富氢生理盐水在豚鼠变应性鼻炎中对CD4＋CD25＋Foxp3＋Treg细胞的调节及影响

Effect of hydrogen-rich saline on the CD4 ＋ CD25 ＋ Foxp3 ＋ Treg cells of allergic rhinitis guinea pigs model

目的 探讨富氢生理盐水（hydrogen-rich saline,HRS）对变应性鼻炎（allergic rhinitis, AR）豚鼠CD4＋ CD25＋ Foxp3＋ Treg细胞的调节作用和机制.方法 18只健康雄性豚鼠,采用随机数表法随机分为3组（对照组/AR组/HRS干预组,各组n=6）.卵清蛋白（ovalbumin,OVA）联合氢氧化铝用于AR组及HRS干预组致敏,HRS干预组致敏后用HRS干预10 d,对照组均同期使用等量生理盐水替代.观察各组豚鼠症状、外周血嗜酸粒细胞计数;酶联免疫吸附试验检测血清中白细胞介素10（IL-10）、转化生长因子β（TGF-β）的水平;免疫组化法检测鼻黏膜IL-I0、TGF-β表达水平;流式细胞仪检测脾组织及外周血中CD4＋ CD25＋ Foxp3＋ Treg细胞比例.采用SPSS17.0软件进行统计学分析.结果 豚鼠症状学评分AR组较对照组高,HRS干预组较AR组低,差异有统计学意义[（6.29±1.79）分比（1.01±0.71）分,（4.50±0.84）分比（6.29±1.79）分,F=24.725,P值均＜0.05].外周血嗜酸粒细胞计数AR组较对照组高,HRS干预组较AR组低,差异有统计学意义[（0.41±0.05）×109/L比（0.25 ±0.03）×109/L,（0.32±0.03）×109/L比（0.41±0.05） ×10g/L,F=70.05,P值均＜0.05].对照组的血清中IL-10及TGF-β含量最高[分别为（86.88±17.17） pg/ml、（598.28±72.70）pg/ml],HRS干预组较AR组的IL-I0及TGF-β含量均明显升高[（72.54±11.75）pg/ml比（53.49±10.07） pg/ml,（530.23±57.15） pg/ml比（482.69±65.96） pg/ml,F值分别为28.357、14.128,P值均＜0.05].对照组的外周血及脾组织中CD4＋ CD25＋ Foxp3＋ Treg细胞比例最高（分别为1.81％±0.10％、1.29％±0.74％）;在外周血中,HRS干预组的CD4＋ CD25＋ Foxp3＋ Treg细胞比例较AR组升高[（1.50％±0.11％）比（1.15％±0.11％）,F=168.96,P＜0.05],但在脾组织中,HRS干预组的CI4＋ CD25＋ Foxp3＋ Treg细胞比例与AR组相比差异无统计学意义[（1.01％±0.08％）比（0.98％±0.09％）,F =97.381,P＞0.05].结论 HRS可诱导CD4＋ CD25＋ Foxp3＋ Treg细胞数量增加,促进IL-10和TGF-β分泌,进而抑制变态反应性炎性反应.

Objective To explore the effect of hydrogen-rich saline on the CI4 ＋ CD25 ＋ Foxp3 ＋ Treg cells in a guinea pig model of allergic rhinitis （AR） and investigate the underling anti-inflammatory mechanism.Methods Using random number table,eighteen guinea pigs were divided into three groups （control group/AR group/HRS group,n =6 of each group）.AR guinea pig model was built with ovalbumin and aluminum.The guinea pigs were injected with hydrogen-rich saline （HRS group） for ten days after sensitation.And control group was injected with eqnal normal saline at the same time.Number of sneezes,degree of runny nose and nasal rubbing movements were scored.Peripheral blood eosinophil count was recorded.The content of interleukin 10（IL-10） and transforming growth factor β3 （TGF-β） in the serum were detected by enzyme-linked immunosorbent assay （ELISA）.Immunohistochemical method was taken to detect IL-10 and TGF-β in nasal mucosa-The proportion of CD4 ＋ CD25 ＋ Foxp3 ＋ T cells in the CI4 ＋ T cells of spleen and peripheral blood were determined with flow cytometry.SPSS 17.0 software was used to analyze the data.Results There was significant difference in symptom scores among them.The scores of AR group preceded control group,and HRS could decrease the scores of AR （（6.29 ± 1.79） vs （1.01 ± 0.71）,（4.50 ± 0.84） vs （6.29 ± 1.79）,F =24.725,all P 〈 0.05）.The highest number of eosinophils in the peripheral blood belonged to control group,and the number of eosinophils were dramatically reduced after HRS administration （（0.41 ±0.05） × 109/L vs （0.25 ±0.03） × 109/L,（0.32 ±0.03） × 109/L vs （0.41 ±0.05） × 109/L,F =70.05,all P 〈0.05）.The content of IL-10 and TGF-β in control group is peak （（86.88 ± 17.17） pg/ml,（598.28 ±72.70） pg/ml,respectively）,and compared with AR group,HRS also increased the expression of IL-I0 and TGF-β of peripheral blood （（72.54 ± 11.75） pg/ml vs （53.49 ± 10.07） pg/ml,（530.23 ± 57.15） pg/ml vs （482.69 ± 65.96） pg/ml,F value was 28.357,14.128,respectively,all P 〈 0.05）.The proportion of CD4 ＋ CD25 ＋ Foxp3 ＋ Treg cells in controls exceeded HRS group and AR group （1.81% ± 0.10%,1.29% ± 0.74%,respectively）,and HRS treatment increased the ratio of CD4 ＋ CD25 ＋ Foxp3 ＋ Treg cells than AR group of peripheral blood （（1.50% ± 0.11%） vs （1.15% ±0.11%）,F =168.96,P 〈0.05）.But there was no significant diferences in splene tissue （（1.01% ±0.08%） vs （0.98% ±0.09%）,F=97.381,P〉0.05）.Conclusion Both the number and the cytokine secretion of CD4 ＋ CD25 ＋ Foxp3 ＋ Treg cells are decreased in AR group,HRS may inhibit inflammatory response and ameliorate AR via improving the number and the cytokine secretion.