| Background and ObjectivesThrombocytopenia, a clinically frequent dose-limited toxicity caused by normal dose of radiotherapy and chemotherapy, can result in radiotherapy and chemotherapy dose reductions or treatment delays, and even therapy termination. Thrombocytopenia is also common in patients of immune thrombocytopenia (ITP), myelodysplastic syndrome (MDS) and post-hematopoietic stem cell transplantation (post-HSCT). Severe thrombocytopenia can lead to death caused by fatal bleeding. Therefore, thrombocytopenia affects clinical efficacy, patient survival time and increases the medical costs. Currently, thrombocytopenia can be managed by platelet transfusion and growth factors injection. The growth factors include recombinant human thrombopoietin (rhTPO), recombinant human interleukin-11 (rhIL-11), eltrombopag and Romiplostim (the latter two drugs are TPO receptor agonists). Currently, only rhTPO and rhIL-11 have been approved for the treatment of tumor therapy-induced thrombocytopenia by the China Food and Drug Administration (CFDA) and both of them are expensive. In addition, repeated platelet transfusions increases the risk of generation of platelet antibody, resulting in the ineffectiveness of platelet transfusion. Consequently, plenty of scientists focused on inventing or discovering new drugs that can promote thrombopoiesis.Melatonin (MLT), with chemical name of 5-methoxy-N-acetyltryptamine, is one of the primary factors regulating nervous system and endocrine system. Italian scholars had indicated that melatonin can reduce the incidence of thrombocytopenia in solid-tumor patients. However, so far the accurate effect and potential molecular mechanism of melatonin on megakaryopiesis or thrombopiesis has not been reported. Our previous studies have demonstrated that serotonin promotes hematopoiesis and megakaryopoiesis. MLT is acetylated product of 5-HT. Therefore, we hypothesized that MLT may affect the formation of megakaryocytes proliferation and platelets formation, which can explain why melatonin has a therapeutic effect in thrombocytopenia. The purpose of this study is to verify our hypotheses through a murine modle of radiation-induced thrombocytopenia in vivo and a megakaryoblastic cell line, CHRF-288-11 in vitro, respectively.Contents and methods1. Radiation-induced thrombocytopenia in mice and outcome measuresA total of 24 male Balb/c mice (6-8 weeks old) were randomly divided into four equal groups including normal group, control group, MLT group (10mg/kg/d) and TPO group (1μg/kg/d). In addition to the normal group, mice in another three groups received 4-Gy irradiation for 3 minites to establish radiation-induced myelosuppression with thrombocytopenic model. Then, intraperitoneal injections of the corresponding materials (control group:normal saline, MLT group:melatonin of lOmg/kg/d, TPO group:TPO of 1 μg/kg/d) were performed for 21 days. Peripheral red blood cell (RBCs), white blood cells (WBC) and blood platelet counts were recorded on days 0,7,14 and 21, respectively. Mice were euthanasized on day 21 and bone marrow samples were harvested and frozen in cryomolds with 5-mm sections. Slides were stained using the Wright-Giemsa staining method and hematoxylin-eosin staining method. On days 0,7,14 and 21, measurement of body weight was performed in each group. On day 21, organs of liver, kindney and spleen in each group were weighted. After euthanasizing mice on day 21, we extracted bone marrow cells from bilateral femur of the mice. These cells were cultured using the methyl cellulose method to analyze CFU-GM, BFU/CFU-E and CFU-GEMM. CFU-MK and CFU-F were cultured using plasma clot colony method and adherent method, respectively.2. Biological characteristics of CHRF-288-11 cell line (CHRF)The slides were stained using the Wright-Giemsa staining method. The cultured cells were counted and the growth curve of CHRF was drew to calculate the population double time. Flow cytometry were used to evaluate the expression level of different CD markers on CHRF cells. The cytogenetical characterization of CHRF cells were analyzed using the G-banding method.3. The effect of melatonin on CHRF proliferation and apoptosisCCK-8 and Typan Blue Exclusion methods were used to detect effect of different concentration of melatonin (0,20,50,100,200,500nM) on CHRF proliferation. Apoptosis was induced by cytokine and serum depletion. Melatonin (200nM) or TPO (50ng/ml) was added to this apoptotic culture, respectively. After 72 hours incubation, cells were collected for flow cytometry analysis. The population of apoptotic cells was measured using AnnexinV-FITC/PI, JC-1 and active caspase-3-PE.4. Detection for the expression level of melatonin receptors on CHRF surfaceReal-time Polymerase Chain Reaction (RT-PCR) and Western blotting were used to detect the expression level of melatonin receptors (MT1 and MT2) on CHRF cells surface.5. Potential molecular mechanisms for the effect of melatonin on megkaryopoiesisWestern blotting method was used to test the effect of melatonin on PI3K/AKT signal pathway and melatonin receptors were detected to confirm whether the receptors involved in the processes.6. Statistical analysisStatistical analysis was performed using the SPSS 13.0 statitical software (Chicago, Illinois, USA). Initially, tests of normality were conducted. If data were in accordance with normal distribution, they were expressed as mean and standard deviation. One-Way ANOVA is adopted to compare the means among different groups. Levene test was used to detect the homogenety of variance. If the variance is homogenety, Bonferroni method was used for multiple comparisons, otherwise Welch method was used and Dunnett’s T3 method was applied for multiple comparisons. Independent t-test was applied to comparing the difference between two independent samples. A P value of ≤0.05 was considered as significant.Results1、Effect of melatonin on the hematopoiesis of irradiated mice.(1) Peripheral blood cell counts① Red blood cell counts (RBCs)Postradiation, red blood cell counts in control, MLT, TPO groups reduced, which showed lowest levels on the 7th day. Then it gradually recoverred to nearly normal value. The mean RBC counts on the day 7 showed statistically significant (F= 4.212, P= 4.212) among the three groups. Further multiple comparisons revealed that mice in TPO group had significantly higher RBCs than those in control group [(7.950 ± 0.596)×1012/L versus (6.717±0.512)×1012/L, n= 6, P= 0.043]. Similarly, on the day 14, significant differences were identified regarding RBCs among three groups (F = 5.349, P= 0.018). Outomes of multiple comparisons showed that no significant difference was found between MLT group and control group. Howver, RBCs level in the TPO group was significantly higher than control group [(9.350 ± 0.880)×1012/L versus (8.067±0.476)×1012/L, n= 6, P= 0.022]. On day 21, statistical difference was also found among the groups(F=14.998,P=0.000).Results of multiple comparisons shpwed that the average RBC levels in MLT and TPO groups were both significantly higher than those in control group[MLT group versus control group: (10.400±0.894)×1012/L versus(8.800±0.469)×1012/L,n=6,P=0.004；TPO group versus control group:(10.917±0.665)×1012/L versus(8.800±0.469)×1012/L,n=6, P=0.000].Based on the above outcomes,we concluded that MLT can enhance the recoveryof RBC in a murine model of radiation-induced myelosuppression.② White blood cells counts(WBCs)After radiation,the peripheral WBCs reduced,with lowest level on the seventh day postradiation and gradually baek to normal value.On days 7 and 14,no significant count differences were identified among the three groups(Day 7,F= 3.369,P=0.062,Day 14,F=2.830,P=0.091).However,significant difference were found among the three groups on day 21(F=7.290,P=0.006).Multiple comparisons showed that WBCs in MLT group and TPO group were significantly higher than those in control group[Day 21,MLT group versus control group:(7.250 ±1.725)×109/L versus(4.750±0.758)×109/L,n=6,P=0.040；TPO group versus control group:(8.000±1.897)×109/L versus(4.750±0.758)×109/L,n=6,P= 0.007].In addition,no significant differenee was found between MLT group and TPO group.Therefore,based on the above outcmes,we concluded that MLT,with similar efficacy as TPO,can promote the recovery of peripheral WBCs.③ Platelets countsSimilar to RBCs and WBCs,peripheral blood platelet counts in mice after radiation declined,with minimum level on day 7 days and gradually back to normal. No significant differences were found regarding platelet counts among the three groups at initial day and the 7th day(Day 0:F=0.638,P=O.542；Day 7:F=1.353,P =0.288).However,significant differenves Were found among the three groups on day 14 (F= 7.820, P= 0.005). Outcomes of the multiple comparisons showd that only TPO group had a statistical higher level of platelet counts than that in control group [385.000 ± 55.045)×109/L versus (261.667 ± 53.541) ×109/L, n= 6, P= 0.004]and no significant difference was found between MLT group and control group (P= 0.200). On day 21, significant differences were identified among the groups (F= 8.005, P= 0.004) and both MLT group and TPO group had significantly higher values of platelet counts than those in control group [MLT group versus control group:(509.167 ± 108.509)×09/L versus (336.667 ± 84.951)×109/L, n= 6, P= 0.013; TPO group versus control group:(520.000 ± 69.282) ×109/L versus (336.667 ± 84.951) ×109/L, n = 6, P= 0.008]. But platelet counts did not differ significantly between MLT group and TPO group. Therefore, we concluded that MLT possesses similar efficacy as TPO, which can promote the recovery of peripheral platelet counts secondary to radiation in mice.(2) Body weight and organs weight of mice① Body weightSimilar to peripheral blood cell counts, mice body weight began to reduce after radiation. By day 7, significant differences were identified among the four groups (F = 5.532, P= 0.006). By day 14, mice body weight gradually increased in all of the groups, however, mice in MLT group still had a statistically lower weight than those in the normal group [(26.267 ± 0.739) g versus (27.833 ± 0.427) g, n= 6, P= 0.001]. By day 21, significant differences were also existed among the four groups (F= 3.507, P= 0.034). Results of the LSD method showed a statistically lower body weight in MLT group than those in normal group [(26.850 ± 0.907) g versus (28.000 ± 0.735) g, n= 6, P= 0.018]. However, the difference did not reveal statistical significance when using the Bonferroni method for multiple comparisons (P= 0.111).② Organs (liver, kindney and spleen) weight in each groupOn day 21 postradiation, all mice in the four groups were euthanasized for detection of organ weight. With respect to liver weight, significant differences were found among the groups (F= 11.003, P= 0.000). Multiple comparisons showed that mice in MLT group had a significantly lower liver weight than those in control group [(1.260 ± 0.044) g versus (1.450 ± 0.092) g, n= 6, P= 0.000]. Liskwise, significant differences were also found regarding the kindney weight among the four groups (F= 7.552, P= 0.001). Results of multiple comparisons using the LSD method showed a significant difference between TPO group and control group (n= 6, P= 0.011). However, this difference was not statistical significance when using the Bonferroni method for multiple comparisons (P= 0.068). Multiple comparisons using the Bonferroni method revealed that mice in MLT group had a significantly lower kindney weight than those in TPO group [(0.449 ± 0.025) g versus (0.490 ± 0.016) g, n= 6, P= 0.001]. Similarly, statistical differences were found among the four groups regarding the spleen weight. Multiple comparisons using the LSD method revealed that the average spleen weight of mice in TPO group were significantly higher than whose in MLT group and normal group [TPO group versus MLT group:(0.142 ± 0.027) g versus (0.109 ± 0.011) g, n= 6, P= 0.012; TPO group versus normal group: (0.142 ± 0.027) g versus (0.113 ± 0.018) g, n= 6, P= 0.025]. Variance existed using different methods for analysis, LSD is more sensitive while Bonferroni is more rigid. Based on the above outcomes, we concluded no obvious protection effect of MLT on mice organs.(3) Effect of MLT on the formation of bone marrow colonyCFU-MK was anzlyzed using the plasma clot colony culture method. Results showed that MLT can significantly promote the formation of CFU-MK [MLT versus Control:(33.000 ± 0.018) versus (16.170 ± 2.714), n= 6, P< 0.001]. Other colonies including CFU-GM, BFU/CFU-E and CFU-GEMM were analyzed using the methyl cellulose method. Compared with control group, MLT can significantly promote the colony formations of CFU-GM [(58.330 ± 6.743) versus (41.170 ± 4.834), n= 6, P< 0.01], BFU-E [(31.330 ± 7.840) versus (12.000 ± 4.243), n= 6, P< 0.001] and CFU-GEMM [(20.830 ± 2.041) versus (10.000 ± 1.897), n= 6, P< 0.001], indicating that MLT can promote the recovery of granulocytes as well as erythron. CFU-F was analyzed using the adherent culture method. Results showed that no significant difference was found regarding the number of CFU-F between MLT group and TPO group, implying that MLT can provide similar protection effect on bone marrow mesenchymal cell as TPO does, significantly better than the control group does [MLT versus Control:(21.670 ±2.422) versus (11.670 ± 3.502), n= 6, P< 0.001; TPO versus Control:(20.830 ± 6.513) versus (11.670 ± 3.502), n=6, P< 0.001].(4) Pathological tests of bone morrow tissuesOn day 21, all mice in the four groups were euthanasized for pathological analysis. Pathological tests showed that the number of hematopoietic cells in the bone marrow tissue in the control group was markedly decreased when compared with that in the normal group. Additionally, the ratios of cell necorosis and apoptosis in control group were higher than those in normal group. The number of bone marrow cells, especially the number of megakaryocytes, in MLT and TPO groups is higher than that in control group. In MLT group, the number of megakaryocytes and its progenitor cells in bone marrow tissues was higher than those in control group. The recovery of megakaryocytes and its progenitor cells were better than granulocytes and erythrocytes.2> Biological characteristics of CHRF-288-11 cell line (CHRF)Wright-Giemsa staining showed that CHRF cells possessd large cell body and large cell nucleus as well. These cells showed irregular shapes with uneven mazarine dyeing cytoplasm. These comply with the characteristics of megakaryocytes. Cell surface molecules were analyzed by FCM. Outcomes of FCM showed that CHRF cells had high expressions of CD61:92.07%, CD41:99.04%, CD42:95.03%, which were in accordance with the specific molecular markers of megakaryocytes. These cells also had high expressions of CD13:99.20% and CD33:98.11%. HLA-DR was positive of 25.010%. These cells had negative expressions of CD3:6.310%, CD34: 2.540%, CD19:5.5%, CD38:2.760%. The chromosome karyotype was 95, XY, with abnormal numbers of multiple chromosomes. CHRF cell population doubling time was 58.88 hours.3、Effect of MLT on CHRF proliferation and apoptosis(1) MLT can promote the proliferation of CHRF cells, which relies on time and concentration. The maximum promotion effect concentration was 200 nM.(2) Anti-apoptotic effect of MLT on CHRF① Annexin V/PIThe percentage of early apoptotic cells did not differ statistically among the four groups (F= 1.714, P= 0.217). However, significant differences were identified regarding the percentage of late apoptotic cells among the four groups (F= 23.087, P = 0.000). Multiple comparisons using Bonferroni method showed that the percentages of late apoptotic cells in MLT and TPO groups were both significantly lower than that in control group [MLTgroup versus control group:(13.143 ± 2.510)% versus (26.105 ± 4.638)%, n= 4, P< 0.01; TPO group versus control group:(10.620 ± 3.145)% versus (26.105 ± 4.638)%, n= 4, P< 0.001]. With regard to the percentage of total apoptotic cells, significant differences were found among the four groups (F= 25.860, P= 0.000). Multiple comparisons revealed that that the percentages of total apoptotic cells in MLT and TPO groups were both significantly lower than that in control group [MLT group versus control group:(14.180 ± 2.492)% versus (27.930 ± 3.828)%, n= 4, P< 0.001; TPO group versus control group:(11.960 ± 3.661)% versus (27.930 ± 3.828)%, n= 4, P< 0.001].(2) Caspase-3 and JC-1The expression of active caspase-3 showed significant differences among the four groups (F= 107.428, P-0.000). Additionally, the expression of active caspase-3 in MLT group was significantly lower than that in control group [(22.833 ± 2.275)% versus (31.200 ± 2.022)%, n= 3, P=0.002]. However, no significant difference was identified between MLT group and TPO group [(22.833 ± 2.275)%versus (19.900 ± 1.179)%, n= 3, P= 0.252].JC-1 outcomes were similar to Caspase-3 outcomes. The proportion of JC-1 positive cells was significantly higher in control group. Furthermore, the proportion of JC-1 positive cells in MLT group was statistically lower than that in control group [(36.553 ±2.259)% versus (44.503 ± 3.033)%, n= 3, P= 0.048]. However, no significant difference was identified between the MLT group and TPO group [(36.553 ± 2.259)% versus (34.873 ± 2.671)%, n= 3, P> 0.05].4x Expressions of MLT recepitors in CHRF(1) Detection of MLT recepitors associated mRNA expressions by real-time PCRWe used independent t-tests to compare the expression levels of MT1-mRNA and MT2-mRNA in CHRF and L-02 cells, respectively. Result of MT1-mRNA revealed that the expression of MT1-mRNA in CHRF cells was significantly lower than that in L-02 cells [CHRF versus L-02:(0.000 ± 0.000) versus (0.824 ± 0.543), Levene test F= 6.550, P= 0.034, t=-3.390, P= 0.028] Result showed similar MT2-mRNA expression levels between the two cell lines [CHRF versus L-02:(0.216 ± 0.111) versus (0.583 ± 0.302), Levene test F= 3.206, P= 0.124, t=-2.282, P= 0.063].(2) Detection of MLT receptors proteins expressions by Western-BlottingOutcomes of the Western-Blotting were in accordance with Q-PCR outcomes that CHRF cells express MT2 receptor.5、 Effect of MLT on PI3K/AKT signal pathway(1) Effect of MLT on expressions of AKT proteinAfter treated by MLT, the level of p-Akt protein increased significantly than that in control group [MLT versus control:(69.370 ± 6.787)% versus (33.479 ± 6.056)%, n= 5, P= 0.000]. The level of p-Akt protein in group intervened by MLT with Wortmannin (100nM) (PI3K inhibitor) was statistically lower than that in MLT group [Melatonin + Wortmannin versus Melatonin:(42.700 ± 13.645)% versus (69.370 ± 6.787)%, n= 5, P= 0.002]. In addition, the level of p-Akt protein in Wortmannin group was significantly lower than that in MLT group [Wortmannin versus Melatonin: (25.767 ±7.734)% versus (69.370 ± 6.787)%, n= 5, P= 0.000]. However, no significant differences were identified regarding the level of p-Akt protein, neither between Wortmannin group and control group, nor between Wortmannin group and MLT with Wortmannin group (P> 0.05).(2) Effect of Luzindole (MLT receptor inhibitor) on expression of p-Akt proteinSignificant differences were found regarding the expressions of p-Akt protein among the four groups (Welch test, P= 0.000, Levene test, F= 5.987, P= 0.006). Outcomes of multiple comparisons using the Dunnett’s T3 mrthod showed that the proportion of p-Akt protein in MLT group was significantly higher than that in control group [Melatonin versus Control:(51.988 ± 10.090)% versus (14.277 ± 6.513)%, n= 5, P= 0.001]. The proportion of p-Akt protein in MLT with Luzindole (1μM) was statistically lower than that in MLT group [MLT + Luzindole versus MLT: (3.436 ± 1.113)% versus (51.988 ± 10.090)%, n= 5, P= 0.002]. However, no significant difference was found between MLT with Luzindole group and control group [MLT+Luzindole versus Control:(3.436 ± 1.113)% versus (14.277 ± 6.513)%, n= 5, P= 0.081]. The proportion of p-Akt protein in Luzindole group was significantly lower than that in MLT group [Luzindole versus MLT:(10.084 ± 4.779)% versus (51.988 ± 10.090)%, n= 5, P= 0.001]. Nonetheless, no significant differences were found regarding the proportion of p-Akt protein, neither between Luzidole group and control group (P= 0.804), nor between Luzidole group and MLT with Luzidole group (P= 0.139).Conclusions1. Melatonin can enhance hematopoietic recovery in a murine model of radiation-induced thrombocytopenia.2. CHRF have the morphological characteristics of the megakaryocyte and highly express the specific megakaryocytic CD markers:CD61, CD42 and CD42.3. Melatonin can enhance CHRF cell proliferation and has a similar anti-apoptotic effect as thrombopoietin on megakaryocytes.4. CHRF express MT2 receptor.5. The anti-apoptostic effect of melatonin is probably mediated via MT2 receptor with subsequent activation of the PI3-k/Akt signal transduction pathway. |
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