PCR). Briefly, total RNA was extracted from cultured PBMCs using RNA extraction kit (Qiagen, Hamburg, Germany) according to the manufacturer’s instructions. Then, cDNA synthesis was synthesized by the usage of a cDNA synthesis kit (Exiqon, Copenhagen, Denmark), based on manufacturer’s instructions. The expression level of each mRNA was measured by real-time PCR and a SYBR Green Real Time PCR kit (Ampliqon), according to manufacturer’s instructions. Finally, the mRNA expression was normalized by the detection of β-actin housekeeping gene. The used primers sequences designed by OLIGO v. 7.56 software (Molecular Biology Insights, Inc., CA, USA) for qPCR assay are listed in Additional file 1: Table S1.
Quantification of cytokine levels
The concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-8 and IFN I α and β cytokines were quantified using ELISA kits (Mybiosource, San Diego, USA), according to the manufacturer’s instruction. Briefly, the wells of micro-titer ELISA plate (Maxisorp, Nunc, Roskilde, Denmark) were coated with anti-TNF-α, anti-IL-1β, anti-IL-8 and anti-IFN I α and β antibodies. Afterward, the supernatant of the cells and biotinylated mouse anti-hTNF-α mAb, anti-hIL-1β mAb, anti-hIL-8 mAb and anti-hIFN I α and β mAb were added to the relative wells. Next, the existence of anti-cytokine antibody was investigated by the addition of a streptavidin alkaline phosphatase conjugated anti-mouse IgG Ab (Sigma). Finally, p-nitrophenyl phosphate (4 mg/ml) was added as substrate and the absorbance was evaluated at 405 nm in an ELISA plate reader (LabSystems, Helsinki, Finland).
All experiments were performed in triplicate. Achieved data were analyzed using GraphPad Prism v.8 (GraphPad, La Jolla, CA) and presented as mean ± standard deviation (SD). One-Way ANOVA followed by Dunnett's T3 multiple comparisons test was done to compare the means between groups. p < 0.05 was considered as statistically significant.