Human peripheral blood B lymphocytes (AHH-1 cells, BNCC331188) were purchased from BNBIO.com (Beijing, China), and human intestinal epithelial cells (HIECs, MZ-0792) were purchased from Mingzhoubio.com (Zhejiang, China). The cells were cultured in DMEM containing 10% fetal bovine serum at 37 °C and 5% CO2.
Cell Counting Kit (CCK)-8 assay
Cells (103 cells/well) were cultured in an incubator containing 5% CO2 at 37°C for 24 hours, and then 10 μL of CCK-8 solution (GipBio, Shanghai, China) was added and mixed well. Then, the cells were incubated for 4 h with oscillated and read in a microplate reader. The absorbance of each well at 450 nm was measured by normalization to the blank control. The cell survival rate was calculated based on the following formula: cell survival rate (%) = [(As-Ab)/(Ac-Ab)]×100.
As = Absorbance of treatment wells
Ab = Absorbance of blank
Ac = Absorbance of Control wells
The Annexin V-FITC/PI Kit (CA1020, Solarbio, Beijing, China) was used to detect cell apoptosis. Cells cultured for 24 hours (1 × 106 cells) were collected and washed with precooled phosphate-buffered saline (PBS). The cells were suspended in 1 mL of 1× binding buffer containing Ca2+ and centrifuged at 300 ×g for 10 mins. Then, the cell concentration was adjusted to 1×106 cells/mL with 1 mL of 1× binding buffer. A total of 100 µL of the cell solution was added to 5 µL of annexin V-FITC and incubated in the dark for 10 min at room temperature. Then, 5 µL of propidium iodide (PI) was added, and the cells were incubated for 5 min. Finally, the volume of the cell suspension was adjusted to 500 µL with PBS and evaluated by flow cytometry (1040, ACEA NovoCyteTM, USA) within one hour. The results were evaluated by Cell Quest software (Version 5.1, BD Biosciences).
Effect of zymosan on cell toxicity
AHH-1 and HIEC cells were administered 0, 20, 40, 80 or 160 μg/ml zymosan (tlrl-zyn, InvoGen). The CCK-8 assay and cell flow cytometry were used to evaluate cell viability and apoptosis 24 h, 48 h, and 72 h after administration to determine the dose-limiting toxicity of zymosan.
Determination of the dose at which zymosan exerts radioprotective effects on cell
Twelve hours before irradiation, cells were treated with 0, 5, 10, or 20 μg/ml zymosan and then irradiated with 4 Gy X-rays at a dose rate of 0.25 Gy/min (Synergy, Elekta, Beijing, China). The CCK-8 assay and flow cytometry were utilized to evaluate cell viability and apoptosis at 24 h to determine the optimal dose of zymosan.
Comparison of the radioprotective effects of lipopolysaccharide and zymosan in vitro
Cells were randomly divided into 4 groups: the normal control (control) group (cells were normally cultured), irradiation only (model) group (cells were irradiated with 4 Gy radiation), LPS group (cells were treated with 20 μg/ml LPS 12 h before being irradiated), and zymosan group (cells were treated with 20 μg/ml zymosan 12 h before being irradiated).
A total of 120 male Sprague Dawley rats weighing 180 ± 20 g (aged 6-8 weeks) were purchased from Jinan Pengyue Experimental Animal Co., Ltd. (scxk (Lu) 20190003). The rats were housed at normal temperature (22 ± 2 °C) and humidity (55 ± 5%). Normal diet and water were freely provided, and the animals were housed under a 12-hour light/dark cycle. The rats were adaptively fed for 1 week. The animal experiments were conducted in accordance with the guidelines of the National Institutes of Health (NIH pub. No. 85-23, revised 1996) and were approved by the Animal Protection and Use Committee of Binzhou Medical University.
Toxic effects of zymosan on rats
Zymosan (0, 5, 10, 20, or 40 mg/kg) was sterilely injected intraperitoneally, which was dissolved in sterile normal saline. Then, the rats were fed routinely and observed for 21 days. Weight changes were observed weekly. The relative weight changes were calculated by (Treatment group-Control)/(Control group) * 100. After 21 days, the rats were euthanized with 3% pentobarbital sodium (150 mg/kg). The spleen, thymus and liver were removed and immediately weighed, and the organ index was calculated by the following formula: Organ index = organ weight/body weight .
Determination of the appropriate dose of zymosan for radioprotection of rats
Twenty-four hours before irradiation, the rats were intraperitoneally injected with 0, 2, 4, 8 or 10 mg/kg zymosan and then irradiated with 7 Gy X-ray at a dose rate of 1.0 Gy/min. Then, they were fed routinely and observed for 21 days. The survival of the rats was assessed each day. If animals were moribund, they were humanely euthanized. After 21 days, the rats were euthanized with 3% pentobarbital sodium (150 mg/kg). The spleen index and thymus index were calculated.
Comparison of the radioprotective effects of lipopolysaccharide and zymosan in vivo
Twenty-four rats were randomly divided into 4 groups: the normal control (control) group (the rats were normally fed), irradiation only (model) group (the rats were irradiated with 7 Gy X-ray at a dose rate of 1.0 Gy/min), LPS group (the rats were intraperitoneally injected with 10 mg/kg LPS 24 h before being irradiated), and zymosan group (the rats were intraperitoneally injected with 10 mg/kg zymosan 12 h before being irradiated). If animals were moribund, they were humanely euthanized. The survival of the rats was assessed each day. After 21 days, the rats were euthanized with 3% pentobarbital sodium (150 mg/kg). The spleen index and thymus index were calculated.
Prism 5.01 statistical analysis software was used for data processing, and the results are expressed as the mean ± standard deviation (`X±SD). Data from multiple groups were assessed by one-way analysis of variance (ANOVA), and Tukey’s test was used for subsequent analysis. p<0.05 indicated a significant difference.