Cell culture
HaCaT cell line and HaCaT keratinocytes (ATCC, Manassas, VA, USA) were cultured using Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA) which contains 10% of fetal bovine serum (FBS; Hyclone) and 1% of penicillin/streptomycin (penicillin 100 IU/mL, streptomycin 100 μg/mL; Invitrogen/Life Techmologies, Carlsbad, CA, USA) for HaCaT culture and cultured it within cell incubator kept under the condition of 5% of CO2 with the temperature at 37 °C.
Sample treatment
I purchased geniposidic acid which is refined in the form of powder from Santa Cruz Biotech (Santa Cruz, CA, USA) and dissolved it in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) in a proper density to use it in the experiment. After I cultured HaCaT cell (1 × 106 cells/well) in the cell medium for 24 h, I added geniposidic acid in a proper density to culture medium and pretreated for 3 h, and for ultraviolet B test on cells, I used UV-B lamp (UVP, Upland, CA, USA) to examine. I used fiber optic Spectrometer System USB2000 (Ocean Optics, Dunedin, FL, USA) to estimate UVB wavelength, and at the UVB test on HaCaT, I washed it twice with phosphate-buffered saline (PBS; (Thermo Fisher Scientific, USA) in a pH of 7.4 to remove culture medium of cell culture dish. To prevent drying of cell, I opened cell culture dish lid for UVB test after adding 1 mL of PBS to cell culture dish, removed PBS after UVB testing, added culture medium again to culture in the culture incubator for 24 h, and then used in the experiment.
Cell viability estimation
I used the principle of WST assay to estimate cell viability. I used EZ-Cytox cell viability assay kit (Itsbio, Seoul, Korea), and after inoculating HaCaT (3 × 103 cells/well) in 96-well plate and cultured for 24 h, I treated HaCaT with geniposidic acid of each density of 5, 10, 20, 30, and 40 μM respectively, tested 40 mJ/cm2 of UVB, and then cultured for 24 h. After I added 10 μL of EZ-Cytox cell viability assay kit reagent (ItsBio) into cultured well and cultured for 1 h, I used microplate reader (Bio-Rad, Hercules, CA, USA). I estimated the absorbance in the scale of 490 nm, repeated each experiments three times separately, and derived average value and standard deviation of cell viability.
Cell cycle analysis
I used an equipment of BD FACS Calibur Flow Cytometer (BD Biosciences, San Jose, CA, USA). I estimated the number of cells in sub-G1, G1, S, and G2/M cell cycle to analyze cell cycle. After I inoculated 2 × 105 cells/well of HaCaT in 60 mm of culture dish, I cultured it for 24 h, and then, I pretreated it with geniposidic acid for 3 h. After testing UVB, I cultured it for 24 h more and obtained cultured cells, and then, I centrifuged it at 5000 rpm with temperature at 4 °C for 5 min and precipitated cells. After I removed the supernatant and dissolved the cell pellet with 300 μL of PBS, I slowly added 700 μL of absolute ethanol (Biopure, Canada) while vortexing, and I refrigerated it with temperature at 4 °C for 3 h to fix cells. I added 1 mL of PBS, centrifuged at 5000 rpm with temperature at 4 °C for 5 min to remove supernatant, dissolved the pellet with 200 μL of propidium iodide (PI) staining buffer (PI 50 μg/mL, RNase 0.1 μg/mL, 0.05% Triton X-100; Sigma Aldrich), and then settled with temperature at 37 °C for 1 h. Afterward, I centrifuged HaCaT cells at 5000 rpm with temperature at 4 °C for 5 min to remove supernatant and washed with PBS, and then, I dissolved the pellet with 1 mL of PBS and through Flow Cytometer estimated the number of cells in each cell cycle.
qRT-PCR analysis
To analyze and find out gene expression occurred within HaCaT by geniposidic acid quantitatively, I used qRT-PCR method. I mixed 0.2 μM of primers, 20 mM of Tris/HCl in a pH of 8.4, 50 mM KCl, 0.8 mM of dNTP, 3 mM of MgCl2, 0.5 U Extaq DNA polymerase, and 1× SYBR green (Invitrogen) in PCR tube to manufacture reaction solution and used Linegene K (BioER, Zhejiang, China). After denaturation with temperature at 94 °C for 3 min, I performed denaturation (94 °C, 30 s), annealing (58 °C, 30 s), and polymerization (72 °C, 30 s) for 40 cycles and proceeded PCR. With melting curve, I verified the effectiveness of PCR, and by standardizing expression of β-actin, I performed the comparative analysis for each gene expression, and the primer used in the experiment is shown as Table 1.
DPPH radical scavenging activity assay
I injected the geniposidic acid diluent of each density into 96-well plate, added 50 μL of 0.2 mM of 1,1-diphenyl-2-picrylhydrazyl (DPPH), and settled it for 30 min. Using a microplate reader (Bio-Rad), I estimated the absorbance in the scale of 514 nm and repeated this estimation three times. I derived the average value and standard deviation of absorbance.
ABTS+ radical scavenging assay
I mixed 7.4 nm of 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and 2.6 mM of potassium persulfate in the final density, reacted in dark room at room temperature for 12 h, and formed ABTS+, and then, I made the value of the absorbance to be 0.706 (± 0.01) in the scale of 732 nm. After adding 100 μL of ABTS+ into 100 μL geniposidic acid in 96-well plate and neglecting for 7 min, I estimated the absorbance in the scale of 732 nm. I repeated this estimation three times and derived the average value and standard deviation of the absorbance.
Caspase-3 activity
Caspase-3 which induces apoptosis is affiliated with cysteine proteinase family, and I used Apoalert caspase-3 colorimetric assay kit (Clontech, Mountain View, CA, USA) as method to estimate pNA (p-nitroanilide) action using its character to decompose Ac-DEVD-pNA (N-acetyl-Asp-Glu-Val-Asp p-nitroanilide) which is a substrate complex. I separated the cells treated by sample from culture dish, centrifuged it at 1500 rpm for 5 min, and added cold cell lysis buffer into cell pellet whose culture medium was removed, and then, I settled it under the condition of the ice for 10 min and centrifuged it at 15000 rpm with temperature at 4 °C for 3 min to separate supernatant only. With regard to the separated supernatant, I used bradford assay to quantify only total 50 μg of protein and apply to caspase-3 activity reaction. I put suspension which I finished protein quantification into 96-well plate, added reaction buffer, and cultured it with temperature at 37 °C for 30 min. Then, I added caspase-3 substrate, cultured with temperature at 37 °C for 1 h, and used microplate reader (Bio-Rad) to estimate the absorbance in the scale of 405 nm.
Comet assay (single-cell gel electrophoresis)
I used CometAssay® Reagent Kits (Trevigen, Gaithesburg, MD, USA) and melted LMAgarose (low melting agarose) completely for 5 min, and then, I covered it with cab, cooled in water bath whose temperature was at 37 °C for 20 min, and mixed 1 × 105 cells/mL of cell with melted LMAgarose (at 37 °C) at the ratio of 1 to 10 (v/v). Afterward, I poured 50 μL of mixed liquor onto Comet slide and kept in slide in the refrigerator with temperature at 4 °C for 10 min, and then, I immersed the slide in alkaline solution at room temperature for 20 min. After I immersed the slide in alkaline electrophoresis solution and covered, I electrophoresed it with voltages at 21 V for 30 min, and after finishing electrophoresis, I immersed it twice in distilled water for 4 min to remove electrophoresis solution and immersed in 70% ethanol for 5 min. I dried it with temperature at 37 °C, put dried agarose gel into 100 μL of SYBR® gold nuclei acid gel stain (Invitrogen), and dyed it in the dark room for 30 min. I rinsed it with water simply to remove dyeing reagent and observed agarose gel which I dried it completely with temperature at 37 °C through fluorescence microscope.
MDA assay
I used colorimetric method to estimate malondialdehyde (MDA) level and used Lipid Peroxidation (MDA) Assay Kit (Abcam, Cambridge, UK). First, I inoculated 1 × 107 cells/well of HaCaT in 6-well plate and pretreated it with reagent, and then, I tested the UV and cultured it for 48 h. I used a microplate reader (Bio-Rad) to estimate in the scale of 586 nm and used the principle that oxidated lipid (MDA) reacts with TBA (thiobarbituric acid) within kit and detects through colorimetric method.
Statistical process
In this research, I performed every experiment more than three times separately under the same condition and obtained experimental result, and used Student’s t test for each experiment and found p value. When p value in every experimental result is less than 0.05, I analyzed that it is statistically significant.