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Analysis of apoptosis in alveolar macrophages induced by periodontopathic bacteria and development of a method of protection from pneumonia

牙周病细菌诱导的肺泡巨噬细胞凋亡分析及肺炎防护方法的开发

基本信息

批准号：
11557169
负责人：
NISHIHARA Tatsuji
金额：
$ 6.27万
依托单位：
Kyushu Dental College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

项目摘要

Infection of murine macrophages in vitro with periodontopathic bacterium Actinobacillus actinomycetemcomitans induces apoptotic cell death. In this study, we investigated the involvement of caspases in apoptotic cell death of A.actinomycetemcomitans-infected macrophages. Two peptide inhibitors of caspases, benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone(Z-VAD-FMK)and benzyloxycarbonyl-Asp-Glu-Val-Asp(OMe)-fluoromethyl katone(Z-DEVD-FMK), inhibited apoptotic cell death of mouse macrophage cell line J774.1 infected with A.actinomycetemcomitans. During the process of apoptosis, interleukin-1β(IL-1β) was detected in the culture supernatants of J774.1 cells. IL-1β secretion was blocked by the caspase-1 inhibitor, Z-VAD-FMK, indicating that caspase-1 is involved in not only the induction of apoptosis but also the IL-1β secretion from A.actinomycetemcomintas-infected J774.1 cells. Immunoblot analysis revealed that the infection of A.actinomycetemcomitans to J774.1 cells induced the cleavage of retinoblastoma protein(Rb), suggesting that caspase-3 was activated by A.actinomycetemcomitans-infection. The cytosol from A.actinomycetemcomitans-infected J774.1 cells induced Rb proteollysis in vitro, which was inhibited by the caspase-3 inhibitor, Z-DEVD-FMK.Furthermore, caspase-3-like activity was markedly increased in J774.1 cells infected with A.actinomycetemcomitans between 12h and 24 h, which was subsequently inhibited by the addition of caspase-3 inhibitor, Z-DEVD-FMK.These findings indings indicate that caspase-3 induces apoptosis in J774.1 cells infected with A.actinomycetemcomitans. Taken together, these results suggest that caspase-1 and caspase-3 are involved in the induction of apoptosis in A.actinomycetemcomitans-infected macrophages.

牙周病细菌放线菌体外感染小鼠巨噬细胞可诱导凋亡细胞死亡。在这项研究中，我们研究了caspase在a.c ray omycetemcomitans感染巨噬细胞凋亡中的作用。两种半胱天冬酶肽抑制剂，苄氧羰基-缬氨酸- α - asp (OMe)-氟甲基酮（Z-VAD-FMK）和苄氧羰基- asp -葡氨酸-缬氨酸- asp (OMe)-氟甲基酮（Z-DEVD-FMK），抑制了小鼠巨噬细胞系J774.1感染a .放线菌temcomitans的凋亡细胞死亡。凋亡过程中，J774.1细胞培养上清液中检测到白细胞介素-1β(IL-1β)。IL-1β的分泌被caspase-1抑制剂Z-VAD-FMK阻断，表明caspase-1不仅参与了a.t actinomycetemcomintas感染的J774.1细胞的凋亡诱导，还参与了IL-1β的分泌。免疫印迹分析显示，a.c ray - omycetemcomitans感染J774.1细胞后，诱导了视网膜母细胞瘤蛋白（retinoblastoma protein, Rb）的裂解，提示a.c ray - omycetemcomitans感染后激活了caspase-3。a .actinomycetemcomitans感染的J774.1细胞胞浆在体外诱导Rb蛋白水解，并被caspase-3抑制剂Z-DEVD-FMK抑制。此外，caspase-3-like活性在感染a .放线菌的J774.1细胞中在12h至24h之间显著增加，随后被caspase-3抑制剂Z-DEVD-FMK抑制。以上结果表明，caspase-3可诱导放线菌感染的J774.1细胞凋亡。综上所述，这些结果表明caspase-1和caspase-3参与了a.t actinomycetemcomitans感染巨噬细胞的凋亡诱导。