1. Identification of saffron by water test
After saffron was immersed in water at room temperature, there would appear obvious physicochemical phenomena, which could be used to identify the authenticity of the variety. The dried stigma of saffron (0.5g), identified by Professor Zhao Kuijun from Beijing Friendship Hospital, Capital Medical University, was weighed and floated on the water, and the color change of water and the morphological change of medicinal material was recorded at different time points.
2. Preparation of sample solutions
Ethanol extract of saffron (EES) was extracted by referring to the method in the criterion of the Pharmacopoeia of the People’s Republic of China, 2015. Specific for, the saffron (1 g) was accurately weighed, pulverized, added with 10 mL of 75% ethanol, then, ultrasonically extracted in an ice bath. After 2 h, the EES Freeze-dried powder was collected by vacuum freeze dryer. All dried samples were powdered to a homogeneous size and sieved through a No. 65 mesh. In each case, 0.1 g of powder was ultrasonic-extracted for 20min with 50 mL of methanol: water (1:1). The supernatant solution was filtered through a 0.2 mm PTFE membrane filter before being injected into the UPLC system.
3. Content determination and component identification of ethanol extract of saffron
Chromatographic experiments were performed as described previously. Briefly, Acquity UPLC system (Waters, Milford, MA) connected to Xevo G2 Q-ToF MS equipped with an electrospray ionization source (Waters, Milford, MA, USA) was performed on an ACQUITY UPLC T3 column (2.1× 100 mm,1.7 μm) by gradient elution. The column temperature was maintained at 45℃ and the flow was 0.4 mL/min. The mobile phase consisted of solution A (water with 0.1% formic acid) and solution B (acetonitrile with 0.1% formic acid). Elution was starting at 5% B which was held constant for 1 min, then increased linearly to 100% from 1 to 16min, and the injection volume was 5 μL.
The ESI-Q-TOF-MS detection was operated using the positive (ESI+) ion mode in the mass range of 50-2000 Da. The optimized mass spectrometer parameters were set as follows: capillary voltage of 2.5 kV, sample cone voltage of 21 V, source and desolvation temperatures were 130°C and 350°C respectively. cone gas flow of 50 L/h, desolvation gas flow of 800 L/h, nitrogen, and argon was used as the cone and collision gases. Data were switched between the low energy (4 V) and elevated energy (10-55 V) acquisition modes every 0.2 s.
4. Cell culture
PC12 cells were obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). They were cultured in RPMI1640 medium (Gibco, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (HyClone, Logan, UT, USA), 100 mg/ml streptomycin, and 100 U/ml penicillin at 37˚C in a humidified atmosphere with 5% CO2.
5. In vitro cytotoxicity of EES
For the cell experiments, EES was dissolved in water, prepared into a 2 mg/mL stock solution (calculated based on the concentration of raw medicinal materials). The screening of cytotoxicity for PC12 cells was assessed by Cell Counting Kit-8 (CCK-8) assay kit (Dojindo, Kumamoto, Japan) as previously described. Briefly, the PC12 cells were plated into a 96-well cell culture cluster at 4x103 per well. Then, the cells were treated with EES in different concentrations for 24 h. At mentioned time points, 10 μL CCK-8 was added to each sample respectively, and the cells were incubated at 37˚C for 1 h. With mixing gently to ensure homogeneous distribution of color, the absorbances of solution were measured at 450 nm performing a microplate reader (Molecular Devices, USA). The cell was calculated using the equation: cell viability (100%) = (ODtreatment/ ODcontrol) x100%.
6. Protective efficacy of EES on corticosterone induced PC12 cell injury
The PC12 cell culture method was the same as above. After attachment, respectively, cells were incubated with different concentrations (10, 20, 40, 80, 160, 320, and 640) of EES for an additional 24h. To validate the experiments, fluoxetine (10 μM, Sigma-Aldrich, St. Louis, MO, USA), a classical antidepressant, was used as a positive control. Then, cells were coincubated with 500 μM of corticosterone (Sigma-Aldrich, St. Louis, MO, USA) for 24 h. Finally, the calculation method of cell viability rate is the same as above. Optimal EES concentrations for use in the following experiments were determined based on preliminary results.
7. Total RNA isolation, library construction, and mRNA sequencing
After corresponding treatment, PC12 cells were lysed in TRIzol reagent (Invitrogen, USA) and total RNA was extracted. A total amount of 2 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation buffer was added for interrupting mRNA to short fragments. First-strand cDNA was synthesized with random hexamers using the fragments as templates. Then, Buffer, dNTPs, RNase H, and DNA polymerase I was added to synthesize the second-strand cDNA. The double-stranded cDNA was then purified, end-repaired, and A-tailed was added for adaptor ligation. DNA fragments of 250–300 bp in length were selected preferentially by purifying the library fragments with the AMPure XP system (Beckman Coulter, Beverly, USA). Finally, PCR amplification was performed to enrich the cDNA libraries, whose quality was assessed on the Agilent Bioanalyzer 2100 system. After cluster generation, the library preparations were sequenced on an Illumina Hiseq 4000 platform and paired-end 150bp reads were generated.
8. Data analysis of differentially expressed genes (DEGs)
The DEGs were analyzed using the DESeq R package (version 1.10.1), which has been a routine statistical approach to determine the digital differential expressions of genes using a model assuming the negative binomial distribution of the data. To control the false positive rate, Benjamini and Hochberg's approach was used for adjusting the P values, and a P value less than 0.05 was considered to indicate a significant difference.
9. Functional enrichment analysis
The DEGs were subjected to enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedias of Genes and Genomes (KEGG). GO functional enrichment and KEGG pathway enrichment were performed by Metascape (https:// metascape.org).
10. Confirmation of RNA-seq results with qPCR analysis
PC12 cells were lysed in TRIzol reagent as above. For PCR analysis, cDNA was Synthesized via FastKing RT Kit (With gDNase) (TIANGEN BIOTECH, Cat: KR116). Amplification and quantitation of PCR were performed using a real-time PCR system with SuperReal PreMix Plus (SYBR Green) (TIANGEN BIOTECH, Cat: FP205). mRNA expression levels were normalized to GAPDH mRNA levels. Fold expression determination, gene-to-GAPDH ratios were determined by using the 2−ΔΔCt method. The thermal cycles were performed with an initial denaturation at 95℃ for 10 min, followed by 40 cycles of 95℃ for 15 s and 60℃ for 1 min.
11. Statistical analysis
As detailed above, statistical analysis and graphs were performed with GraphPad prism statistical software (GraphPad Software). Results were demonstrated as mean± standard deviation (SD). Statistical significance was determined by one-way analysis of variance followed by Tukey's secondary test for significance. P< 0.05 was considered to be statistically significant.