Miyazawa Masaharu, Suzuki Hidekazu, Masaoka Tatsuhiro, Kai Akemi, Suematsu Makoto, Nagata Hiroshi, Miura Soichiro, Ishii Hiromasa
Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan.
Free Radic Biol Med. 2003 Jun 15;34(12):1621-30. doi: 10.1016/s0891-5849(03)00218-1.
Deregulated cell turnover in Helicobacter pylori (H. pylori)-colonized gastric mucosa has been suggested to be linked to the gastric carcinogenesis pathway. We previously reported attenuation of apoptosis and enhancement of cellular proliferation in the H. pylori-colonized gastric mucosa of Mongolian gerbils as compared to that in mice, which might reflect a specific link between H. pylori colonization and carcinogenesis in the Mongolian gerbils; the difference between the two strains could be attributable to differences in the host genetic background. Inducible-type nitric oxide synthase (iNOS) is thought to participate in not only the inflammatory response, but also in the regulation of gastric mucosal cell turnover in H. pylori-colonized gastric mucosa. Thus, the present study was designed to examine gastric leukocyte activation and epithelial cell apoptosis in the gastric mucosa following H. pylori inoculation in iNOS-knockout mice.
iNOS-knockout mice (iNOS(-/-)) and their iNOS(+/+) littermates were orally inoculated with the Sydney strain of H. pylori (SS1, 10(8) colony-forming units [CFU]). H. pylori infection was confirmed by microaerobic bacterial culture. The stomach of each mouse was evaluated 14 weeks and 30 weeks after the inoculation. Gastric mucosal accumulation of polymorphonuclear leukocytes (PMN) was assessed by determining the myeloperoxidase (MPO) activity and histological score based on the updated Sydney system. The level of apoptosis was determined by estimation of the cytoplasmic levels of mono- and oligonucleosomes and by the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method.
The SS1-inoculated mice showed persistent H. pylori colonization for 12 weeks. While gastric mucosal PMN infiltration increased following SS1 inoculation in both iNOS(+/+) and iNOS(-/-)strains, enhanced DNA fragmentation was observed in only SS1-colonized iNOS(+/+) mice, and not in the iNOS(-/-) mice. In conclusion, although the recruitment of PMN in response to H. pylori was evoked even in the gastric mucosa of iNOS(-/-) mice, epithelial cell apoptosis induced by H. pylori was attenuated in this strain. These data suggest that iNOS may play an important role in promoting apoptosis in the H. pylori-infected inflamed gastric mucosa, and that persistent inflammation without apoptosis in iNOS(-/-) mice with H. pylori infection may be linked to preneoplastic transformation.
幽门螺杆菌（H. pylori）定植的胃黏膜中细胞更新失调被认为与胃癌发生途径有关。我们之前报道，与小鼠相比，蒙古沙鼠幽门螺杆菌定植的胃黏膜中细胞凋亡减弱而细胞增殖增强，这可能反映了幽门螺杆菌定植与蒙古沙鼠致癌作用之间的特定联系；这两种品系之间的差异可能归因于宿主遗传背景的不同。诱导型一氧化氮合酶（iNOS）不仅被认为参与炎症反应，还参与幽门螺杆菌定植的胃黏膜中胃黏膜细胞更新的调节。因此，本研究旨在检测iNOS基因敲除小鼠接种幽门螺杆菌后胃黏膜中的胃白细胞活化和上皮细胞凋亡情况。
iNOS基因敲除小鼠（iNOS(-/-)）及其iNOS(+/+)同窝小鼠经口接种幽门螺杆菌悉尼株（SS1，10(8) 菌落形成单位 [CFU]）。通过微需氧细菌培养确认幽门螺杆菌感染。在接种后14周和30周对每只小鼠的胃进行评估。通过测定髓过氧化物酶（MPO）活性和基于更新后的悉尼系统的组织学评分来评估胃黏膜中多形核白细胞（PMN）的积聚情况。通过估计单链和寡核小体的细胞质水平以及采用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记法来确定凋亡水平。
接种SS1的小鼠显示幽门螺杆菌持续定植12周。虽然在iNOS(+/+)和iNOS(-/-)品系中接种SS1后胃黏膜PMN浸润均增加，但仅在定植SS1的iNOS(+/+)小鼠中观察到DNA片段化增强，而在iNOS(-/-)小鼠中未观察到。总之，尽管即使在iNOS(-/-)小鼠的胃黏膜中也能引发对幽门螺杆菌的PMN募集，但该品系中幽门螺杆菌诱导的上皮细胞凋亡减弱。这些数据表明，iNOS可能在促进幽门螺杆菌感染的炎症性胃黏膜中的细胞凋亡中起重要作用，并且在感染幽门螺杆菌的iNOS(-/-)小鼠中无细胞凋亡的持续性炎症可能与肿瘤前转化有关。
