Reagents and test kits
Cordyceps militaris were obtained commercially from Nanjiang Hongxing Biological Company, Bazhong City, Sichuan Province of China. Dulbecco’s Modified Eagle’s Medium (DMEM). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), acetonitrile and dimethyl sulfoxide (DMSO)were purchased from Sigma–Aldrich, Inc (St. Louis, MO, USA). Annexin V-FITC Apoptosis Detection Kit was obtained from ALPCO (Salem, NH, USA). Fetal bovine serum (SV3087), penicillin, streptomycin (SV3010), and trypsin (0.25 % concentration) were recruited from Biotechnology Hyclone Inc (Los Angeles, U.S.A). and HP Macroporous Adsorption Resin was purchased from Mitsubishi Chemical Holdings (Japan). Superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), malondialdehyde (MDA) and total antioxidant capacity test kit were obtained from Jiancheng Institute of Bioengineering. (Nanjing, China). The four principal antibodies were present Bax, Bcl-2, caspase-3 and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Isolation and purification of Carotenoids from Cordyceps militaris
The fresh fruit bodies of Cordyceps Sinensis was air-dried at 60°C at a constant weight, and then the dried fruit bodies were ground into powder by passing through a 60mesh sieve. Weigh (2g) with acetone-ethanol (2:1,20ml), then add complex enzyme (0.5%) to adjust the pH of the extraction system to 4. The enzymatic digestion time was 45 min at 50°C, and then the mixture was sonicated for 1.5h. The supernatant was taken by centrifugation for 10 min at 4500 rpm and 100 times diluted. The resulting solution was the crude extract of Cordyceps carotenoids (CMC).
The crude extract CMC pigment supernatant was loaded onto an HP-20 macroporous resin adsorption column for further purification by complete adsorption (particle size 0.3-1.2 mm) ≥ 90%, and the eluate was desorbed with 60% ethanol as the carotenoid purification (CMCT) pigment purification solution. The eluent was placed on a rotary evaporator and concentrated under reduced pressure to a paste (60 rpm for min at 50℃), collected the vacuum freeze-dried CMCT purify, which were used for further studies. Bintong Total Antioxidant Capability Kit (T-AOC) to detected the total antioxidant activitied of pigments.
Antioxidant analysis and UV spectrum analysis
The DPPH was removed by colorimetric method using pyrogallol auto-oxidation to remove O2 in combination with sodium salicylate complexation to remove OH. The total antioxidant activity of the extracted pigments was determined by colorimetric method (T-AOC) test kit and using a microplate reader, the absorbance was determined. (Bio-Tek, Winooski, VT, USA) In the wavelength range of 380-600 nm, in the wavelength range for UV-Vis spectral scanning.
Determination of pigment composition
The composition of carotenoids of Cordyceps Sinensis was determined by ultra-performance liquid chromatography (UPLC). The carotenoids of Cordyceps Sinensis were dissolved in pure methanol through a 0.22µm filter protected from light. A Waters C18 reversed-phase column with reversed-phase specifications (2.1×50mm, 1.7μm) was used, the column temperature was held constant at 30°C, and the flow rate was (0.25Ml/min), the injection volume (2µl), the elution gradient temperature (32-40min), and the detection wavelength was 445nm. The drying temperature for HRMS detection was (450℃). The DAD wavelength was (380nm-600nm) The drying gas flow rate was 40L/min. For HRMS detection. The pigment was mixed with potassium bromide at 1:100 to a powder to a wavelength of 500-4000 cm-1.
ARPE-19 Cell Culture
Human retinal epithelial cells ARPE-19 cells were purchased from the American (ATCC) model culture collection and Dulbecco's Modified Eagle Medium (DMEM)/F12 preserved the ARPE-19 cells. Taken 10% heat-inactivated fetal bovine serum (Los Angeles, U.S.A.) 100 U/ml of penicillin (Los Angeles, U.S.A.) and 100 µg/ml of 10% streptomycin (SV3010 Los Angeles, U.S.A.) Cells were kept in an incubator and monitored in a humidified environment at 37 ℃ and 5% CO2 and RPE cells were selected during the first 5-7 passages when they reached 90% confluence passed with 0.25% Trypsin (Biotechnology Hyclone Inc Los Angeles, U.S.A), every 3-4 days, and placed into appropriate culture plates for each experiment.
Induction of oxidative stress using H2O2.
Oxidative stress tests in serum-free or serum-containing media were conducted. After fresh serum-free base cell processing, ARPE-19 cells were plated on 96-well plates at a concentration of 1-105 cells/mL and enabled cell replication to cling to the bottom of the board and fusion overnight. Add DMEM/F12 medium with a final concentration of H2O2 (0-500μM) and incubate for 12h. Untreated cells served as controls to obtain final concentrations depending on the experiments, followed by a 12h exposure of different concentrations of CMCT.
MTT assays
The MTT test was used as the cell viability test measure. Briefly, the viability of ARPE-19 was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (St. Louis, MO, USA). The spread of cells ARPE-19 cells was plated at 1×105 cell/mL at 96-well plates. ARPE-19 cells cultivated in DMEM/F12 were pretreated for 12 hours at various CMCT concentrations. The medium was discarded, washed twice with PBS, and continuously exposed to H2O2 for 1h. After various treatments, MTT solutions were prepared in advance, stored in keeping in darkness, and then added to 96-well plates with 20μm of 5mg/mL MTT solution per well to reflect the mitochondrial activity of the cells through the transformation and dissolution of eszopiclone MTT methyl crystals to form particles, and combed at 37°C for 4h. After incubation the medium solution in each well was discarded 150μl of dimethyl sulfoxide (DMSO) was added, and the reaction was stopped and placed on an electric shaker for 10 min. The wells' crystals were then fully dissolved. The absorption of each well with a microplate reader at 492 nm was calculated (Model Devices SpectraMaxi3X; American Valley Molecular Instruments, Inc.), and all experiments were performed in triplicate. The average of the results was read for calculation. The absorbance values represent cell viability normalized to the untreated control and represent 100% cell viability the cells' relative viability as a percentage of the control.
Detection of cell death by ARPE-19
Apoptosis assays were evaluated by Annexin V-FITX/PI kit according to the manufacturer's instructions. ARPE-19 cells were incubated on 1 × 105-well plates with or without CMCT treatment for 12h after exposure to H2O2 for 12h. Afterward, cells were collected by washing twice in ice-cold PBS liquid by centrifugation at 4°C for 5 min and resuspended in fresh medium and 5μl Annexin V-FITX was added for staining in the dark for 15 min. Immediately after staining, Flow cytometry was used to analyze the cells, and the number of apoptotic cells was measured using Cell Quest analysis tools. Results were expressed as Annexin-V negative-PI negative for normal cells, Annexin-V positive-PI negative for cells in early apoptosis, and Annexin-V positive-PI positive for cells in late apoptosis or necrotic cells and all assay data were performed in triplicate.
Intracellular ROS levels are measured
A fluorescent probe DCFH-DA staining determined the number of reactive oxygen species in cells. Briefly, ARPE-19 cells in 94-well plates were re-stained with H2O2 (400μM) for 12h, extended and excess liquid discarded then ARPE-19 cells were pretreated with CMCT for 12h. Cells were incubated with 10μM DCFH-DA reagent from the kit for 30min at 37°C in the dark, and serum-free cell culture medium was washed twice to remove excess DCFH-DA and resuspended in PBS. The cells' absorbance wavelength was recorded immediately with a microplate reader. Flow cytometry was used to record the intracellular fluorescence strength during therapy at 488 nm excitation and 525 nm emission wavelengths.
Detection of SOD activity, CAT and GSH, MDA levels
Upon various treatments, the effects of the CMCT on oxidative stress in different ARPE-19 cells were measured with four specific oxidative biomarkers including SOD, MDA, CAT and GSH. Briefly, dismutase (SOD), malondialdehyde (MDA) and catalase (CAT) activity were observed using the test kit obtained, refer to instructions of the manufacturer.
SOD activity assay, the cells were pretreated at 37°C and attached to the plate, two cells were collected and washed with PBS and centrifuged for 5 minutes to separate the sediment the resulting cell pellets were incubated in the buffer (25 mM NaCl, 0.5% Triton X-100, 60 mM Tris-HCl (pH7.4), 1mM Na3VO4, 20 mM NaF, 10 mM Na4P2O7. For 60 min, the mixture was incubated with ice water, with Centrifuge at 4°C for 30 minutes. The supernatant was collected, and the package operation for superoxide dismutase has measured the maker's directions.
CAT activity assay, the method was based on detecting the reaction decomposition of CAT in the sample under the adaptive H2O2 concentration. In this procedure, prepare pelleted cells and cell supernatants according to the previous activity detection analysis, di- dilute the volume to 50μl with phosphate buffer solution (pH 7.0), and added with 20ul H2O2 matrix solution and carry out the enzyme-catalyzed reaction at 25°C for 15 min, then detect the sample at 528 nm before. A standard bovine liver CAT curve was used for determining the function of the enzyme.
GSH activity assay, the same operation was used to prepare 20μl of cell-free supernatant in the sample for SOD activity determination. The detection kit was used to analyze the total protein, and the supernatant was centrifuged for glutathione determination. (4℃ 15000*9 10min). Read the absorbance at a wavelength of 405nm according to the manufacturer's instructions. The quantification of intracellular glutathione was based on its use solution as a standard.
The impact of CMCT on the identification of oxidation biomarkers in cells was calculated using the MDA activity assay. Briefly, cells were treated with H2O2 (100μM) for 1h and CMCT overnight. After completion of treatment, the cells were then washed with PBS, and the oxidation biomarkers were obtained by centrifugation and identified using the test set as directed by the manufacturer. For both antioxidant enzymes and MDA amounts, nmol per mg of protein has been expressed.
Western blot analysis
Cells were pretreated. Briefly, two washes of ice-cold PBS were performed on the cells. After treatment, the cells were collected that the cells were laced with RIPA buffer for nuclear tissue. At 4°C for 15min the lysate was centrifuged at 12,000 × g and the supernatant were extracted. The protein content in the samples was detected in the test kit and according to the directions of the manufacturer. 20μg protein samples were electrophoresed on polyacrylamide (PAGE) 10% sodium dodecyl sulfate (SDS), moved to the membrane of polyvinyl fluoride (PVDF), and sealed with 5% skim milk at a temperature of 4°C. Incubate the antibody characterizing overnight, wash with PBS and then incubate the eggs at room temperature for 1h. with the following secondary antibody. The ECL Western blotting assay kit is used to detect protein bands.
Statistical Analysis
All experiments were repeated at least three times, and data are shown as mean ± standard deviation or percentages. origin9.0 was used for plot analysis, and variance analysis between groups was compared using one-way ANOVA or ordinary two-way ANOVA, and variability with *P<0.05, **P<0.01 and #P<0.05, ##<0.01. was considered statistically significant.