Human peripheral blood B lymphocyte (AHH-1, BNCC331188) was purchased from BNBIO.com (Beijing, China) and human intestinal epithelial (HIEC, MZ-0792) cells were purchased from Mingzhoubio.com (Zhejiang, China). The cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 °C and 5% CO2.
Effect of zymosan on cell toxicity
AHH-1 and HIEC cells were respectively administered to zymosan (tlrl-zyn, InvoGen) at 0, 20, 40, 80 and 160 μg/ml. CCK-8 and cell flow cytometry were used to detect the cell activity and apoptosis at 24 h, 48 h, and 72 h after administration to determine the dose-limiting toxicities of zymosan.
Determination of the best dose of zymosan on cell radiation protection
Twelve hours before irradiation, cells were treated with zymosan at 0, 5, 10, and 20 μg/ml, and then irradiated with 4Gy X-rays. CCK-8 and cell flow cytometry were utilized to detect cell activity and apoptosis at 24 h to determine the optimal dose of zymosan.
Cells are randomly divided into 4 groups: normal control group (Control, cells are normally cultured), only irradiation group (Model, cells are irradiated with 4Gy radiation dose), positive control group (lipopolysaccharides, LPS, cells were treated with 20 μg/ml LPS 12 h before irradiation), the best dose group of (zymosan, cells were treated with 20μg/ml zymosan 12 h before irradiation.
Cells (103-104 cells/well) were cultured in an incubator containing 5% CO2 at 37℃ for 24 hours, then added with 10 μl CCK-8 solution (GipBio, Shanghai, China) and mixed well. Then were continued to be incubated for 4 h and oscillated in a microplate reader. The absorbance of each hole at 450 nm was measured by zero adjustment of blank control hole. The cell survival rate (%) =[(As-Ab)/(Ac-Ab)]×100.
As = Absorbance of experimental pores containing cells, medium, CCK-8 and compounds to be tested.
Ab = Absorbance of blank pores containing medium and CCK-8.
Ac = Absorbance of control pores containing cells, medium and CCK-8.
Annexin V-FITC/PI kit (CA1020, Solarbio, Beijing, China) was used to detect cell apoptosis. Cells cultured for 24 hours (1 × 106 cells/time) were collected and washed with precooled PBS. Cells were suspended in 1 mL of 1× binging buffer and centrifuged at 300 ×g for 10 mins. Then the cell concentration was adjusted to 1×106 cells/mL with 1 mL of 1× binging buffer. 100 µL of cells were added with 5 µL of annexin V-FITC and incubated in dark for 10 mins at room temperature, then added with 5 µL of propidium iodide (PI) and incubated for 5 mins. Finally, the volume of cell incubation suspension was adjusted to 500 µL by PBS and tested on the computer within one hour.
A preliminary study on the protective mechanism of zymosan
To determine whether zymosan exerts radiation protection through the TLR2/4-MyD88-G-CSF/GM-CSF/IL-12/IL-6 pathway, we used MyD88 inhibitors (10µM，ST2825, MedChemExpress, Shanghai, China) to treat cells , and analyzed the expression of this pathway related proteins.
The expression of TLR2, TLR4, MyD88, G-CSF, IL-12 and IL-6 in each cell line was detected by Western-blot method. 40 μg of samples were added with 10% SDS-PAGE buffer and electrophoresed using Bio-Rad electrophoresis device. Then they were transferred to the PVDF membrane for 30-60 mins, blocked, and incubated with the following primary antibody overnight: anti-rabbit TLR2 (1:1000, PA5-17492, ThermoFisher, Shanghai, China), TLR4 (1:200, 48-2300, ThermoFisher), MyD88 (2 µg/mL, PA5-19919, ThermoFisher), G-CSF (1:1000, PA5-86821, ThermoFisher), IL-12 (0.8µg/mL, PA5-18741, ThermoFisher), IL-6 (1:1000, P620, ThermoFisher) and β-actin (1:8000, PA1-16889, ThermoFisher). After rewarming, the membranes were incubated with secondary antibody Ig G (0.3µg/mL, A32731, ThermoFisher) at room temperature for 1 h. Then they were washed and colored with ECL luminescent substrate for 3-5 mins. Protein expression levels were scanned, quantified and finally normalized relative to β-actin using the Image J (NIH) software.
Statistical analysis method
Prism5.01 statistical analysis software was used for data processing, and the results were expressed as mean ± standard deviation (`X±SD). The data analyses among multiple groups were assessed by one-way analysis of variance (ANOVA), and Tukey test was exerted for subsequent analysis. p<0.05 indicates that the difference is statistically significant.