探討Lunasin在肥胖微環境下對A549肺泡上皮細胞發炎反應與其可能影響機制

Abstract

肥胖是過多能量累積造成脂肪組織增加並伴隨發炎反應，長期控制不佳者可能導致肺部纖維化。本研究欲探討在肥胖微環境下，lunasin是否能降低肺泡上皮細胞的發炎反應和纖維化的作用。本研究包含A549細胞以外加葡萄糖與棕櫚酸palmitic acid (PA)做為體外肥胖模式，與動物實驗以高油高果糖飲食誘導小鼠肥胖模式。首先在建立實驗模型的結果中，最終以7 mM正常葡萄糖與35 mM高葡萄糖 、PA 500 μM、lipopolysaccharide (LPS) 10 μg/mL和TGFβ 5 ng/mL外加入培養基中作為肥胖微環境。結果顯示，在發炎模式中，lunasin可抑制由PA或LPS誘發的低糖培養細胞之IL-6和MCP-1分泌；而低劑量的lunasin可抑制由LPS誘發高糖培養細胞之MCP-1和TGFβ分泌。在Surfactant protein (SP)的結果顯示， LPS刺激會減少正常糖培養及增加高糖培養細胞中SPD蛋白表現量，而lunasin則能夠調節其含量。分析可能作用路徑，lunasin可抑制由LPS刺激的NFκB p65的表現。在TGFβ 誘發纖維化模式中，再以刮傷癒合測定細胞遷移能力，並測量epithelial-mesenchymal transition (EMT) marker的表現，結果顯示TGFβ誘導可增加vimentin並降低E-cadherin的表現，而lunasin則無法影響TGFβ刺激後的細胞遷移與EMT表現。在肥胖小鼠實驗中，自小鼠六週大開始餵食實驗飲食，實驗分組包含正常飲食、高油高果糖飲食、腹腔注射lunasin，以及飲食添加lunasin，分析鼠肺臟組織均質液中發炎性細胞激素含量。在22週齡結果顯示，高油高果糖飲食添加lunasin可降低肺臟TNFα和TGFβ的生成。而在33週齡結果中，高油高果糖飲食的肺臟IL-6、IL-1β和IL-17a顯著降低，在腹腔注射lunasin則能提升此細胞激素生成，但飲食補充lunasin則是降低IL-6、IL-1β和TGFβ的生成。綜合上述，lunasin能夠抑制肥胖微環境下發炎肺泡中的促發炎性細胞激素IL-6、MCP-1和TGFβ分泌，且可能是透過抑制NFκB路徑的作用，但對於纖維化的影響則不顯著。未來將進一步分析，發炎與免疫防護相關的表面蛋白與其可能影響的訊息路徑。

Obesity is an increase in adipose tissue caused by excessive energy accumulation and is accompanied by an inflammatory reaction. Subsequently, uncontrol and long-term inflammation extends pulmonary fibrosis development. Aim of our study is to investigate whether lunasin reduces inflammation and fibrosis in A549 pulmonary epithelial cells under obese conditions. The palmitic acid (PA) and glucose were used to mimic obese conditions in vitro. The mice fed with high-fat and fructose diet were used as obese animal model. In the results of establishing the experimental model, 7 mM normal glucose and 35 mM high glucose, PA 500 μM, lipopolysaccharide (LPS) 10 μg/mL, and TGFβ 5 ng/mL were added to the culture medium as an obesity microenvironment. In inflammation, lunasin inhibited IL-6 and MCP-1 secretions induced by PA or LPS in normal glucose medium; while the low dose of lunasin inhibited MCP-1 and TGFβ secretions induced by LPS in the high glucose medium. Surfactant protein D (SP-D) executes immune response and defense. Lunasin treatment modulated SP-D levels stimulated by LPS, especially the effect was difference between normal glucose and high glucose medium. In fibrosis, the migration was determined by wound healing assay and epithelial-mesenchymal transition (EMT) marker that was stimulated by TGFβ. However, lunasin did not effect cell migration and EMT expression. In vivo, mice were divided as normal diet (ND), high-fat high-fructose diet (HF), intraperitoneal injection of lunasin (HF-IL), and dietary supplementation with lunasin (HF-DL). 33-week-old mice lung homogenate of IL-6, IL-1β, and IL-17a expression were significantly reduced in HF group, and HF-IL increased the production of these cytokines, but HF-DL group were reduced. In conclusion, lunasin inhibited cytokines IL-6, MCP-1 and TGFβ through NFκB pathway in inflamed alveoli, but this effect was not showed in fibrosis.