| ObjectiveBenzene is a colorless or light yellow transparent oily liquid with aromatic odor which is an important raw material for production and organic solvent,and often used in pharmaceutical,synthetic rubber plastics,detergent,dye synthesis and paint,viscose and other processes.Benzene is regarded as a common occupational poison and pollutants in the environment.Benzene is a recognized carcinogen and was identified as an environmental carcinogen by the International Agency for Research on Cancer(IARC)in 1982.In China,chronic benzene poisoning has been the leading cause of chronic occupational poisoning for many years.Benzene pollution has become an urgent public health problem.Chronic benzene poisoning mainly damage to the hematopoietic system,Haematological diseases,such as cytopenia,aplastic anaemia,leukopenia and leukaemia are associated with benzene exposure,and the early symptom is characterized by leukopenia.Benzene poisoning has no specific antidote.At present,the treatment of benzene poisoning mainly adopts symptomatic and supportive therapy,correcting anemia,raising white blood cells and anti-tumor chemotherapy drugs,but long-term use can cause bone pain,fever,fatigue,and even stimulate the growth of malignant cells and a series of side effects.Therefore,it is very important to find an intervention drug to prevent benzene poisoning.Allicin is rapidly metabolized at room temperature to produce a variety of organic sulfur compounds with biological effects,such as diallyl-thio-(DAS),-diallyl dithio-(DADS),-diallyl trisulfide(DATS)and other organic sulfur compounds.Whether DAS can effectively resist benzene-induced leukopenia in peripheral blood of mice has not been reported.Therefore,in this experiment,DAS was selected as the intervention drug,and the model of peripheral leukopenia was established by oral exposure to benzene.Two-part experiments were conducted to try to confirm whether DAS has antagonistic effect on peripheral blood leukopenia induced by benzene in mice?What is the antagonistic mechanism?In the first part of the experiment,90 male ICR mice were randomly divided into blank control group(n?15),benzene model group(n=15),DAS low,middle and high dose intervention groups(n=15)and DAS control group(n=15).The low,middle and high dose of benzene+DAS intervention group and DAS control group were given the same volume of corn oil,respectively.The other two groups of DAS,of 160mg/kg were given the same volume of corn oil.After 2 hours,the animals in the benzene model group and DAS intervention group were given benzene(1.3g/kg.bw)corn oil suspension orally,and the other two groups were given the same volume of corn oil.Once a day for 4 weeks.On the 14th and 29th day,blood was taken from the medial canthus of the eye,and anticoagulant blood was collected to detect blood routine.29 days later,the animals were killed,spleen and thymus were taken,weighed and organ coefficient was calculated,and paraffin sections of spleen and thymus were prepared for pathological examination.It was found that:1.examination of blood routineCompared with the blank control group,the peripheral blood leukocyte count of mice in the benzene model group decreased by 68.99%on the 14th day after exposure,and The difference of significant was statistically(P<0.01),in which lymphocytes and monocytes decreased by 71.72%and 53.19%respectively(P<0.01).The difference of significant was statistically(P<0.01).Compared with the 14th day,the lymphocytes,monocytes and neutrophils in the benzene model group decreased by 83.00%,81.03%and 89.37%respectively on the 29th day,and The difference of significant was statistically(P<0.01 or 0.05).Red blood cells and hemoglobin decreased by 20.84%and 19.25%respectively,and The difference of significant was statistically(P<0.01 or 0.05).During the intervention period,there were significant differences in the mean value of peripheral blood cells among all groups(P<0.01).Compared with the benzene model group,the white blood cell count in the,DAS high dose intervention group increased by 270.30%on the 14th day after intervention,and The difference of significant was statistically(P<0.01).Among them,monocytes and lymphocytes increased by 136.36%and 260.00%respectively,and The difference of significant was statistically(P<0.0lor 0.05).Compared with the 14th day of the intervention,the effect of the intervention on the 29th day was obvious.The white blood cells of the low,middle and high dose groups of DAS increased by 95.76%,167.80%and 453.39%,respectively,and The difference of significant was statistically(P<0.01).Among them,the lymphocytes of the low,middle and high dose groups of DAS increased by 114.67%,157.33%and 553.33%,respectively,and The difference of significant was statistically.The monocytes in the middle and high dose groups of DAS increased by 154.55%and 472.73%respectively,and The difference of significant was statistically(P<0.0lor 0.05),DAS.The middle and high dose neutrophils increased by 281.82%and 713.64%respectively,The difference of significant was statistically(P<0.0lor 0.05),DAS).The difference of significant was statistically(P<0.01 or 0.05).2.Pathological examinationNaked eye observation of the blank control group spleen capsule intact,smooth surface,crisp and soft,dark red color,showing a long oval shape.In the benzene model group,the spleen color became lighter,showed a short oval shape,and the volume became smaller.Compared with the benzene model group,the spleen color and size were recovered in different degrees in the low,middle and high dose DAS intervention groups.In the blank control group,the thymus capsule was intact,soft,milky white,short and thick.In the benzene poisoning model group,the thymus became lighter,smaller and flattened.Compared with benzene model group,the color and size of thymus in low,middle and high dose DAS intervention groups were improved in different degrees.No obvious pathological changes were found in the organs of the DAS positive control group.Under microscope,the normal spleen capsule was pink,the red pulp was crimson,and the white pulp was dense purplish blue,which were splenic nodules.The boundary between red pulp and white pulp is clear.Compared with the blank control group,the pathological tissue color of spleen in the benzene model group became lighter,the red pulp was light pink and the number was more,the white pulp was loose and the number of lymphocytes was sparse and less,and the boundary between red and white pulp was blurred.Compared with the benzene model group,the color and number of white pulp and red pulp were improved in different degrees in the low,middle and high dose groups of DAS.Compared with the blank group,there was no obvious visible lesion in the DAS control group.Compared with the blank control group,the white pulp area ratio of the benzene model group decreased significantly(P<0.01),and compared with the benzene model group,the white pulp area ratio of the low,middle and high dose groups of DAS increased significantly(P<0.01or 0.05).The thin layer capsule on the surface of normal thymus was pink,the cortical thymocytes were dense and dark purple-blue,the medulla was lighter,and the boundary between cortex and medulla was clear.Compared with the blank control group,in the benzene model group,the thymic cortex became thinner,the thymocyte sparse color became lighter,and the boundary between cortex and medulla was blurred.Compared with the benzene model group,the cortical thickness and color were improved in the low,middle and high dose groups of DAS,and the number of thymocytes increased in different degrees.Compared with the blank control group,the cortical area of the benzene model group was significantly decreased(P<0 01),and the thymic cortical area of the low,middle and high dose groups of DAS was significantly higher than that of the benzene model group(P<0.01,0.05).The cortical area of thymus decreased in DAS positive control group(P<0 01).3.Determination of organ coefficientThere were significant differences in spleen,thymus weight and organ coefficient among all groups,and the spleen weight,thymus weight,spleen coefficient and thymus coefficient in the benzene model group were significantly lower than those in the blank control group(P<0.01).Compared with the benzene model group,the spleen weight and spleen coefficient in the middle and high dose DAS intervention groups were significantly higher than those in the benzene model group(P<0.01,0.05).The thymus weight and thymus coefficient in the high dose),DAS intervention group were significantly higher than those in the benzene model group(P<0.01r 0.05mol/L).In the second part of the experiment,rats were selected to establish a leukopenia model to further explore the effect and mechanism of DAS on leukopenia.The animal grouping and treatment methods were the same as those in chapter 1.At the end of the 4th week,the rats were anesthetized by intraperitoneal injection of pentobarbital(50mg/kg.bw).The blood was taken from the abdominal aorta,and the thymus,spleen and bilateral femurs were taken for blood routine,bone marrow leukocyte count,organ coefficient,bone marrow polychromatic erythrocyte micronucleus,y H2AX in BMCs and PBLs,oxidation and antioxidation indexes and related protein expression.It was found that:1.examination of blood routineCompared with the blank control group,the peripheral blood leukocyte count in the benzene model group decreased by 59.76%(P<0.01),in which the proportion of monocytes and neutrophils decreased by 69.90%and 73.97%respectively(P<0.01).The proportion of lymphocytes increased by 32.28%(P<0.01),and red blood cells,platelets and hemoglobin decreased by 2.52%,57.74%and 5.02%respectively(P>0.05).Compared with the benzene model group,the white blood cell count in the middle and high dose DAS intervention groups increased by 164.86%and 132.97%(P<0.01),and the proportion of monocytes and neutrophils in the low,middle and high dose DAS intervention groups increased(P<0.01),while the proportion of lymphocytes decreased(P<0.01).2.examination of bone marrow leukocyte countThere was significant difference in the mean value of bone marrow leukocytes among different groups.Compared with the blank control group,the bone marrow leukocyte count of rats in the benzene model group decreased by 33.11%(P<0.01),in which the proportion of monocytes and neutrophils decreased by 39.61%and 28.64%respectively,and the proportion of lymphocytes increased by 15.79%.Compared with the benzene model group,the white blood cell count in the low,middle and high dose DAS intervention groups increased significantly by 137.92%,273.56%and 378.66%(P<0.01),and the proportion of neutrophils in the low,middle and high dose DAS intervention groups all increased(P<0.01).3.examination of micronucleus rate of bone marrow erythrocytesThere were significant differences(P<0.05)in MPCE number and micronucleus rate(‰)among different groups.Compared with the blank control group,the MPCE number and micronucleus rate(‰)in the benzene model group increased(P<0.05),and the MPCE number and micronucleus rate in each dose of DAS intervention group decreased compared with the benzene model group.There was no significant difference(P>0.05)in MPCE number and micronucleus rate(‰)between the blank control group and the DAS control group(P<0.05).4.Effects of DAS and benzene on the expression of ? H2AX in BMCs and PBLs of ratsThe target cell group with active state was selected after the parameters were adjusted by flow cytometry.See figure 2(a),figure 2(b).The average fluorescence intensity and relative fluorescence intensity of y H2AX in BMCs of rats in each group were significantly different(P<0.05).Compared with the blank control group,the average fluorescence intensity and relative fluorescence intensity of y-H2AX in the benzene model group increased(P<0.05),and the average fluorescence intensity and relative fluorescence intensity of y-H2AX in the middle and high dose DAS intervention groups decreased significantly(P<0.05)compared with the benzene model group.The average fluorescence intensity and relative fluorescence intensity of y-H2AX in PBLs of rats in each group were significantly different(P<0.05).Compared with the blank control group,the average fluorescence intensity and relative fluorescence intensity of y-H2AX in the benzene model group increased(P<0.05),and the average fluorescence intensity and relative fluorescence intensity of ?-H2AX in the low,middle and high dose DAS intervention groups decreased(P<0.05)compared with the benzene model group.5.Effects of DAS and benzene on serum MDA concentration,T-SOD activity,GSH content,GSH/GSSG ratio and T-AOC levelThere was significant difference in the mean value of serum MDA concentration among different groups.Compared with the blank control group,the concentration of MDAin the benzene model group increased(P<0.01),and the concentration of MDA in the high dose DAS intervention group decreased(P<0.01)compared with the benzene model group.There were significant differences in serum T-SOD activity among different groups.Compared with the blank control group,the activity of T-SOD in the benzene model group decreased(P<0.01),and the activity of T-SOD in the middle and high dose DAS intervention groups increased compared with the benzene model group(P<001or 0.05).There were significant differences in serum GSH content and GSH/GSSG ratio among different groups.Compared with the blank control group,the GSH and GSH/GSSG in the benzene model group decreased(P<0.01),and the GSH and GSH/GSSG in the high dose DAS intervention group increased compared with the benzene model group(P<0.01or 0.05).There was significant difference in the mean value of T-AOC in each group.Compared with the blank control group,the T-AOC in the benzene model group decreased(P<0.05),and compared with the benzene model group,the T-AOC in the low,middle and high dose DAS intervention groups increased(P<0.01 or 0.05).6.Effects of DAS and benzene on MDA concentration,T-SOD activity,GSH content,GSH/GSSG ratio and T-AOC level in spleen homogenateThere was significant difference in the mean concentration of MDA in spleen homogenate among different groups.Compared with the blank control group,the concentration of MDA in the benzene model group increased(P<0.05),and the concentration of MDA in the low,middle and high dose DAS intervention groups decreased compared with the benzene model group(P<0.01).There was significant difference in the mean value of T-SOD activity in spleen homogenate among different groups.Compared with the blank control group,the activity of T-SOD in the benzene model group decreased,and the activity of T-SOD in the high dose DAS intervention group increased compared with the benzene model group(P<0.05).There were significant differences in the content of GSH and the ratio of GSH/GSSG in spleen among different groups.Compared with the blank control group,the ratio of GSH and GSH/GSSG in the benzene model group decreased(P<0.01 r 0.05),and the ratio of GSH and GSH/GSSG in the low,middle and high dose DAS intervention groups increased compared with the benzene model group(P<0.01 0.05).There was significant difference in the mean value of T-AOC in spleen among different groups.Compared with the blank control group,the T-AOC in the benzene model group decreased(P<0.05),and the T-AOC in the low,middle and high dose DAS intervention groups increased compared with the benzene model group(P<0.05).7.Effects of DAS and benzene on MDA concentration,T-SOD activity,GSH content,GSH/GSSG ratio and T-AOC level in BMCsThere was significant difference in the mean concentration of MDA in bone marrow cells among different groups.Compared with the blank control group,the concentration of MDA in the benzene model group increased(P<0.05),and the concentration of MDA in the low,middle and high dose DAS intervention groups decreased compared with the benzene model group(P<0.01).Compared to the blank control group,the activity of T-SOD in the benzene model group decreased,and the activity of T-SOD in the low dose DAS intervention group increased compared to the benzene model group(P<0.05).Compared i the model group with the blank control group,the ratio of GSH and GSH/GSSG decreased,while the ratio of GSH and GSH/GSSG in the high dose DAS intervention group increased compared with the benzene model group(P<0.05).Compared with the model group with the blank control group,the capacity of T-AOC was decreased,while the T-AOC in the low,middle and high dose DAS intervention groups increased compared to the benzene model group.8.Effects of DAS and benzene on MDA concentration,T-SOD activity,GSH content,GSH/GSSG ratio and TAOC level in PBMCsThere was significant difference in the mean concentration of MDA in peripheral mononuclear cells among different groups.Compared with the blank control group,the concentration of MDA in the benzene model group increased(P<0.01),and the concentration of MDA in the low,middle and high dose DAS intervention groups decreased compared with the benzene model group(P<0.01).Compared with the blank control group,the activity of T-SOD in the benzene model group decreased,while the activity of T-OD in the low,middle and high dose DAS intervention groups increased compared with the benzene model group.Compared with the blank control group,the ratio of GSH and GSH/GSSG in the benzene model group decreased,while the ratio of GSH and GSH/GSSG in the middle and high dose DAS intervention groups increased compared with the benzene model group(P<0.01 or 0.05).Compared with the blank control group,the T-AOC in the benzene model group decreased(P<0.05),and the T-AOC in the low,middle and high dose DAS intervention groups increased compared with the benzene model group.9.expression of oxidative stress-related proteins in bone marrow cellsCompared with the blank control group,Keapl and Nrf2 in the benzene model group decreased by 50.21%and 51.09%,respectively.The downstream protein ?-GCSC,HO-1,SOD-1 decreased by 38.12%,50.09%and 65.12%,respectively.Compared with the benzene model group,the expression level of histone was improved after intervention with each dose of DAS.Conclusion1.Continuous intragastric administration of benzene for four weeks led to peripheral blood leukopenia in the benzene model group,which can be judged that the animal model of peripheral blood leukopenia was established successfully.2.Each dose of DAS can increase the number of peripheral blood leukocytes and bone marrow leukocytes,which proves that it has the effect of antagonizing blood toxicity and bone marrow toxicity.3.DAS can improve the level of oxidative stress in animal blood system by activating antioxidant enzymes and increasing the level of non-enzymatic antioxidants,thus reducing lipid peroxidation,which is related to activating Keapl/Nrf2 pathway and up-regulating the expression of downstream protein-related proteins NQO1,?-GCSc and SOD-1.4.Benzene-induced chromosome breakage is the main mode of genotoxicity in bone marrow.Benzene-induced DNA damage can lead to changes in(HSC)of hematopoietic stem cells,resulting in leukemic clones.DAS can relieve the inhibitory effect of benzene on bone marrow by regulating bone marrow genotoxicity,thus further antagonizing the decrease of peripheral blood cells induced by benzene.The activation of Keap1/Nrf2 pathway may be an important mechanism for DAS to regulate the injury of bone marrow nucleated cells induced by oxidative stress. |
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