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PPARγ Agonist Attenuates Vocal Fold Fibrosis in Rats via Regulation of Macrophage Activation

Kaba, Shinji 京都大学 DOI:10.14989/doctor.k24137

2022.07.25

概要

声帯線維化は不可逆的な発声障害をもたらすため、コミュニケーション機能を損ない、社会的にも精神的にも大きなハンディキャップとなる。その多くは炎症や損傷の結果生じ、特に声帯手術や頸部放射線治療後に医原性にも発生するため、実臨床において重要な課題となっている。これまでの声帯線維化に対する研究の多くはコラーゲンの産生源である線維芽細胞を主なターゲットとしてきた。近年、さまざまな臓器でマクロファージが線維化に関わることが示されてきたが、損傷後の声帯におけるマクロファージの動態はほとんど知られていない。そこでまず、マウス声帯損傷モデルにおけるマクロファージの表現型の変化を間接蛍光抗体法で確認したところ、無処置の声帯では主にCD206 陽性の修復性マクロファージが常在し、損傷後 5 日目以内に iNOS 陽性の炎症性マクロファージが一過性に増加することが明らかになった。

そこで、損傷後一過性に増加する炎症性マクロファージが線維化を制御しているという仮説をたて、炎症性マクロファージをターゲットとした薬剤治療を試みた。Peroxisome proliferator-activated receptor-γ (PPARγ) は、創傷治癒の炎症期に主にマクロファージに発現し、その表現型を制御している。PPARγアゴニストであるピオグリタゾンを損傷後のラット声帯に投与し、声帯におけるマクロファージの表現型への影響をqRT-PCR と間接蛍光抗体法で評価したところ、ピオグリタゾンは炎症期である損傷後 4 日目における Ccl2 と Cd68 の遺伝子発現を PPARγ依存的に抑制し、iNOS陽性の炎症性マクロファージの増加を抑制した。以上より、ピオグリタゾンは炎症期のCcl2 の発現上昇を抑制することにより炎症性マクロファージの浸潤を抑制することが示唆された。次にピオグリタゾンの声帯線維化への影響をqRT-PCR、間接蛍光抗体法、組織特殊染色法で評価した。ピオグリタゾンは炎症期において Tgfb1、Fn1、Acta2、Has1 の発現上昇を抑制し、成熟期である損傷後56 日目においてコラーゲン・Ⅰ型コラーゲン・フィブロネクチンの増加、ヒアルロン酸と粘膜固有層断面積の減少をPPARγ依存的に抑制した。以上より、ピオグリタゾンが損傷後のラット声帯の線維化・組織拘縮を減弱させることが示された。

最後に、ヒト単球系細胞株であるTHP-1 細胞からマクロファージを誘導後、LPS とIFN-γの刺激で誘導した炎症性マクロファージにおいて、ピオグリタゾンはPPARγ非依存的にCcl2 の発現上昇を抑制することがqRT-PCR で確認された。PPARγアゴニストはM1 刺激下のマクロファージにおけるNFκB 標的遺伝子の発現を抑制するが、その効果は薬剤濃度や M1 刺激の種類によって PPARγ依存的にも非依存的にも発現しうることが報告されており、この結果はin vivo 実験の結果を否定するものではない。

本研究によりピオグリタゾンは炎症性マクロファージにおける Ccl2 発現を直接的もしくは間接的に抑制することで炎症性マクロファージの声帯への浸潤を抑制し、成熟期の声帯線維化を制御することが示され、炎症性マクロファージが声帯損傷時の線維化に関わっていることが示唆された。 またピオグリタゾンは PPARγ依存的に声帯線維化を抑制したことから、PPARγが声帯損傷時の線維化抑制に関わっていることが示唆された。これらの結果が、マクロファージやPPARγをターゲットとした声帯線維化に関する研究の礎となり、新規予防法の開発につながることが期待される。

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