2.1. Materials
Optical rotations were measured on a JASCO P-1020 digital polarimeter. UV spectra were recorded on a HITACHI UH 5300 UV spectrophotometer. ECD data were acquired on a J-815-150S Circular Dichroism spectrometer. IR spectra were recorded on a Nicolet-Nexus-470 spectrometer using KBr pellets. 1H and 13C NMR spectra were acquired by a JEOL Eclips-500 spectrometer at 500 MHz for 1H and 125 MHz for 13C in CDCl3, using TMS as internal standard. HREIMS were measured on a Thermo MAT95XP high resolution mass spectrometer, and EIMS spectra on a Thermo DSQ EImass spectrometer. Semi-preparative HPLC was performed on a Hitachi L-2000 HPLC system coupled with a Hitachi L-2455 photodiode array detector and using a semi-preparative C18 column (Kromasil 250 × 10 mm, 5 µm). The column temperature was set at 30 °C, and the flow rate was 2 mL/min. Silica gel (Qing Dao Hai Yang Chemical Group Co.; 300 − 400 mesh) was used for column chromatography (CC). Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.; G60, F-254) were used for thin-layer chromatography. LPS from Escherichia coli O111:B4 and AChE from Electrophorus electricus were purchased from Sigma (Sigma-Aldrich, USA). Primary antibodies against COX-2 (#12282), iNOS (#13120), TLR4 (#14358), p38 (#8690), ERK (#4695), JNK (#9252), phosphor-ERK1/2 (Thr202/Tyr204, #4370), phosphor-p38 (Thr180/Tyr182, #4511), phosphor-JNK (Thr183/Tyr185, #4668) and NF-κB p65 (#8242) were purchased from Cell Signaling Technology (Beverly, MA, USA).
2.2. Substrate
Cryptotanshinone (1) was purchased from Macklin Biochemical Co., Ltd in Shanghai, China and authenticated by comparing its physical and spectroscopic data with the reported values. Its purity was determined to be 98% by HPLC analysis.
2.3. Fungal Strain
The fungus C. elegans AS3.2028 was purchased from China General Microbiological Culture Collection Center, Beijing, China.
2.4. Biotransformation Medium
The biotransformation experiment was carried out in modified Czapek–Dox medium, which consisted of the following ingredients: glucose (15 g/L), sucrose (15 g/L), MgSO4∙7H2O (0.5 g/L), K2HPO4∙3H2O (1 g/L), FeSO4∙7H2O (0.01 g/L), peptone (5 g/L), KCl (0.5 g/L) and distilled water.
2.5. Biotransformation Procedure
A spore suspension of the fungus grown on PDA was transferred to 500 mL Erlenmeyer flasks containing 100 mL biotransformation medium, and incubated at 28 °C, 180 rpm on a rotary shaker for 24 h. Subsequently, the substrate cryptotanshinone (1) dissolved in methanol was added to each flask to achieve a final concentration of 0.1 mg/mL in a sterile condition. The cultures were incubated for another 72 h.
2.6. Extraction and Isolation
The large-scale cultures (10 L) were filtered and the filtrate was extracted with an equivalent volume of EtOAc for three times. The organic layer was collected and concentrated under vacuum at 40 °C. The EtOAc extract was subjected to silica gel CC eluted with petroleum ether − acetone (10:1 to 1:4) to give five fractions (Fr.1 − Fr.5). Fr.4 was subjected to semi-preparative HPLC (MeOH − H2O, 55%) to provide metabolites 2 (6.2 mg), 3 (56.0 mg), and 4 (17.8 mg).
2.7. Bioassay for NO Production Inhibitory Activities
The bioassay for NO production inhibitory activities was performed as described by Xia et al.24. The BV-2 microglia cells were seeded in 96-well plates. In each well, LPS (1 µg/mL) was added after treating with or without compounds at various concentrations for 24 h. The NO production in the supernatant was detected by the Griess reaction. The absorbance at 540 nm was measured in a microplate reader. The NO concentration and the inhibitory rate were calculated through a calibration curve. Quercetin was used as the positive control. Experiments were operated in triplicate, and the data were described as mean ± SD of three independent experiments.
2.8. ELISA Measurement
The BV-2 microglial cells were cultured in 96-well plates, grown overnight, incubated with compounds for 1 h, and then stimulated with LPS (1 µg/mL) for 24 h. The supernatant (50 µL) from the culture was collected to determine the concentrations of IL-6, IL-1β and TNF-α with ELISA kits according to the protocols.
2.9. Western blot
BV-2 cells were seeded in 6-well plates, incubated with or without compounds for 1 h, then treated with or without LPS (1 µg/mL) for 16 h. The cells were lysed with RIPA lysis buffer. The concentrations of protein were determined by Pierce Rapid Gold BCA Protein Assay Kit. The quantified protein of each sample was electrophoresed in 10% SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) western membrane. The membranes were blocked using 5% (W/V) skim milk in TBST (Trisbuffered saline with 0.1% Tween 20) for 2 h. Then the membranes were incubated with primary antibodies at 4 °C overnight. After being washed by TBST three times, the membranes were incubated with corresponding secondary antibody at room temperature for 2 h. Finally, immunoreactive signals were detected using a chemiluminescence imager (GE Amersham Imager 600, USA). Intensities of band signals were quantified using the densitometric ImageJ software (National Institutes of Health, MD).
2.10. Transcriptome sequencing
The BV-2 cells were treated with 10 µM of compound 2 (sample group) or DMSO (model group) for 1 h followed by stimulated with LPS (1 µg/mL) for 24 h. The control group was without LPS stimulation. Total RNAs were extracted with Trizol (Invitrogen, USA) from the treated BV-2 cells. Three biological replicates were sequenced using the Illumina Hiseq × 10 platform for each group. The differentially expressed genes (DEGs) were screened out with a q value (p-adjust value) ≤ 0.01 and fold change ≥ 2 and further analyzed with KEGG.
2.11. Cell Viability Assay
The mouse hippocampal neuron cell line (HT22) was used in glutamate toxicity model to evaluate the neuroprotective activities of compounds25. The HT22 cells were cultured in 96-well plates and pretreated with tested compounds (1 µM and 0.1 µM) for 1 h. To each well, 5 mM glutamate was added. After treatment for 12 h, the culture medium was replaced with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (0.5 mg/ml) and incubated in the dark at 37 °C for 4 h. The MTT solution was carefully removed. Dimethyl sulfoxide was added to dissolve the formazan crystals. The absorbance was detected at 540 nm using a microplate reader. Experiments were operated in triplicate. The cell survival rates to vehicle were calculated with Graphpad prism 7.
For BV-2 cells, after being seeded into a plate, the cells were incubated with various concentrations of the compounds for 24 h. The MTT assay was used to evaluate their cytotoxic activities, consistent with the above operations.
2.12. Immunofluorescence Assay.
The immunofluorescence assay was performed as described by Xia et al.24. Briefly, BV-2 cells were pre-treated with DMSO, LPS, and 10 µM compound 2 before LPS, respectively. The cell-seeded glass coverslips were subjected to treatment with cold 4% paraformaldehyde and 0.2% Triton X-100 (in PBS). Then, the coverslips were blocked with 5% BSA (in PBS) for 1 h and incubated with a primary antibody NF-κB p65 at 4 °C overnight, followed by addition of a secondary antibody labeled with Alexa Fluor 594 for 1 h. After being stained with DAPI, the coverslips were washed and sealed. Images were obtained by fluorescence microscope (Leica, Solms, Germany).
2.13. Bioassay for AChE Inhibitory Activities
The AChE inhibitory activities of compounds were evaluated in 96 well microplates using a spectrophotometric method26,27. The inhibition rates were calculated by comparing the rates of enzyme reaction of samples relative to that of blank (DMSO). Huperzine A was used as a positive control. All experiments were performed in triplicates independently.
2.14. Physicochemical Properties of Compounds Predicted in silico
The prediction of physicochemical properties, including the number of H-bond donor (HBD) and H-bond acceptor (HBA), 1-octanol/water (Log P), and water solubility (Log S), was conducted using PhysChem Module software in ACD/Labs Percepta 14.0.028.