Simvastatin可以藉由調節成骨細胞中mitophagy的機制來減緩發炎性骨吸收

缺氧是面對發炎性疾病時所需處理的治療標的，而細胞中應付缺氧的主要胞器就是粒線體。Mitophagy是指粒線體經由自噬作用而降解的機制，細胞會透過這項作用藉以清除代謝失去功能的粒線體，進而維持細胞內粒線體的平衡和基因完整性。Simvastatin是臨床上一廣為被使用作為降血脂的藥物，它同樣也具備抗發炎的功能。在我們先前的研究已經證明simvastatin可以藉由調控成骨細胞中細胞自噬和細胞凋亡的作用進而減緩根尖病變的進展。然而，其中詳細的機轉仍待闡明。在本次的研究中，我們將目標集中在粒線體機制的探討。我們利用西方點墨法去觀察，以simvastatin處理過後的MC3T3-E1細胞，在缺氧刺激下對於MC3T3-E1細胞內PTEN-induced putative kinase 1（PINK1）/ parkin（聯手調控mitophagy）和poly (adenosine phosphate ribose) polymerase（PARP）片段（細胞凋亡的標記）的蛋白質表現。我們也以免疫螢光法去監測PINK1與parkin於細胞內的實際位置。在誘導根尖病變的大鼠模型，我們設計不同的投藥途徑，利用影像學分析以及組織免疫染色去探討simvastatin最佳的治療方式。研究結果發現，缺氧刺激同樣可以提高PINK1/parkin與PARP cleavage的表現，而simvastatin更促使缺氧刺激下PINK1/parkin的表現，但卻會抑制PARP cleavage的量。我們在共軛焦顯微鏡下可觀察到，於缺氧刺激下PINK1可以穩定聚集在粒線體上，parkin也從細胞質被誘導轉至粒線體，而simvastatin更是強化了PINK1/parkin這樣的集中粒線體分布。影像學分析的結果顯示simvastatin可以有效減小根尖病變的範圍，尤其是將之作為根管局部用藥時治療效果更加顯著。組織免疫染色的結果也確認了simvastatin會增強成骨細胞中PINK1/parkin的表現。總結以上所述，我們發現缺氧會刺激成骨細胞啟動mitophagy。此外，無論在in vitro或in vivo的研究，simvastatin都會增強mitophagy的進行。這樣的結果暗示simvastatin會藉由促使成骨細胞中mitophagy機制以防止細胞走向凋亡，進而減緩根尖病變中的發炎性骨吸收。

關鍵字

simvastatin ； mitophagy ； PINK1 ； parkin ；細胞凋亡

並列摘要

Mitochondria are the organelles that deal with hypoxia, a potential therapeutic target for the treatment of inflammatory diseases. Through mitophagy, a process of mitochondrial degradation by autophagy, cells rid themselves of dysfunctional components to maintain mitochondrial homeostasis and genetic integrity. Simvastatin is a widely used anti-hyperlipidemia drug that also possesses anti-inflammatory function. We had previously demonstrated that simvastatin diminished the progression of periapical lesions by modulating autophagy and apoptosis in osteoblasts. However, the underlying mechanisms remained to be elucidated. In this study, we focused on mitochondria. We examined the effect of simvastatin on hypoxia-stimulated expression of PTEN-induced putative kinase 1（PINK1）/ parkin（team players in promotion of mitophagy）and poly (adenosine phosphate ribose) polymerase (PARP) fragmentation (an apoptosis marker) in MC3T3-E1 by western analysis. We also monitored the subcellular localization of PINK1 and parkin with immunofluorescence. In rat models of induced periapical lesions, different routes of administration of simvastatin were assessed radiographically and immunohistochemically for its therapeutic potential. Western blot showed elevated levels of PINK/parkin and PARP cleavage after hypoxia stimulation. Simvastatin enhanced hypoxia-induced PINK1/parkin and decreased PARP fragmentation. We detected that PINK1 accumulated in the mitochondria and parkin was recruited to the mitochondria when treated in hypoxia and simvastatin promoted the redistribution under confocal microscope. Radiography revealed that simvastatin reduced the extension of periapical lesions in rats, especially when simvastatin used as local application. Immunohistopathology confirmed that the number of PINK1/parkin–synthesizing osteoblasts increased after simvastatin treatment. In conclusion, we found that hypoxia stimulated mitophagy mechanism in osteoblasts. In addition, simvastatin enhanced mitophagy both in vitro and in vivo. Our data implied that simvastatin might alleviate the progression of inflammatory bone resorption in periapical lesions by promoting mitophagy to protect osteoblasts from going to apoptosis.

並列關鍵字

simvastatin ； mitophagy ； PINK1 ； parkin ； apoptosis

參考文獻

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Simvastatin可以藉由調節成骨細胞中mitophagy的機制來減緩發炎性骨吸收

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