探討Lunasin在肥胖微環境下對RAW264.7細胞與EL-4 T細胞免疫反應之影響

Abstract

全球的肥胖盛行率在流行病學調查中依然居高不下，肥胖常常連帶許多代謝疾病的發生，原因在於肥胖過程中，脂肪組織會有多種免疫細胞浸潤，伴隨著促發炎性細胞激素的分泌，形成慢性發炎的環境，進而發展出許多疾病，包含第二型糖尿病、代謝症候群、心血管疾病及癌症等。Lunasin為一種天然種子胜肽，存在於許多穀類中，由43個胺基酸所組成，已被證實具有抗腫瘤、抗發炎、抗氧化、降膽固醇及免疫調節等功用。本實驗目的為探討肥胖微環境下，lunasin對於RAW264.7巨噬細胞與EL-4 T細胞免疫功能的影響。細胞培養時以添加3T3-L1脂肪細胞培養液（adipocyte condition medium, Ad-CM）與LPS (Lipopolysaccharide)刺激來模擬肥胖微環境。分析3T3-L1細胞分化過程中細胞激素的變化，隨著分化進行adiponectin、leptin、VEGF和PAI-1含量逐漸增加，顯示Ad-CM可作為肥胖微環境的模型，結果顯示在肥胖微環境下，lunasin處理能夠降低RAW264.7巨噬細胞經LPS或Ad-CM誘發的IL-1β及MCP-1的分泌量，亦降低其遷移能力；然而，在RAW264.7巨噬細胞極化實驗下，lunasin的處理並無影響。Lunasin處理能降低因LPS誘發的ROS生成量。此外增加Ad-CM模式下健康細胞的比例，以及降低其低活性細胞比例，另外，lunasin處理亦能增加RAW264.7巨噬細胞胞飲及胞吞活性。最後，使用共培養來探討3T3-L1脂肪細胞與EL-4 T細胞之間的相互作用，EL-4 T細胞與受TNF-活化3T3-L1脂肪細胞共培養的模式下，會刺激IL-6、TNF-α和IL-4分泌量增加，而lunasin的介入可以降低3T3+EL4共培養模式下的IL-4分泌量，降低3T3 (TNF-α)+EL4共培養中TNF-α分泌，但使IL-1β增加以恢復到基準值。綜合而論，lunasin可以降低RAW264.7巨噬細胞的發炎反應和氧化反應，並抑制遷移能力，且增加吞噬功能，在EL-4 T細胞與3T3-L1脂肪細胞共培養下具有調節細胞激素的效果。Obesity is characterized by a chronic low-grade inflammation leading to various pathological developments. In adiposity, several types of immune cells infiltrate into adipose tissues, especially macrophages, and the process accompanies pro-inflammatory cytokines secretions, forming a chronic inflammatory environment. Lunasin, a natural seed peptide, exhibits several biological activities, such as anti-carcinogenetic, anti-inflammatory, and anti-oxidative activities. Adipocyte-conditioned medium (Ad-CM) and lipopolysaccharide (LPS) were used in cell culture to mimic the physiology of obesity. The secretion of adiponectin, leptin, VEGF and PAI-1 in Ad-CM increased in the process of differentiation, so Ad-CM might mimic the obese microenvironment. The results have showed that the secretion of interleukin-1β (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) was inhibited by lunasin treatment in RAW264.7 cells stimulated by LPS or Ad-CM. Moreover, lunasin suppressed the chemotaxis of RAW264.7 cells stimulated by LPS, but had no effect on M1/M2 gene expression. In addition, lunasin reduced the generation of reactive oxygen species (ROS) induced by LPS, while increased the proportion of healthy cells and reduced the proportion of low-vitality cells in Ad-CM challenge. In cell functions assay, lunasin increased the pinocytosis and phagocytosis activity in LPS or Ad-CM challenge. Lunasin downregulated the secretion of IL-4 and TNF-α in EL-4 T cells co-cultured with 3T3-L1 adipocytes, but upregulated the secrcetion of IL-1β in 3T3 (TNF-α)+EL4. These results have demonstrated that lunasin could suppresses obesity-related inflammation, oxidative stress and migration in RAW 264.7 macrophages, and regulated the secretion of cytokines in EL-4 T cells co-cultured with 3T3-L1 adipocytes. In summary, lunasin acts as a potential anti-inflammatory agent.