Effects of chlorella in OVA-sensitized BALB/c mice

Chlorella has been reported to have various physiological activities, including antiarteriosclerotic, cholesterol-lowering, anti-inflammatory, and immunoregulatory effects. However, there has been no report on the long-term effects of chlorella ingestion on immunity. In the present study, 4- to 10-week-old (young) and 4- to 50-week-old (old) female BALB/c mice were sensitized or not with ovalbumin (OVA), and given basic diet containing chlorella powder at 2% or basic diet alone. The effects of chlorella ingestion on immunity were investigated by measurement of splenic cytokines and immunoglobulin (Ig), analysis of T- and B-cells in the spleen and small intestine by flow cytometry, and analysis of the liver by DNA microarray. Results were compared between the young and old, OVA-sensitized and -nonsensitized, and chlorella and non-chlorella ingestion groups. Production of interferon-γ (IFN-γ) was maintained in the nonsensitized old groups, and ratios of T-helper type 1 (Th1) to T-helper type 2 (Th2) cells were similar in the young and old groups. In addition, overproduction of OVA-specific Igs due to OVA sensitization was strongly suppressed, and significant immunotolerance was exhibited irrespective of age. In addition, suppression of T-cell decreases in the spleen due to aging and suppression of changes in T- and B-cells due to OVA sensitization in the small intestinal lymph were demonstrated on flow cytometric analyses. On DNA microarray analysis, immune-related terms including IL11 and major histocompatibility complex (MHC) class 1 were detected, and expression of genes was shown, which were related to IL1-linked genes and complex involving macrophages from the pathways of cytokines and inflammatory response. In addition, suppressions of declined lipid metabolism and energy production were also suggested. Although how the ingredients in chlorella were involved in these changes is unclear, our findings suggest that prevention of decrease in acquired immunity by aging and induction of strong immunotolerance occurred following chlorella ingestion. The Young Control group and Old Control group were fed normal food, and the Old Chlorella intake group was fed food including 2.0% Chlorella powder. The total RNA was isolated from liver and samples was pooled for each group (n = 6 for each group). A table of normalized log2 ratios (where numerator is sample expression data, and denominator is all sample median data) is linked below as a supplementary file.已有研究报道，小球藻（Chlorella）具有多种生理活性，包括抗动脉硬化、降胆固醇、抗炎及免疫调节作用。然而，目前尚无关于长期摄入小球藻对机体免疫功能影响的相关报道。本研究以4~10周龄（青年）、4~50周龄（老年）的雌性BALB/c小鼠为实验对象，分别用卵清蛋白（ovalbumin, OVA）致敏或不致敏，同时给予含2%小球藻粉的基础饲料或单纯基础饲料。通过检测脾脏细胞因子与免疫球蛋白（immunoglobulin, Ig）、流式细胞术（flow cytometry）分析脾脏与小肠内T、B淋巴细胞亚群，以及DNA微阵列（DNA microarray）分析肝脏组织，探究小球藻摄入对免疫功能的影响。实验分别比较了青年与老年小鼠、OVA致敏与未致敏小鼠、小球藻摄入与未摄入组间的实验结果。研究发现，未致敏的老年小鼠组中γ干扰素（interferon-γ, IFN-γ）的产生得以维持，且青年与老年小鼠组的辅助性T细胞1型（T-helper type 1, Th1）与辅助性T细胞2型（T-helper type 2, Th2）细胞比例无显著差异。此外，OVA致敏诱导的OVA特异性Ig过度产生被显著抑制，且无论小鼠年龄如何，均表现出显著的免疫耐受。流式细胞术分析还显示，小球藻摄入可抑制衰老导致的脾脏T细胞数量减少，以及OVA致敏引起的小肠淋巴组织中T、B淋巴细胞亚群的异常变化。DNA微阵列分析检测到与免疫相关的条目，包括白细胞介素11（IL11）与主要组织相容性复合体（major histocompatibility complex, MHC）I类分子，同时发现与细胞因子与炎症反应通路中白细胞介素1（IL1）相关基因及巨噬细胞相关复合物的基因表达发生改变。此外，研究还提示小球藻摄入可改善衰老导致的脂质代谢与能量产生能力下降。尽管目前尚不明确小球藻的具体活性成分如何介导上述变化，但本研究结果表明，长期摄入小球藻可预防衰老引起的获得性免疫功能下降，并诱导产生强效免疫耐受。本实验中，青年对照组与老年对照组小鼠饲喂普通饲料，老年小球藻摄入组小鼠饲喂含2.0%小球藻粉的饲料。从各组小鼠肝脏中提取总RNA并混合（每组n=6）。标准化log2比值表（其中分子为样本表达数据，分母为所有样本的中位数数据）作为补充文件附后。