The mechanism for the radioprotective effects of zymosan‐A in mice

Abstract It proved that Zymosan‐A protected the haematopoietic system from radiation‐induced damage via Toll‐Like Receptor2 in our previous study. In this study, we investigated the potential mechanism for the radioprotective effects of Zymosan‐A. The mice were treated with Zymosan‐A (50 mg/kg, dissolved in NS) via peritoneal injection 24 and 2 hours before ionizing radiation. Apoptosis of bone marrow cells and the levels of IL‐6, IL‐12, G‐CSF and GM‐CSF were evaluated by flow cytometry assay. DNA damage was determined by γ‐H2AX foci assay. In addition, RNA sequencing was performed to identify differentially expressed genes (DEGs). Zymosan‐A protected bone marrow cells from radiation‐induced apoptosis, up‐regulated IL‐6, IL‐12, G‐CSF and GM‐CSF in bone marrow cells. Zymosan‐A also protected cells from radiation‐induced DNA damage. Moreover, RNA sequencing analysis revealed that Zymosan‐A induced 131 DEGs involved in the regulation of immune system process and inflammatory response. The DEGs were mainly clustered in 18 KEGG pathways which were also associated with immune system processes. Zymosan‐A protected bone marrow cells from radiation‐induced apoptosis and up‐regulated IL‐6, IL‐12, G‐CSF and GM‐CSF. Moreover, Zymosan‐A might also exhibit radioprotective effects through regulating immune system process and inflammatory response. These results provided new knowledge regarding the radioprotective effect of Zymosan‐A.

Zymosan-A were dependent on the TLR2 signalling pathway. 19 In addition, we found that Zymosan-A mitigated the damage of haematopoietic system and accelerated the recovery of haematopoiesis in mice. However, the potential mechanism is still unclear.
In this study, the molecular mechanism of radioprotection of Zymosan-A was studied using flow cytometry, c-H2AX foci assay and RNA-seq. Our experiments provided new knowledge regarding the radioprotective effect of Zymosan-A.

| Cell culture and treatment
Human B lymphocyte (AHH-1)) was obtained from American Type Culture Collection, and cultured in RPMI 1640 with 10% FBS at 37°C in a 5% CO2 humidified chamber. Cells were treated with Zymosan-A (40 lg/mL) 12 and 2 hours before irradiation.

| Antibody staining and flow cytometry
Bone marrow cells (BMCs) were isolated freshly. Then, cells were strained through a 40-lm strainer in the presence of phosphate-buffered saline and red blood cells were removed. Cells were stained with antibody for 20 minutes at 4°C. The cell apoptosis was analysed using the apoptosis detection kit according to the manufacturer's instructions. PI and Annexin V were used to stain BMCs.
BMCs were fixed, permeabilized and labelled with anti-G-CSF, anti-GM-CSF, anti-IL-6 and anti-IL-12 and then subjected to flow cytometry analysis.

| Immunofluorescence analysis
Immunofluorescence analysis was used to detect c-H2AX foci. AHH-1 cells were seeded in 6-well plates at the concentration of 2*10 5 per well. Then, cells were treated with Zymosan-A (40 lg/mL) 12 and 2 hours before 6 Gy irradiation. Then 0, 0.5, 2 hours later, cells were fixed in 4% paraformaldehyde for 20 minutes and permeabilized in 0.5% Triton X-100 for 10 minutes. After blocked in BSA, cells were stained with c-H2AX, and then stained with the secondary antibody  Guangzhou Ribo Bio Co., Ltd. with the Illumina HiSeq 2500. Prior to sequencing, the raw data were filtered to produce high-quality clean data. All the subsequent analyses were performed with the clean data. All the differentially expressed genes (DEGs) were used for heat map analysis, Gene Ontology Analysis and KEGG ontology enrichment analyses. For KEGG enrichment analysis, a P-value < .05 was used as the threshold to determine significant enrichment of the gene sets.

| Statistical analysis
Data were expressed as means AE standard deviation (SD).
Two-tailed Student's t test was used to analyse the difference between 2 groups. These data were analysed using SPSS ver. 19 (IBM Corp., Armonk, NY, USA). P < .05 was considered statistically significant.

| Zymosan-A inhibited BMCs apoptosis caused by radiation
The mortality of mice after radiation was associated with a serious and continuous BMCs loss. 16 In previous study, we showed that Zymosan-A significantly improved the number of BMCs after ionizing radiation. 19 To explore the potential mechanism, we detected the apoptosis of BMCs 24 hours after radiation. The results showed that the BMCs apoptosis rate increased after radiation, while the apoptosis rate was decreased significantly in BMCs from mice which treated with Zymosan-A ( Figure 1).

| Zymosan-A up-regulated the levels of GM-CSF, G-CSF, IL-12 and IL-6 in BMCs
The protective effects of GM-CSF, G-CSF, IL-12 and IL-6 have been proven in several studies. [20][21][22] Those cytokines play important roles in the haematopoietic system. 23,24 Using flow cytometry, we found    IR + Zymosan-A groups, compared to IR + NS groups (Table 1).
DEGs expression heat map was shown in Figure 4.

| DEGs gene ontology analysis between IR + NS and IR + Zymosan-A groups
Gene ontology analysis was used to investigate changes in the patterns of genes between IR + NS and IR + Zymosan-A groups.
The significantly enriched GO analysis of DEGs was shown in Table 2 and Figure 5.

| Signalling pathway enrichment analysis of DEGs between IR + NS and IR + Zymosan-A groups
To further study the biological functions of the DEGs, KEGG analysis was used to evaluate the functions of the DEGs. The DEGs were mapped to 18 pathways in the KEGG database, as shown in Table 3.
Moreover, the DEGs were classified into 5 classifications including cellular processes, environmental information, human diseases, metabolism and organismal systems ( Figure 6). Within the environmental information group, TNF signalling pathway and NF-kappa B signalling pathway were significantly enriched.

| DISCUSSION
Radiation-induced death is a complex pathophysiological process. 6,25 The loss of BMC is the major reason of mice death after radiation.
In previous study, we showed that the number of BMCs in mice treated with Zymosan-A was higher than that in mice treated with NS. 19 Using flow cytometry, we proved that Zymosan-A inhibited apoptosis of BMCs induced by radiation, which contributed to the increased number of BMCs. Next, we detected the levels of GM-CSF, G-CSF, IL-6 and IL-12 in BMCs. Those cytokines play important roles in haematopoiesis. 26,27 For example, G-CSF, which can stimulate the production of proteases that cleave many interactions including CXCR4/SDF-1, has been used to induce HSC mobilization in current clinical practice. 28 In addition, our unpublished experimental data revealed that Zymosan-A also increased the number of LSK cells, which play critical roles in reconstitution of haematopoietic system after radiation. 29,30 Ionizing radiation can lead to DNA damage through direct and indirect effects, including double-strand breaks(DSB), single-strand breaks(SSB), base mutation and deletion. 31 Among them, DSB is the most serious consequence of cell, which can directly cause cell death and induce a series of inflammatory responses. 32,33 In our previous study, we found that Zymosan-A can protected AHH-1 form radiation-induced apoptosis. 19 By detecting c-H2AX, the marker of DSB damage, we showed that the DSB damage can be significantly reduced in Zymosan-A-treated group. This date reminded us that the radioprotection of Zymosan-A can be achieved by reducing DSB. In conclusion, Zymosan-A exhibited great protective effects against ionizing radiation. Zymosan-A protected bone marrow cells from radiation-induced apoptosis, up-regulated IL-6, IL-12, G-CSF and GM-CSF in BMCs. In addition, Zymosan-A also protected cells from radiation-induced DNA damage in vitro. Moreover, Zymosan-A treatment induced 131 DEGs which were related to F I G U R E 5 Gene ontology analysis and significant enriched GO terms of differentially expressed genes (DEGs) between IR + NS and IR + Zymosan-A groups. GO analysis classified the DEGs into 3 groups (biological process group, molecular function group and cellular component group).
T A B L E 3 Signalling pathway enrichment analysis of differentially expressed genes between IR + NS and IR + Zymosan-A groups