Cell Culture

Human skin fibroblasts CCD-1079Sk (CRL-2097™) were purchased from American Type Culture Collection (ATCC, VA, USA), and cultured in DMEM-F12 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (Gibco, USA). Sub-culturing was done every 2–3 days.

Treatment

Emodin and aloe-emodin were purchased from Sigma-Aldrich (MO, USA) and dissolved in DMSO. The studied concentrations were chosen according to the cytotoxicity assay which was done in the range of 0-1000 µM with the ATP assay. According to the IC50 values, for the evaluation of the gene expression levels 25 µM, 12.5 µM, and 6.25 µM were selected for emodin; 10 µM, 5 µM, and 2.5 µM for aloe-emodin. The control group was the solvent containing the same percentage of DMSO (0.1%) with the treatment groups.

ATP Bioluminescence Assay

ATP content of the cells was determined with recording the bioluminescence using CellTiter-Glo® 2.0 (Promega, USA). The luminescent signal is proportional to the ATP content of the cells which is an indicator of cell viability [11]. Opaque white 96-well plates were used. CCD-1079Sk cells were seeded 104/well and treatment was conducted after a 24 h incubation. Following 24 h treatment with emodin and aloe-emodin, the cells were shaken for 2 min with CellTiter-Glo® 2.0 Reagent which was added the same amount with the culture medium present in each well. After 10 min incubation in room temperature, the luminescence was recorded using a BioTek Synergy H1 (Epoch, Germany) microplate reader. The results are given as a percentage relative to the solvent control group.

In vitro wound healing (scratch) assay

For evaluating the migration of the CCD-1079Sk cells. The CytoSelect™ 24-well Wound Healing Assay Kit (Cell Biolabs Inc, USA) was used. The inserts were placed inside every well of the 24 well plate using a sterile forceps with their “wound field” aligned in the same direction. 500 µL of medium containing 2 × 105 cells was added to the gaps of the inserts. After 24 h incubation in a cell culture incubator overnight for the cells to provide a monolayer, the inserts were removed, and the images of each well were taken before treatment. After taking the images, emodin (6.25 µM, 12.5 µM, and 25 µM) and aloe-emodin (2.5 µM, 5 µM, and 10 µM) treatments were done for 24 h and the images of the same area of the wounds were taken. For calculating the wound healing percentage, the program ImageJ (National Institutes of Health, USA) was used, and comparison was done between the treatment groups with the vehicle control group.

Evaluation of gene expression levels

Gene expressions of JNK, P38, and ERK were evaluated by reverse transcription polymerase chain reaction (RT-PCR) and Beta actin (βactin) was used as house-keeping gene. Total RNA extraction was done according to the manufacturer’s instructions of Quick RNA Mini Prep Kit (Zymo Research). cDNA synthesis was done using SensiFAST™ cDNA Synthesis Kit (Bioline). RT-PCR was performed with the SensiFAST™ SYBR No-ROX Kit (Bioline). Gene expression levels were calculated by the 2-ΔΔCT method [12]. The studied primer sequences can be found in Table 1.

Table 1 Primer sequences for determination of gene expression levels Molecular docking study

The docking study was carried out using the Schrödinger Software Suite (Maestro Schrödinger Release 2022-2: Meastro, Schrödinger, LLC, New York, NY, 2021). The crystal structure of N-terminus kinase-1 (JNK1) complexed with its inhibitor quercetagetin at 2.70 A ̊ resolution and the crystal structure of p38alpha complexed with pyrazolobenzothiazine inhibitor COXH11 (9Y5) at 2.50 A ̊ (PDB ID: 3V3V and 5OMH, respectively) was retrieved from the RCSB Protein Data Bank [13, 14]. The crystal structures of the enzymes were prepared using the multi-step Protein Preparation Wizard Module of Schrödinger Software Suite (Schrödinger Release 2022-2: Protein Preparation Wizard; Epik, Schrödinger, LLC, New York, NY, 2021; Impact, Schrödinger, LLC, New York, NY; Prime, Schrödinger, LLC, New York, NY, 2021) [15]. The retrieved crystal structure was optimized by removing water molecules, heteroatoms, and co-factors. The hydrogens, missing atoms, bonds, and charges were computed and corrected through Maestro. The studied compounds (emodin and aloe-emodin) and the co-crystallized ligands (quercetagetin and 9Y5) were also prepared and optimized (generating various tautomers and ring conformations, assigning bond orders, and stereochemistries) using the LigPrep module of Schrödinger Software Suite (Schrödinger Release 2022-2: LigPrep, Schrödinger, LLC, NY, 2021). All the conformations generated were minimized using the OPLS4 force field. The receptor grid was generated around the co-crystalized ligand of the enzyme to specify the binding site, where the grid box size was determined with the receptor Grid Generation implemented in Glide (Schrödinger Release 2022-2: Glide, Schrödinger, LLC, NY, 2021) [16,17,18]. The Glide docking was performed in the Standard Precision (SP) mode.

C. elegans Survival (Thermotolerance) assay under heat stress

C. elegans used in this experiment were obtained from the Caenorhabditis Genetics Center CGC. The type used was the wild N2 strain and was maintained in the Nematode Growth Medium (NGM). One-liter volume of NGM was prepared as follows: First agar 17 g, NaCl 3 g, and peptone 2.5 g were mixed and autoclaved at 121 °C for 15 min. After the flask cooled down to 55 °C; 1 M MgSO4.7H2O 1 mL, 1 M CaCl2.2H2O 1 mL, 5 mg/mL cholesterol in ethanol 1 mL, 1 M KPO4 buffer 25 mL, and 100 µg/mL penicillin-streptomycin 1 mL were added under the laminar flow cabinet using sterile techniques with a constant mixing to make sure the mixture was homogeneous, and later it was poured into 100 mm diameter Petri plates and left for 24 h under 22 ± 2 °C temperature conditions. After synchronizing, a total of 210 synchronized worms were prepared, and 35 were divided into each plate separately. For this experiment, 15 plates were prepared and divided into five groups, three as the control group and three as the experimental groups. The NGM plates were seeded with 500 µL of Escherichia coli OP50-1 strain (OD:0.4; treated at 65 °C for 10 min to kill) under sterile conditions as a food source; the control groups only contained the E. coli and a concentration of 75 µM and 150 µM emodin and aloe-emodin was added to the experimental groups. DMSO was included in the control group. The seeded plates were left overnight at room temperature to allow the E. coli OP50-1 to grow.

Our experiment used the thermotolerance test that is part of survival assays to evaluate and screen the stress evolution of the experimental worms exposed to emodin and aloe-emodin compared to the control group on worm’s lifespan within hours. The experiment started at the end of the L4 larval stage; the plates were incubated at 35°±0.8 °C for 12 h in an incubator, and images were captured with a high-resolution scanner (Epson Perfection, V800 Photo) starting from T0 the plates were scanned in every 20 min, till every worm dies. The worms that were not moving in two consecutive scans were accepted as dead. The results were scored and analyzed using a statistical method, The Kaplan-Meier, to estimate the survival function.

Statistical analysis

Statistical analysis was performed using Graphpad Prism 6. Statistical differences of the treatment groups vs. the untreated control group were evaluated with one-way ANOVA followed by the Tukey test. The results were represented as mean ± standard deviation (SD)., *p < 0.05 and **p < 0.01 versus the control group.