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[要旨] 機能性食品成分は, 標的分子を事前に予測することが困難であり, また複数の成分が異なる標的に作用する可能性があることから, 機能性発揮の分子機構を完全に解明することは困難である. そのため, 食品成分が細胞に与える影響を包括的にとらえる手法の開発が求められている. 本研究では, まず, 大腸菌由来リポ多糖 (LPS) で活性化したマクロファージ様Raw264.7 細胞を用いて, マンゴー葉のメタノール抽出物 (MLE) の抗炎症作用の詳細を解析した. さらに, 細胞内のタンパク質変動をSWATH質量分析法で解析することで, MLEの抗炎症作用の分子機構を解明できるか試みた. 1.0 ng/mL のLPS で活性化したRaw264.7 細胞にMLE を添加すると, 培養上清中のNO とIL-6 濃度はMLE 0.56 μg/mL で, TNFα 濃度は2.3 μg/mL で有意に減少し, 18 μg/mL でいずれの物質も検出限界以下にまで低下した. 同様に, iNOS の発現量はMLE 2.5 μg/mL で, TNFα, IL-6, IL-1β の発現量は10 μg/mL で有意に減少し, 40 μg/mLで, いずれの遺伝子の発現量もLPS 未処理のレベルにまで低下した. 順相クロマトグラフィーでMLE を分画したところ, NO 産生抑制作用は濃縮されず, 全ての画分に弱い作用が確認された. ここから, MLE 中の複数の成分が複数の受容体に作用することで, 協奏的に強い抗炎症作用を呈すると考えられた. SWATH 質量分析法で得たタンパク質定量データを主成分判別分析 (PC-DA) にかけると, 未処理の細胞 (LPS-群), 10 ng/mLのLPSで刺激した細胞 (LPS+群), LPS に加えて, 10 μg/mL のMLE で処理した細胞 (MLE 群) の3 群に明確に分離され, それぞれ特徴的なタンパク質プロファイルを有していることが示された. その中でも, 細胞の恒常性を保ち, 炎症を収束へと導くAnnexinA1 やAnnexin A2 がMLE 群で増加していた. また, LPS-activated MAPK signaling 経路に関連する因子に注目すると,LPS+群に比べて, MLE 群では, 抗炎症に関わるSQSTM1 が増加し, 経路の中心因子であるp38MAPK や, ERK1/2 が減少していた. これらの因子がMLE の抗炎症作用と関わっていると考えられた.[Abstract] It is often difficult to fully elucidate the molecular mechanism of functional foods, since foods generally contain multiple functional substances, which may act on different targets. Therefore, there is a need to develop a method that comprehensively captures the effects of food ingredients on cells. In this study, we first analyzed the details of the anti-inflammatory effect of mango leaf ethanol extract (MLE) using macrophage-like Raw264.7 cells activated with lipopolysaccharide (LPS). Secondary, we attempted to elucidate the molecular mechanism of the anti-inflammatory effect of MLE by analyzing intracellular protein fluctuations by SWATH mass analysis. Addition of MLE to Raw264.7 cells activated with 1.0 ng/mL LPS significantly reduced the concentrations of NO and IL-6 in the culture supernatant at 0.56 μg/mL, and that of TNFα at 2.3 μg/mL. All of them decreased to below the detection limit at 18 μg/mL of MLE. Similarly, the expression level of iNOS was significantly reduced at 2.5 μg/mL of MLE, and that of TNFα, IL-6, and IL-1β was at 10 μg/mL. At 40 μg/mL of MLE, the expression level of all genes was reduced to the level of LPS-untreated cells. When MLE was fractionated by normal phase chromatography, the NO production inhibitory effect was not concentrated, and weak effects were detected on all fractions. This indicated that multiple components in MLE may act on multiple receptors to exert a strong anti-inflammatory effect. When the quantitative data obtained by SWATH-MS was analyzed by PC-DA, it was clearly distinguished into three groups, untreated cells (LPS -), those treated with 10 ng/mL LPS (LPS+), and those treated with LPS and 10 μg/mL MLE (MLE), indicating that the three groups possess distinctive proteome profiles. Among them, Annexin A1 and Annexin A2, which have been reported to maintain cell homeostasis and contribute to the termination of inflammation, were increased in the MLE group. Focusing on the factors related to the LPS-activated MAPK signaling pathway, SQSTM1 involved in anti-inflammation increased, and p38MAPK and ERK1/2, which are the central factors of the pathway, decreased. These factors were thought to be involved in the anti-inflammatory effec of MLE. |
