| Objective: The immune system is the most important barrier of the body,shouldering the functions of body defense,surveillance,stability,etc.Normal immune function is an important cornerstone of the body's health.Benzene is a toxic environmental pollutant that can not only affect the body's immune system but cause hematopoietic malignancies.Previous studies have shown that multiple pathways may be involved in this process,including oxidative stress,DNA damage,cell cycle regulation,and programmed cell death,but the specific mechanism of benzene-induced immunotoxicity has not been fully elucidated.This research intends to study the immunotoxicity of benzene exposure to peripheral blood mononuclear cell and spleen in mice from various levels of immune organs,immune cells and immune molecules by constructing a mouse model of benzene exposure,providing experimental research basis for further research on the immunotoxic effects and its mechanism of benzene exposure.Methods:1.Forty BALB/c mice aged 6-8 week without specific pathogens were randomly divided into 4 groups: control group(corn oil),low-dose group(50 mg/kg),medium-dose group(150 mg/kg)and high-dose group(500 mg/kg),with 10 mice in each group.The mice were orally administered once a day for 5 days a week in 4 consecutive weeks.The body weight changes of the mice were recorded weekly during the exposure period,and the indicators were tested 24 hours after the last exposure.2.The peripheral blood of mice was used for routine blood analysis.The levels of immunoglobulin G,interleukin-2 and interleukin-6 in plasma were determined by enzyme-linked immunosorbent assay.Mito SOX fluorescent dye combined with flow cytometry were used to determine mitochondrial ROS content in peripheral blood mononuclear cell.ATP assay kit and BCA protein assay kit were used to determine ATP content in peripheral blood mononuclear cell of mice.Thiobarbituric acid method was used to determine malondialdehyde content.The activity of glutathione peroxidase was determined by dithio-bis-nitrobenzoic acid method.And the activity of total superoxide dismutase was determined by hydroxylamine method.3.Aseptically dissecting the mice,extracting the spleen and calculating the spleen coefficient,and performing pathological examination on the spleen of the mice.The TUNEL test kit was used to detect the apoptosis of the spleen cells.The Cell Counting Kit-8 was used to measure the proliferative capacity of T and B lymphocytes in the spleens.4.Isotope-labeled relative and absolute quantitative techniques were used to compare the spleen protein expression differences between the spleen of mice in high-dose group and control group.Bioinformatics analysis of differential expressed proteins was performed using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes database to identify key proteins.The crucial differential expressed proteins and their corresponding mRNA expression levels were verified using the Wes fully automated protein quantification system and quantitative Real-time PCR.Results:1.During the experiment,there was no case of direct death of the mice,but the growth of body weight of the mice in low,medium and high-dose groups was inhibited by the exposure.The body weights of the mice in high,medium and low-dose groups were significantly lower than those in control group since the first,second and third weeks of exposure period respectively.At the end of the exposure,the peripheral blood routine counts of the mice in low,medium and high-dose groups were significantly affected.The peripheral blood leukocytes,lymphocytes,red blood cells and hemoglobin counts of the mice in low,medium and high-dose groups were significantly lower than those in control group,and the peripheral blood platelet counts of the mice in middle and high-dose groups were significantly lower than those in control group.2.The content of immunoglobulin G,interleukin-2 and interleukin-6 in peripheral blood of the mice in low,medium and high-dose groups were significantly lower than those in control group.The mitochondrial ROS content of peripheral blood mononuclear cell of the mice in low,medium and high-dose groups was significantly higher than that in control group.The ATP content of peripheral blood mononuclear cell of the mice in high-dose group was significantly lower than that in control group.The plasma MDA content of the mice in middle and high-dose groups was significantly higher than that in control group.The total superoxide dismutase and glutathione peroxidase content of the mice in middle and high-dose groups were significantly lower than those in control group.3.The spleen quality of the mice in low,medium and high-dose groups was significantly lower than that in control group.The spleen coefficient of the mice in middle and high-dose groups was significantly lower than that in control group.The spleen tissues of the mice in low,medium and high-dose groups showed obvious pathological changes,causing damages to mitochondria,endoplasmic reticulum and chromatin in cells of the spleen tissues.The spleens of the mice in middle and high-dose groups were more obviously damaged.The exposure also caused a large amount of apoptosis in spleen tissues of the mice.The proliferative capacity of spleen B cells of the mice in middle and high-dose groups induced by lipopolysaccharide was significantly lower than that in control group,and the inhibition percent reached 18.38±4.56 and 36.08±3.04,respectively.The proliferative capacity of spleen T cells of the mice in middle and high-dose groups induced by concanavalin A was significantly lower than that in control group,and the inhibition percent reached 16.13±2.30 and 29.88±3.06,respectively.4.A total of 251 differential expressed proteins(change fold > 1.5)were screened between the spleens of the mice in high-dose and control mice.These proteins were enriched in 83 significantly different GO Terms and 5 significantly different KEGG pathways.Quantitative analysis revealed that the expression levels of CD22 and NF-?B p65 were significantly decreased at the mRNA level,and the expression of BCL10 was almost unchanged.But at the protein level,the expression of BCL10 was significantly decreased.Conclusion: This research successfully constructed a short-term repeated benzene mouse model.The growth of body weight was significantly inhibited,and the peripheral blood routine index decreased significantly.The levels of plasma immunoglobulin G,interleukin-2 and interleukin-6 in benzene-induced mice were significantly decreased,indicating that the ability of benzene-exposured mice to secrete cytokines was significantly decreased.Peripheral blood mononuclear cells have a significant increase in mitochondrial ROS production and a significant decrease in ATP content,indicating significant abnormalities in mitochondrial function in peripheral blood mononuclear cell of benzene-exposed mice.The results of plasma oxidation/antioxidant levels showed that benzene exposure caused significant oxidative stress in peripheral blood of mice.These results suggest that benzene exposure leads to oxidative stress in peripheral blood of mice,which induces a decrease in mitochondrial function in peripheral blood mononuclear cell,resulting in impaired function.Benzene exposure caused the pathological changes of spleen tissues of the mice,apoptosis of spleen tissue cells,and inhibited the proliferation of lymphocytes.CD22,BCL10 and NF-?B p65 in BCR and NF-?B signaling pathway may be the key regulators of benzene-induced impaired immune function in mouse spleen.In conclusion,benzene cause adverse effects on immune function of peripheral blood mononuclear cell and spleen in mice.The experimental results have laid a theoretical foundation for further study on the immunotoxicity and its mechanism of benzene exposure. |
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