牙齦卟啉單胞菌之外膜囊泡經由活化牙齦蛋白酶加劇根尖病變的進展

Abstract

根尖病變是牙髓感染後引發之慢性免疫性炎症，其臨床上以根尖骨吸收為主要表現。近年研究指出，牙周病菌 Porphyromonas gingivalis（Pg）於其生長過程中可釋放外膜囊泡（outer membrane vesicles, OMVs），其中富含牙齦蛋白酶（gingipains），被認為是影響遠端組織的重要致病因子。本研究旨在探討Pg-OMVs是否可藉由gingipains活化粒線體相關凋亡路徑，促進成骨細胞凋亡，進而加劇根尖病變之發展。 實驗設計包括以PCR確認臨床樣本中gingipain之表現，並自野生型Pg及gingipains缺損突變株KDP136中純化OMVs。於MC3T3-E1細胞中檢測粒線體膜電位變化、活性氧產生與細胞凋亡標記之變化，並以大鼠動物模式評估Pg-OMVs對根尖病變體積與組織病理之影響。 結果顯示，Pg-OMVs能明顯誘導成骨細胞粒線體膜電位下降與活性氧累積，伴隨cytochrome C釋放及caspase-9與caspase-3活化，誘發細胞凋亡，惟KDP136產生之OMVs不具此效應。在動物實驗中，Pg-OMVs處理組之根尖病變面積與體積皆呈上升趨勢，組織染色亦顯示明顯細胞死亡與發炎浸潤。 綜合而言，Pg-OMVs可藉由gingipains活化粒線體路徑引發成骨細胞凋亡，進而促進根尖病變之進程。未來研究將進一步以免疫組織化學法（IHC）驗證病灶中細胞凋亡情形，並分析KDP136動物實驗結果，同時引入已知gingipains抑制劑（KYT-1、KYT-36、KYT-41）進行治療實驗，以評估其抑病潛力。

Periapical lesions are chronic inflammatory conditions initiated by pulp infection, typically characterized by apical bone resorption. Recent evidence suggests that Porphyromonas gingivalis (Pg), a key periodontal pathogen, secretes outer membrane vesicles (OMVs) enriched in gingipains—its principal virulence factors—which may affect distant tissues. This study aimed to investigate whether Pg-OMVs promote osteoblast apoptosis via gingipain-mediated mitochondrial pathways, thereby aggravating the progression of apical lesions. PCR was conducted to assess gingipains expression in clinical specimens. OMVs were isolated from wild-type Pg and a gingipains-deficient mutant strain KDP136. In vitro, MC3T3-E1 osteoblasts were evaluated for mitochondrial membrane potential, reactive oxygen species, and apoptotic markers. In vivo, a mouse model was employed to analyze the impact of Pg-OMVs on periapical lesion volume and histopathology. Pg-OMVs significantly induced mitochondrial dysfunction in osteoblasts, including membrane potential loss, ROS accumulation, cytochrome C release, and caspase-9/caspase-3 activation, all contributing to apoptosis. In contrast, OMVs from KDP136 did not elicit such effects. In vivo, Pg-OMV treatment led to increased apical lesion volume, and histological staining revealed enhanced cell death and inflammatory infiltration. In conclusion, Pg-OMVs aggravate apical lesion progression by inducing osteoblast apoptosis via gingipain-triggered mitochondrial pathways. Future studies will utilize immunohistochemistry to verify apoptotic activity in tissues, complete analysis of ongoing KDP136 animal models, and assess the therapeutic efficacy of gingipain inhibitors (KYT-1, KYT-36, KYT-41).