| ObjectiveChemotherapy and radiotherapy are nowadays two routine therapies in tumor treatment, showing high efficiency in killing tumor cells. However, chemotherapy and radiotherapy meanwhile result in heavy toxic effects on normal tissues, especially rapidly proliferating ones (e.g., bone marrow, follicles). Among these side effects, leukopenia, induced by bone marrow inhibition, is one common and threatening complication, as a result of which the process of chemotherapy or radiotherapy is often interrupted and fails, and sometimes causes heavy infections or even deaths. Recently, it is becoming a hot topic that chemotherapy or radiotherapy is tried to combine with some types of herbs or their extracts to reduce side effects.Garlic oil (GO), a commercially available function food in many countries, is the product of garlic prepared by steam distillation, and accounts for 0.2%-0.5% of the garlic contents, including more than 30 organosulfur compounds such as DAS and DADS. GO has proved to possess the capacity of anti-oxidative damage. Previous studies in our laboratory also showed that GO can increase the number of blood leukocytes in mice treated with CP, the lack of which is just the side effect of chemotherapy or radiotherapy. Therefore, we investigated whether GO could antagonize leukopenia caused by chemotherapy or radiotherapy without weakening their anti-tumor effect.Methods1. Model H22 tumor-bearing mice and animal treatmentMouse Hepatoma 22 (H22) tumor cells were passed in the peritoneal cavities of Kun-Ming mice obtained from Medicine institute of Shandong Medical Academy of Science (Shandong, China). H22 ascites tumor cells (about 3×107/ml) were inoculated subcutaneously at the right axilla of mice.In part I experiment, sixty tumor-bearing mice were randomized into four groups with 15 in each:Tumor Model group (Model), CP group (CP), CP+GO group 1 (CP+GO 1) and CP+GO group 2 (CP+GO 2). One day after inoculation, CP+GO 1 and CP+GO 2 were orally administrated with 25 mg/kg and 50 mg/kg GO combined with 50 mg/kg CP i.p. for 13 days, while CP group were only treated with 50 mg/kg CP. Model group were orally given the same volume of vehicle (corn oil) as CP+GO 1 and CP+GO 2 group. All procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Animals.In partⅡexperiment, sixty tumor-bearing mice were randomized into five groups with 12 in each:Tumor Model group (Model), radiotherapy group, GO1 group (25 mg/kg GO), GO2 group (50 mg/kg GO) and GO3 group (100 mg/kg GO). One day after inoculation, GO 1, GO 2 and GO 3 group were orally administrated with GO combined with single radiation of 500 rad, while radiotherapy group was only treated with single radiation of 700rad. Model group were orally given the same volume of vehicle (corn oil) as GO group. All procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Animals.2. Experimental indicesAt the end of the experiment, mice were sacrificed by cervical dislocation. Blood was sampled from ophthalmic veins into Eppendorf tube coated with EDTA2K as anticoagulant. Then tumor mass and spleens were excised and weighted. Sternum and two femurs were removed for obtaining bone marrow. Leukocytes, erythrocytes and platelets were counted using CA800 hematocytometer. Spleen index, spleen nodes count, bone marrow nucleated cells count, DNA content of bone marrow and micronucleus frequency were obtained.Data were expressed as mean±SD and analyzed using one-way ANOVA, followed by Dunnett post hoc test. Differences were considered to be statistically significant at p <0.05 level (two-sided). SPSS 16.0 was used for statistical analysis.Results1. Effects of GO on leukopenia induced by chemotherapy1.1. Body weight and antitumor effectNo significant abnormality in body weight was detected in Model group. However, the growth of CP group was markedly decreased after receiving 13 days'i.p. administration of CP. Tumor weight in CP group was reduced by 60.5% in comparison with Model group. At the end point of the experiment, the body weight of CP+GO 1 and CP+GO 2 was slightly higher than that of CP group. Meanwhile, CP+GO 1 and CP+GO 2 caused 58.1% and 62.0% tumor inhibition rate, respectively. There is no significant difference among CP, CP+GO 1 and CP+GO 2 in tumor inhibition rate (p>0.05).1.2. Peripheral blood profileCyclophosphamide significantly decreased the number of leukocytes in CP group by 65.9%(p<0.05, vs. Model group), but not the numbers of erythrocytes and platelets (p>0.05). However, statistical differences in leukocytes were obvious between CP group and CP+GO 1, CP+GO 2 group (p<0.05). Erythrocytes and platelets were not changed in CP+GO 1 and CP+GO 2.1.3. Spleen weight, spleen histology, and numbers of spleen nodesCP significantly reduced spleen index by 34.8%(p<0.05, vs. Model). However, combining GO with CP prevented the depression in spleen index (p<0.05, GO+CP 2 vs. CP). Histology examination was carried out at the same time, and the similar results were found in spleen nodes count. The number of spleen nodes in CP+GO2 was approximately 166% higher than that in CP group.1.4. Bone marrow micronucleus, nucleated marrow cell, and the content of DNABone marrow nucleated cells (BMNC) were counted to investigate the effect of GO on bone marrow hemopoietic function. Cyclophosphamide severely decreased the amount of BMNC by 64.3%(p<0.05, CP vs. Model), whereas CP+GO1 and CP+GO2 reversed the inhibitory effects of CP on BMNC (p<0.05). DNA content of bone marrow cells was examined to further verify the result shown by BMNC, and the same result was obtained.The frequency of micronucleated PCE in CP group was higher than that in Model group (p<0.05). The frequencies in CP+GO1 and CP+GO2 were lowered compared with CP group (p<0.05), but were not recovered to the level of Model group (p<0.05).2. Effects of GO on leukopenia induced by radiotherapy2.1. Body weight and antitumor effectThe growth of radiotherapy group was markedly decreased. Tumor weight in radiotherapy group was reduced by 68.7% in comparison with Model group. Meanwhile, GO 1, GO 2 and GO3 caused 69.0%,70.2% and 67.8% tumor inhibition rate, respectively. There is no significant difference among radiotherapy group, GO 1, GO 2 and GO3 in tumor inhibition rate (p>0.05).2.2. Peripheral blood profileRadiotherapy significantly decreased the number of leukocytes in by 75.0%(p<0.05, vs. Model group), but not the numbers of erythrocytes and platelets (p>0.05). Statistical differences in leukocytes were obvious between radiotherapy group and GO2, GO3 group (p<0.05).2.3. Spleen weight, spleen histology, and numbers of spleen nodesRadiotherapy significantly reduced spleen index by 72.7%(p<0.05, vs. Model). Combining GO with radiotherapy prevented the depression in spleen index (p<0.05, GO 3 vs. CP). Histology examination was carried out at the same time, and the similar results were found in spleen nodes count.2.4. Bone marrow micronucleus and the content of DNA Radiotherapy severely decreased the amount of the DNA content of bone marrow by 78.7%(p<0.05, vs. Model), whereas GO2 and GO3 reversed the inhibitory effects of radiotherapy on DNA content (p<0.05). The frequency of micronucleated PCE in radiotherapy group was higher than that in Model group (p<0.05). The frequencies in GO2 and GO3 group were lowered compared with radiotherapy group (p<0.05), but were not recovered to the level of Model group (p<0.05).Conclusions1. Garlic oil is able to antagonize leukopenia on tumor-bearing mice induced by chemo-therapy and radiotherapy without weakening their antitumor effect.2. Garlic oil alleviates genotoxicity induced by chemotherapy or radiotherapy, and its antioxidative effect may be the principle mechanism, which is responsible for its antagonizing leukopenia.3. Observed from the experiment, effective dose range of Garlic oil is 50～100 mg/kg bw (oral). The data will help to design dose for following experiments and clinical application researches. |
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