Cao Chong, Ji Xiaowei, Luo Xin, Zhong Liangjun
Department of Periodontology, The First Affiliated Hospital of Xinjiang Medical University Urumqi 830054, China.
Department of Prosthodontics, The First Affiliated Hospital of Xinjiang Medical University Urumqi 830054, China.
Int J Clin Exp Med. 2015 Oct 15;8(10):18327-34. eCollection 2015.
The aim of the present study was to ascertain the effect of Porphyromonas gingivalis cysteine protease gingipain on the proliferation of rat aortic smooth muscle cells (RASMCs). Gingipains were isolated and purified from the supernatant of P. gingivalis W83, which was cultured under standard anaerobic conditions; primary RASMCs were also cultured. RASMCs were exposed to 200, 100, 50, 25, 12, 6, 3, 1, and 0 μg/mL activated gingipains and the proliferation was evaluated using a cell counting kit-8 (CCK-8) assay after 48 h. α-Smooth muscle actin (α-SMA) and osteopontin (OPN) expression were measured by immunohistochemical staining. In addition, RASMCs were stimulated with 5, 10, 20, and 40 μM KYT-1 (arg-gingipain inhibitor) and KYT-36 (lys-gingipain inhibitor) in combination with the gingipain extracts. Different concentrations of gingipains significantly promoted the proliferation of RASMCs, except those treated with 1 μg/mL, compared to the untreated controls. The proliferation was sustained at a concentration above 12 μg/mL. Immunohistochemical staining showed OPN expression after gingipain stimulation. The proliferative effects of gingipains on RASMCs were blocked after treatment with 10 μM KYT-1 or 10 μM KYT-36 (P < 0.0001); however, the difference between KYT-1 and KYT-36 groups was not statistically significant. These results demonstrated that gingipains can promote phenotypic transformation and proliferation of RASMCs and their effects were blocked by KYT-1 and KYT-36, which help us to ascertain whether Rgp or Kgp contributes to the development of atherosclerosis.
本研究的目的是确定牙龈卟啉单胞菌半胱氨酸蛋白酶牙龈蛋白酶对大鼠主动脉平滑肌细胞（RASMCs）增殖的影响。牙龈蛋白酶从在标准厌氧条件下培养的牙龈卟啉单胞菌W83的上清液中分离和纯化；同时也培养原代RASMCs。将RASMCs暴露于200、100、50、25、12、6、3、1和0μg/mL的活化牙龈蛋白酶中，48小时后使用细胞计数试剂盒-8（CCK-8）测定法评估细胞增殖。通过免疫组织化学染色测量α-平滑肌肌动蛋白（α-SMA）和骨桥蛋白（OPN）的表达。此外，用5、10、20和40μM的KYT-1（精氨酸-牙龈蛋白酶抑制剂）和KYT-36（赖氨酸-牙龈蛋白酶抑制剂）与牙龈蛋白酶提取物联合刺激RASMCs。与未处理的对照组相比，除了用1μg/mL处理的细胞外，不同浓度的牙龈蛋白酶显著促进了RASMCs的增殖。在浓度高于12μg/mL时增殖持续存在。免疫组织化学染色显示牙龈蛋白酶刺激后OPN表达。用10μM KYT-1或10μM KYT-36处理后，牙龈蛋白酶对RASMCs的增殖作用被阻断（P<0.0001）；然而，KYT-1组和KYT-36组之间的差异无统计学意义。这些结果表明牙龈蛋白酶可以促进RASMCs的表型转化和增殖，并且它们的作用被KYT-1和KYT-36阻断，这有助于我们确定Rgp或Kgp是否促成动脉粥样硬化的发展。
