| BackgroundAsthma is one of the most common chronic diseases among children and youth population.In recent years,the prevalence of asthma in children shows the upward trend in China.The onset of asthma is affected by many factors,among which tobacco smoke exposure of gestational mothers is one of the most common inducing factors.Gestation period is the critical period for the tissue and organ development of offspring.Studies have shown that the tobacco smoke exposure during gestation period of F0 generation may increase the risk of asthma in F1 generation,F2 generation and F3 generation through the transgenerational transmission,but its mechanism remains to be unclear at present,so it is of great significance to study the impact and mechanism of tobacco smoke exposure during gestation period on the development of asthma in offspring.ObjectiveThis study aims to study the transgenerational genetic effects of tobacco smoke exposure during gestation period of F0 female mice on the development of asthma in offspring by using the tobacco smoke exposure model of BALB / c mice during gestation period.Specifically,the changes in bronchoalveolar lavage fluid,cell counting and Th1 / Th2 type cytokines in spleen tissues were investigated in F1,F2 and F3 generations of mice with tobacco smoke exposure during gestation period,so as to clarify the changes of asthma susceptibility in offspring.In addition,the changes of T-bet and GATA-3(Th1 / Th2 type cell-specific transcription factors)protein expression of in F1,F2 and F3 generations of mice were investigated,so as to further explore the possible molecular mechanism of transgenerational inheritance.Methods1.Preparation of asthma model in BALB / c miceThe pups in the TSE group were sensitized by intraperitoneal injection of OVA sensitizing fluid,while the pups in the control group were sensitized by intraperitoneal injection of PBS solution instead of ova solution,to prepare of mouse asthma model.2.Preparation of broncho-alveolar lavage fluid and cell countingMice in each group were anesthetized and the trachea was cut,intubated and fixed.Inject 1 ml of pre-cooled sterile PBS solution each time and recovery of bronchoalveolar lavage fluid,to measure the total number of cells and count eosinophils.3.Detection of cytokine levels3.1 Levels of cytokine IL-4 and IFN-γ in spleen monocyte suspensionAfter the last atomization,the spleens of mice in the control group and the tobacco smoke exposure group were taken out by laparotomy under sterile conditions,and the suspension of spleen cells was prepared by grinding.After treatment,the supernatant was taken,and the levels of IL-4 and IFN-γ cytokines were detected by using the mouse cytokine ELISA kit.3.2 levels of cytokine IL-4 and IFN-γ in peripheral serumAfter the last atomization,the eyeballs of mice in the control group and the tobacco smoke exposure group were removed to collect blood,and the serum samples were obtained.The levels of IL-4 and IFN-γ cytokines were detected by using the mouse cytokine ELISA kit.4.Detection of transcription factor expression in lung tissueAfter the last atomization,the lung tissues of mice in the control group and the tobacco smoke exposure group were taken out.Western blotting was used to detect the expression of transcription factor GATA-3 and T-bet.The effect of maternal exposure during gestation period on the expression of transcription factors in offspring was observed.Results1.Symptom analysis of mouse asthma modelThe F1,F2 and F3 generation mice in the tobacco smoke exposure group showed upright body hair,scratching mouth and nose,shaking body,polypnea,unsteady gait,blind leaping and other symptoms after being atomized and stimulated with 20 μ g / ml OVA solution,which met the standard of asthma model preparation.Therefore,it could be concluded that the allergic reaction of mice in the tobacco smoke exposure group showed strongly positive,while the behavior of mice in the negative control group was normal.2.The total number and differential counting of bronchoalveolar lavage fluid cellsCompared with the control group,the total number of bronchoalveolar lavage fluid cells,neutrophil count,eosinophil count and lymphocyte count in F1,F2 and F3 generations of tobacco smoke exposure group were significantly higher than those in the control group(P<0.05),and showed a decreasing trend with the increase of generations.3.The level of cytokines in spleen monocyte suspension3.1 Levels of cytokine IL-4 and IFN-γ in spleen monocyte suspensionCompared with the control group,the levels of IL-4 in the spleen monocyte suspension of F1,F2 and F3 generations of mice in tobacco smoke exposure group were 80.26±21.55 ng/L,64.38±10.32 ng/L and 8.24±5.91 ng/L respectively,which were significantly higher than those in the control group(4.20±1.37 ng/L,P<0.05),and decreased with the increase of generations.Compared with F1 generation,the level of IL-4 in F3 generation of mice was significantly lower(P<0.01).The levels of IFN-γ in F1 and F2 generation were 23.74±8.66 ng/L and 35.27±7.47 ng/L respectively,which were significantly lower than those in the control group(45.53±10.44 ng/L,P<0.01),and the level of IFN-γ in F3 generation(42.71±8.17 ng/L)was significantly higher than that in F1 generation(P<0.05),but there was no significant difference between F3 generation and the control group(P>0.05).3.2 Levels of IL-4 and IFN-γ in peripheral serumCompared with the control group,the levels of IL-4 in the peripheral serum of F1,F2 and F3 generations of mice in tobacco smoke exposure group were 42.19±5.74 ng/L,36.52±5.07 ng/L and 21.22±4.21 ng/L respectively,which were significantly higher than those in the control group(15.27±2.43 ng/L,P<0.05),and decreased with the increase of generations.Compared with F1 generations,the levels of IL-4 in F3 generations of mice were significantly lower(P<0.01).The levels of IFN-γ in F1 generation and F2 generation were 47.33±9.17 ng/L and 56.24±20.15 ng/L respectively,which were significantly lower than those in the control group(60.85±7.05 ng/L,P<0.05),and the level of IFN-γ in F3generation(62.35±23.54 ng/L)was significantly higher than that in F1 generation(P<0.05),but there was no significant difference between F3 generation and the control group(P>0.05).4.Detection of transcription factor expression in lung tissueThe expression of GATA-3 protein in lung tissue of F1,F2 and F3 generation of mice in tobacco smoke exposure group was significantly higher than that in the control group(P<0.05),while the expression of T-bet was significantly lower than that in the control group(P<0.05).Conclusions1.Offspring exposed to tobacco smoke during pregnancy are at increased risk of asthma after allergen sensitization.2.Tobacco smoke exposure during pregnancy can change the expression of transcription factors in the immune system and show the characteristics of intergenerational inheritance. |
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