| Myelosuppression is the most common adverse effect of cytotoxic chemotherapy and is a major limiting factor in the clinical treatment of cancer. Therefore, promotion of hematopoiesis remains an extremely important challenge in cancer therapy. This paper studies the effects and mechanism of interleukin 1 receptor antagonist in bone marrow hematopoietic system of mice and preclinical research on prevention of chemotherapy-induced myelosuppression. We used 5-fluorouracil (5-Fu) mouse chemotherapy model to identify bone marrow (BM) damage and regeneration. After 5-Fu injection, the mouse BM is severely damaged and regeneration is initiated. Within 14 days, the damaged BM is fully recovered through active hematopoiesis. For Affymetrix Gene Chip analysis, five total RNA samples were extracted from bone marrow cells collected at days 0, 3, 7, 11, and 14 days post-5-Fu treatment. The chip data shows that IL-1Ra is upregulated after 5-Fu treatment on day 7 and then downregulated to the baseline on day 14. To validate the gene chip results, protein expression was evaluated using an ELISA kit for IL-1Ra. ELISA analysis of mouse serum after 5-Fu treatment showed that IL-1Ra protein level in serum was increased obviously at day 7 after 5-Fu injection, which is consistent with the gene expression data. Both ELISA and microarray data indicated that IL-1Ra may be a hematopoietic inhibitor and the overexpression of endogenous IL-1Ra is likely responsible for the observed suppression of hematopoiesis after chemotherapy.The function of IL-1Ra in regulation of BM regeneration was examined by two methods: (1) transient overexpression of the protein through plasmid muscle electroporation in normal mice; (2) injection of the recombinant murine IL-1Ra in normal and 5-Fu treated mice. The effects of IL-1Ra on BM regeneration were analyzed by measuring the peripheral white blood cell, platelet and BM cell counts, cell cycle analysis, hematopoietic colony assay, and mouse survival rate after 5-Fu.We constructed the eukaryote expression plasmid pcDNA-IL-1Ra, which contain the cDNA sequence of the mouse IL-1Ra gene. The plasmid was introduced to the mouse tibia muscle by electroporation. The expression level of the mouse IL-1Ra in serum after electroporation was measured by ELISA method. We found that IL-1Ra expression lasted for about 10 days. The mice with pcDNA-IL-1Ra showed some significant differences in peripheral blood and BM cell counts compared to the control pcDNA3.1 mice. There were significant decreased in bone marrow cell counts in the pcDNA-IL-1Ra mice on day 5. At day 10 and 17, pcDNA-IL-1Ra group showed lower peripheral blood cell counts than control group. Hematological data analysis suggests that IL-1Ra overexpression may have some effects on bone marrow hematopoietic system in mice.To further study the role of IL-1Ra in protection of BM from chemotherapy damage, we produced the recombinant murine IL-1Ra protein. The murine IL-1Ra is a low molecular protein of 17KDa with no glycosylation. The prokaryotic expression system was chosen to make the protein using the pET expression system. The vector was pET28a and the host strain was E.coli BL21. IPTG was used to induce the expression of recombinant protein. We constructed the prokaryotic expressing plasmid pET28-IL-1Ra, which contained the coding sequence of mature murine IL-1Ra protein. Then we induced the expression of the rMuIL-1Ra by 1mM IPTG. The collected bacteria went through sonication, Urea denaturation, dilution before the final anion-exchange chromatography by Q Sepharose. When the conductivity reached 60mS/cm, rMuIL-1Ra was eluted from the colume. SDS-PAGE showed the purity was above 96%. The bioactivity assay demonstrated that the rMuIL-1Ra had good activity and may be used to do next functional experiments.We first examined the role of rMuIL-1Ra in normal mice. We tried 3 doses of rMuIL-1Ra 0.01, 0.1 and 1mg/kg. PBS of equal volume was injected in control group. All mice of the 4 groups were injected for 5 days and once a day. The date starting the injection was noted as day 0 and data of the mice were collected at day 0, 5, 10 and 15. At day 5, rMuIL-1Ra groups of the highest doses showed decreased cells counts compared to the control group, but that was recovered rapidly at day 10. These results indicate that the administration of rMuIL-1Ra at various dosages results in decreased BM and peripheral WBC production in mice.Next, we studied the time-effect of rMuIL-1Ra in normal mice. This study shows the effects of rMuIL-1Ra on the number of bone marrow cells in response to the injection of rMuIL-1Ra for different time periods. Mean bone marrow cells counts show a steady decrease over the treated time period, compared with the PBS group. These differences achieved statistical significance at day5.We further studied the effect of rMuIL-1Ra on the 5-Fu treated mice. Four parts of experiments were taken: (1) injection of rMuIL-1Ra for two or five days prior to 5-Fu treatment; (2) injection of rMuIL-1Ra for five days after chemotherapy; (3) administration of rMuIL-1Ra for two days before and two days after 5-Fu treatment; (4) administration of rMuIL-1Ra at 7～10 days after 5-Fu treatment. In cohort 1, platelets data shows that rMuIL-1Ra groups recovered better than the control PBS group at day 3, 7 and 11. The control group had lower BM cells counts than rMuIL-1Ra group at 3, 7, and 11. The survival data demonstrate that pretreatment of mice for 2 days with rMuIL-1Ra could protect 95% of the mice from the subsequent sub-lethal dose of 5-Fu treatment. In cohort 2, platelets data shows that at days 7, 11, and 14, the rMuIL-1Ra groups recovered better than the control group. A significant difference appeared between the rMuIL-1Ra group and the control group in BM cells counts at day 11, 14 and 21. Survival was scored daily for 14 days. Survival rate was increased to 90% of the mice compared to the PBS group which was 20%. This chemoprotection by rMuIL-1Ra may allow an increased doses of 5-Fu without adverse effects on survival. In cohort 3, data demonstrated that the use of rMuIL-1Ra before and after chemotherapy has no much effect on bone marrow hematopoiesis. To investigate the interaction between IL-1Ra and IL-1β,rMuIL-1Ra was injected at 7～10 days after 5-Fu treatment to block the IL-1βaction. The increase of platelet was completely suppressed. The data indicated that endogenous upregulation of IL-1βis responsible for the recovery (rebound) of platelet counts after 5-Fu treatment.To further study the mechanism of the protection of effect of rMuIL-1Ra, we administrated rMuIL-1Ra to normal mice for 5 days and killed these animals within 24h. We analyzed the cell cycle of BMN,Lin-,Lin--c-kit+ (KL) and Lin--c-kit+-Sca-1+ (SKL) cells through FACS. The results showed that rMuIL-1Ra can reduce the BMN and Lin- cells into S and G2/M phase. For more primitive KL and SKL cell cycle analysis, we found that in addition to S phase and G2 / M phase of the ratio decreased, KL and SKL cells has a significant increase in G0 of the G0/G1 phase. Other words, the injection of IL-1Ra can slow down the cell cycle and proliferation. This mechanism can explain the preventive application of rMuIL-1Ra before 5-Fu (cell cycle specific drug) was able to reduce the number of bone marrow cells into S phase. Therefore, the bone marrow damage induced by chemotherapy can be eased.The expression levels between IL-1Ra and IL-1βis a dynamic process. Plasma samples from mice of several cohorts following 5-Fu administration were assayed for IL-1Ra and IL-1βexpression. The serum of mice was analyzed at 3, 7, 11 and 14 days post injection using ELISA analysis. In cohort 1, 3 and control group, the level of IL-1βexpression was steady until day 11, after which IL-1βexpression increased―reaching a peak at day 11. However, in cohort 2, the level of IL-1βexpression reached the peak at day 7 and then decreased the baseline. Therefore, these results suggest that the use of IL-1Ra after chemotherapy would allow for greater doses of chemotherapeutic agents to be administratered.In summary, this work purified the recombinant murine IL-1Ra protein, studied the function of IL-1Ra in regeneration of bone marrow after chemotherapy, which suggest that IL-1Ra may be an important BM protector against chemotherapy induced myelosuppression.
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