多肽SJMHE1调控Th17/Treg细胞平衡以及对哮喘小鼠治疗作用的研究

Regulation of Th17/Treg Cell Balance by Polypeptide SJMHE1 and Its Therapeutic Effect on Asthmatic Mice

目的探索日本血吸虫热激蛋白来源多肽SJMHE1对辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡的调控以及在小鼠过敏性哮喘治疗中的作用。方法免疫磁珠法分选正常小鼠脾脏CD4^(+)T细胞,分别加入0.5、1和1.5μg/mL的SJMHE1进行体外培养。流式细胞术检测不同SJMHE1浓度作用下Th17细胞和Treg细胞的比例,实时荧光定量聚合酶链反应(PCR)检测视黄酸相关核孤儿受体γt(RORγt)和叉头框蛋白P3(FoxP3)mRNA的表达水平。将15只6~8周龄雄性健康BALB/c小鼠依据随机数字法分为磷酸盐缓冲溶液(PBS)组、卵白蛋白(OVA)组和OVA+SJMHE1组,每组5只。构建哮喘小鼠模型,苏木精-伊红染色观察小鼠肺组织病理学变化,实时荧光定量PCR检测小鼠肺组织中白细胞介素(IL)-17A和IL-10 mRNA的表达水平。结果在一定范围内,随着SJMHE1浓度的增加,Th17细胞的比例逐渐降低,Treg细胞的比例逐渐升高。1μg/mL和1.5μg/mL SJMHE1组RORγt mRNA低于0μg/mL SJMHE1组(0.623±0.091、0.347±0.102比1.000±0.087)(P<0.05),FoxP3 mRNA高于0μg/mL SJMHE1组(2.310±0.307、3.010±0.582比1.040±0.133)(P<0.05)。与PBS组相比,OVA组小鼠肺部支气管管壁明显增厚,周围出现大量炎症细胞浸润,而OVA+SJMHE1组小鼠肺部病理学改变较OVA组明显减轻。OVA组IL-17A mRNA高于PBS组(2.500±0.266比0.998±0.054)(P<0.05),OVA+SJMHE1组低于OVA组(1.100±0.188比2.500±0.266)(P<0.05);OVA组IL-10 mRNA低于PBS组(0.498±0.085比1.050±0.141)(P<0.05),OVA+SJMHE1组高于OVA组(2.230±0.455比0.498±0.085)(P<0.05)。结论多肽SJMHE1可能通过调控Th17/Treg细胞平衡缓解哮喘小鼠气道炎症。

Objective To explore the regulation of helper T cell 17(Th17)/regulatory T cell(Treg)balance by schistosoma japonicum HSP-derived polypeptide SJMHE1 and its role in the treatment of allergic asthma in mice.Methods CD4^(+)T cells in spleen of normal mice were collected by immunomagnetic beads method,SJMHE1 was added with 0.5,1 and 1.5μg/mL for in vitro culture.The proportion of Th17 cells/Treg cells was detected by flow cytometry.The relative expression levels of transcription factors retinoic acid-related orphan receptorγt(RORγt)and forkhead box protein P3(FoxP3)mRNA were detected by real-time fluorescence quantitative polymerase chain reaction(PCR).Fifteen healthy male BALB/c mice aged 6-8 weeks were divided into a phosphate buffer(PBS)group,an ovalbumin(OVA)group,and an OVA+SJMHE1 group according to random number method,with 5 mice in each group.The mouse model of asthma was established.Hematoxylin-eosin staining was used to observe the histopathological changes in the lungs of mice,and the relative expression levels of interleukin(IL)-17A and IL-10 mRNA in the lung tissues of mice were detected by real-time fluorescence quantitative PCR.Results In a certain range,with the increase of SJMHE1 concentration,the proportion of Th17 cells decreased gradually,while Treg cells increased gradually.The relative expression levels of RORγt mRNA in 1μg/mL and 1.5μg/mL SJMHE1 groups were lower than that in 0μg/mL SJMHE1 group(0.623±0.091,0.347±0.102 vs 1.000±0.087)(P<0.05),and FoxP3 mRNA higher than that in 0μg/mL SJMHE1 group(2.310±0.307,3.010±0.582 vs 1.040±0.133)(P<0.05).Compared with the PBS group,the bronchial wall in the lungs of mice in the OVA group was significantly thickened with a large amount of inflammatory cell infiltration around,while the pathological changes in the lungs of mice in the OVA+SJMHE1 group were significantly reduced compared with those in the OVA group.IL-17A mRNA in the OVA group was higher than that in the PBS group(2.500±0.266 vs 0.998±0.054)(P<0.05),and that in the OVA+SJMHE1 group was lower than that in the OVA group(1.100±0.188 vs 2.500±0.266)(P<0.05).IL-10 mRNA in the OVA group was lower than the PBS group(0.498±0.085 vs 1.050±0.141)(P<0.05),and that in the OVA+SJMHE1 group was higher than that in the OVA group(2.230±0.455 vs 0.498±0.085)(P<0.05).Conclusion Polypeptide SJMHE1 may alleviate airway inflammation in asthmatic mice by regulating Th17/Treg cell balance.