双歧杆菌对中国对虾原肌球蛋白致敏小鼠的免疫调控作用

Immunological regulation of Fenneropenaeus chinensis tropomyosin-stimulated sensitization by Bifidobacterium infantis in mice

【目的】比较研究婴儿双歧杆菌(Bifidobacterium infantis 13.085)对中国对虾原肌球蛋白致敏BALB/c小鼠预防与治疗过敏反应的差异,探究其对致敏小鼠Treg/Th17细胞平衡及相关细胞因子的影响。【方法】采用硫酸铵盐析及等电点沉淀法纯化中国对虾原肌球蛋白(TM),将中国对虾TM和弗氏佐剂混合液腹腔注射诱发BALB/c小鼠致敏,建立动物过敏模型。将实验小鼠随机分为正常对照组、治疗对照组、双歧杆菌治疗组、预防对照组和双歧杆菌预防组。观察分析小鼠过敏症状(腹泻、肺组织HE染色比较、称重法测定小鼠体重和脾脏脏器系数变化),采用ELISA测定小鼠血清中特异性IgE、IgG2a和组胺的含量,采用流式细胞术测定脾脏T淋巴细胞亚群(Treg、Th17)数量,采用荧光定量PCR测定脾脏中Treg型和Th17型细胞因子和转录因子的表达量。【结果】纯化得到中国对虾原肌球蛋白纯度为84.93%,得率为60.88%。体内试验表明,双歧杆菌治疗组和预防组相比于对照组,腹泻和过敏症状均有明显的缓解;不同时期的双歧杆菌干预均对过敏小鼠肺组织症状有明显的改善作用,且可降低过敏小鼠的脾脏脏器系数。第56天实验周期结束后发现,相比预防对照组和治疗对照组,双歧杆菌预防组和治疗组小鼠血清中特异性IgE和组胺含量显著降低(P<0.05),脾脏Treg/Th17比值显著升高(P<0.05),Th17型细胞因子IL-17A mRNA表达水平显著降低(P<0.01);双歧杆菌治疗组相对于治疗对照组,Treg型细胞因子CD25mRNA表达水平显著升高(P<0.01)。此外,双歧杆菌治疗组血清特异性IgE及IL-17A mRNA转录水平显著低于双歧杆菌预防组(P<0.05),而Treg/Th17比值及CD25 mRNA转录水平显著高于预防组(P<0.05)。【结论】双歧杆菌13.085能有效缓解小鼠过敏症状,且治疗免疫调控效果优于预防效果,其作用可能通过平衡Treg/Th17细胞亚群数量,促进Treg型细胞因子表达而抑制Th17型细胞因子分泌,从而阻断炎性抗体及组胺释放。

[Objective] This work aims to compare the preventive and therapeutic effects ofBifidobacterium infantis 13.085 on suppression of shrimp tropomyosin （TM） sensitization in BALB/c mice. The balance of Treg/Th17 ratio and expression of Treg- and Th17-associated cytokines were also studied. [Methods] Shrimp tropomyosin was purified by ammonium sulfate and isoelectric precipitation. Four groups （Positive 1, B. infantis therapy, Positive2 and B. infantis prevention） of BALB/c mice were immunized by intraperitoneal （IP） injection of purified TM together with incomplete Freund＇s adjuvant. Negative control animals received equal amounts of sterile PBS with Freund＇s adjuvant at each sensitization and challenge point. Allergic symptoms were evaluated by diarrhea, anaphylactic symptoms, HE staining analysis of lung tissue, changes of body weight and splenic viscera coefficient. The levels of specific IgE, IgG2a and histamine in sera were determined by ELISA. The amount of Treg and Th17 cell subsets were analyzed by flow cytometry. The mRNA expression levels of Treg- and Th17-associated cytokines and transcriptional factors were detected by quantitative PCR. [Results] The purity and yield of TM were 84.93% and 60.88%, respectively. In vivo study showed that B. infantis treatment ameliorated the development of diarrhea, anaphylactic symptoms and inflammation of lung tissue. The splenic viscera coefficient also decreased in B. infantis treatment groups compared to positive controls. At the end of experimental period （day 56）, the levels of specific IgE and histamine （P〈0.05） as well as IL-17A mRNA expression （P〈0.01） significantly reduced, while the Treg/Th17 ratio remarkably increased （P〈0.05） in B. infantis treatment groups compared to positive controls. The level of CD25 mRNA expression significantly enhanced （P〈0.01） in B. infantis therapy group compared to Positivel. In addition, the levels of specific IgE and IL-17A mRNA expression in B. infantis therapy group were much lower than those in B. infantis prevention group （P〈0.05）, while the Treg/Th17 ratio and CD25 expression level were higher in B. infantis therapy group compared to B. infantis prevention group （P〈0.05）. [Conclusion] Therapeutic administration of B. infantis 13.085 can effectively alleviate TM-stimulated allergic inflammation and symptoms, which may attribute to the balance of Treg/Th17 ratio, increase of Treg-associated cytokines expression, inhibition of Treg-associated cytokines secretion, and thus leading to blockade of inflammatory antibodies and histamine release.