低浓度二氧化硫对哮喘大鼠CD4^+CD25^+调节性T细胞及转录因子Foxp3表达的影响

Effect of low concentration sulfurdioxide on regulatory T cells and transcription factor Foxp3 in asthmatic rats

目的探讨低浓度二氧化硫(sulfurdioxide,SO2)暴露对哮喘大鼠CD4+CD 25+调节性T细胞(regulatory T cells,Treg)数量及转录因子Foxp3表达的影响。方法将60只雄性Wistar大鼠按随机数字表法分为正常对照组、低浓度SO2暴露组、哮喘组和低浓度SO2暴露哮喘组,每组15只;哮喘组采用卵清白蛋白(OVA)致敏并吸入激发制备哮喘模型,正常对照组吸入雾化生理盐水,低浓度SO2暴露组采用吸入低浓度SO2,低浓度SO2暴露哮喘组于每日雾化激发OVA前给予低浓度SO2吸入。采用流式细胞术检测外周血CD4+CD 25+Treg细胞占CD4+T细胞的比例,酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测外周血和肺组织γ-干扰素(interferon-γ,γ-INF)以及白细胞介素-4(interleukin-4,IL-4)含量;Western blot检测肺组织Foxp3蛋白表达水平。结果哮喘组大鼠CD4+CD 25+Treg为(5.78±1.26)%,低于正常对照组的(9.63±1.37)%;低浓度SO2暴露哮喘组为(3.15±0.82)%,低于正常对照组和哮喘组,差异有统计学意义(P均<0.01)。哮喘组大鼠血浆和肺组织中IL-4含量[(23.51±4.86)ng/L和(0.93±0.16)ng/L]高于正常对照组[(11.24±2.53)ng/L和(0.29±0.06)ng/L],差异有统计学意义(P均<0.01);低浓度SO2暴露哮喘组血浆和肺组织中IL-4含量[(36.73±7.46)ng/L和(1.67±0.41)ng/L]高于正常对照组和哮喘组,差异有统计学意义(P均<0.01);哮喘组血浆和肺组织中γ-INF含量[(63.82±21.78)ng/L和(0.49±0.15)ng/L]低于正常对照组[(156.98±60.23)ng/L和(1.58±0.18)ng/L],差异有统计学意义(P<0.01),低浓度SO2暴露哮喘组γ-INF含量[(10.02±1.68)ng/L和(0.43±0.12)ng/L]低于正常对照组和哮喘组,差异有统计学意义(P<0.01)。哮喘组Foxp3蛋白表达为(8.23±0.56),低于正常对照组的(11.87±0.65)(P<0.05);低浓度SO2暴露哮喘组为(6.05±0.36),低于正常对照组和哮喘组(P<0.05)。结论低浓度SO2暴露可能通过下调CD4+CD 25+Treg数量并抑制Foxp3蛋白表达,进一步加重哮喘的Th1/Th2比例失衡,可能是诱发哮喘发病的因素。

Objective To investigate the effect of low level sulfurdioxide （ SO2 ） exposure on the amount of CD4 ~ CD 25 ~ regulatory T cells （Treg） and expression of transcription factor Foxp3 in asthmatic rats. Methods Sixty male Wistar rats were divided into 4 groups （n = 15 for each group） according to randomdigits table, they were normal control group, SO~ exposure group, asthmatic group （by inhaleing aerosolized ovalbumin） and combined OVA and SO2 exposure group. 8 weeks later, the percentage of CD4＋ CD 25＋ T cells was determined by flow cytometry analysis, the levels of interleukin-4 （IL-4） and interferon- （-,/-INF） in peripheral blood and lung homogenates were measured by enzyme linked immunosorbent assay（ ELISA）, while the protein expression of Foxp3 in the lung tissues was detected by Western blot. Results The percentage of CD4＋ CD 25＋ T cells in asthmatic group （5.78 ±1.26）%was significantly lower than that of normal control group [ （9. 63± 1.37）%, P 〈0. 01 ], this percentage in low level SO2 exposure plus asthmatic group （3.15 ± 0. 82）% was remarkably lower than that of normal control group and asthmatic group （P 〈0.01 ）. IL-4 levels both in plasma （23.51±4. 86 ） ng/L and lung （0.93±0. 16） ng/L were significantly increased in asthmatic rats compared with normal control group [ （11.24 ± 2. 53） ng/L and （0. 29 ± 0. 06） ng/L respectively, P 〈0. 01 ] ; the levels mentioned above in SO2 exposed group [ （36. 73±7.46） ng/L and （1.67 ±0. 41 ） ng/ L, respectively ] were also significantly higher than that of control and asthmatic groups （P 〈 0. 01 ）. The γ-INF levels of plas- ma （63.82±21.78 ） ng/L and lung tissues （0. 49 ± 0. 15 ） ng/L in asthmatic group significantly decreased compared with the normal control group [ （ 156.98 ± 60. 23 ） ng/L and （ 1.58± 0. 18） ng/L, respectively, P 〈 0.01 ], and these levels in asthmatic plus SO2 group [ （ 10. 02 ~ 1.68） ng/L and （0. 43 ±0. 12） ng/L] were all significantly lowered compared with asth- matic group and normal control group （P 〈0. 01 ）. As for protein expression of Foxp3, the level in asthmatic group （8. 23 ±0. 56） was lower than that of normal control group （ 11.87±0. 65, P 〈 0. 05 ）, while the level in asthmatic plus SO2 group （6. 05 ± 0. 36） was also lower than that of normal control group and asthmatic group （ P 〈0. 05 ）. Conclusion The results suggested that low level SO2 exposure may down regulate the amount of CD4＋ CD 25＋ Treg ceils and inhibite the expression of Foxp3, thereby further aggravate the disbalance of Thl/Th2 ratio in asthma, which may be one of the important factors to trigger asthma seizures.