介白素-1β對人類牙髓細胞的細胞間黏附因子-1和單核細胞趨化蛋白-1的影響:PI3K/Akt和MEK/ERK 1/2訊息路徑與環氧酶的角色

Effect of interleukin-1β on the ICAM-1 and MCP-1 expression of human dental pulp cells : Role of PI3K/Akt and MEK/ERK 1/2 Signaling and Cyclooxygenase

洪琇品(Hsiu-Pin Hung)

指導教授 ： 鄭景暉

國立臺灣大學/牙醫專業學院/臨床牙醫學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.00706

摘要

實驗目的: 在牙髓中，介白素-1β（IL-1β）是一種重要的發炎介質。 IL-1β會刺激環氧酶-2（COX-2）並產生前列腺素 (prostanoids)，進而影響牙髓的發炎和癒合過程。然而，當人類的牙髓組織發炎時， IL-1β對於細胞間黏附分子-1（ICAM-1）和單核細胞趨化蛋白-1 (MCP-1)的影響以及其與PI3K/Akt (也叫做Protein Kinase B)和MEK/ERK 1/2訊息傳遞路徑和COX活化之關係仍不完全清楚。
實驗方法: 在不同的時間點使用aspirin (COX抑制劑)或是LY294002 (PI3k/Akt抑制劑)或是U0126 (MEK/ERK 1/2抑制劑)加在人類的牙髓細胞中，檢查IL-1β是否經由這幾種的訊息傳遞路徑來調控牙髓細胞ICAM-1與MCP-1。用MTT的方法檢查是否這些IL-1β和這些抑制劑是否有細胞毒性。用反轉錄聚合酶連鎖反應的方法(RT-PCR)和西方點墨法(western blot)檢查IL-1β是否有刺激牙髓細胞ICAM-1和MCP-1的基因表現和蛋白質產生。用磷酸化的Akt和ERK 1/2抗體以西方點墨法確認IL-1β是否可活化這幾種的訊息傳遞路徑。
實驗結果: IL-1β會刺激牙髓細胞ICAM-1和MCP-1的基因表現和蛋白質產生。Aspirin同時處理會讓IL-1β刺激之牙髓細胞ICAM-1和MCP-1的基因表現增加。而這些訊息傳遞路徑有經過PI3K/Akt和MEK/ERK 1/2。另外， LY294002和U0126都可抑制IL-1β刺激之牙髓細胞ICAM-1和MCP-1的基因表現增加。
實驗結論: 這些結果表示，IL-1β在牙髓發炎和修復的過程中會產生ICAM-1，MCP-1的基因表達與蛋白質產生。這些過程都和PI3K/Akt和MEK/ERK 1/2的訊息傳遞訊息和COX的活化有關。這些結果對於牙髓發炎的治療都是有幫助的。

關鍵字

PI3K/Akt ； MEK/ERK 1/2 ； 環氧酶 ； 牙髓細胞 ； 發炎 ； 介白素-1β ； 細胞間黏附分子-1 ； 單核細胞趨化蛋白-1

並列摘要

Aim: Interleukin-1β (IL-1β) is an important inflammatory mediator of the dental pulp. IL-1β may stimulate cyclooxygenase-2 (COX-2) and prostanoids production of pulp cells and affect the inflammatory and healing processes of dental pulp. However, the effects of IL-1β on Intercellular Adhesion Molecule-1 (ICAM-1) and Monocyte Chemotactic Factor-1 (MCP-1) of human dental pulp cells and its relation to PI3K/Akt and MEK/ERK 1/2 signaling and COX activation are still not clear.
Materials and Methods: Human dental pulp cells were treated with IL-1β with/without pretreatment and co-incubation by aspirin (a COX inhibitor) or LY294002 (a PI3K/Akt inhibitor) or U0126 ( a MEK/ERK 1/2 inhibitor) for different time periods. Viable cell number was evaluated by 3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The expression of ICAM-1 and MCP-1 mRNA was tested by reverse transcriptase – polymerase chain reaction (RT-PCR). ICAM-1 and MCP-1 protein expression was examined by western blotting. Phosphorylation of Akt and ERK 1/2 was measured by western blot.
Results: IL-1β showed little cytotoxicity to pulp cells. It stimulated ICAM-1 and MCP-1 mRNA and protein expression. Aspirin enhanced the IL-1β-induced ICAM-1 and MCP-1 mRNA expression and protein production. IL-1β rapidly activated Akt and ERK 1/2. Both LY294002 and U0126 attenuated the IL-1β-induced ICAM-1and MCP-1 mRNA expression.
Conclusions: These results reveal that IL-1β may involve in the pulpal inflammatory and healing processes by stimulation of ICAM-1and MCP-1 mRNA expression and protein production. These events are associated with differential activation of PI3K/Akt and MEK/ERK 1/2 signaling and COX. These results are useful for future treatment of pulpal inflammatory diseases.

並列關鍵字

PI3K/Akt ； MEK/ERK 1/2 ； Cyclooxygenase (COX) ； Dental pulp cells ； Inflammation ； Interleukin-1β(IL-1β) ； Intercellular adhesion molecule-1 (ICAM-1) ； Monocyte Chemotactic Factor-1 (MCP-1)

參考文獻

Ansai, T., Yamamoto, E., Awano, S., Yu, W., Turner, A. J. & Takehara, T. (2002) Effects of periodontopathic bacteria on the expression of endothelin-1 in gingival epithelial cells in adult periodontitis. Clin Sci (Lond) 103 Suppl 48, 327S-331S.

Bachelor, M. A., Cooper, S. J., Sikorski, E. T. & Bowden, G. T. (2005) Inhibition of p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase decreases UVB-induced activator protein-1 and cyclooxygenase-2 in a SKH-1 hairless mouse model. Mol Cancer Res 3, 90-99.

Bagis, B., Atilla, P., Cakar, N. & Hasanreisoglu, U. (2009) An immunohistochemical evaluation of cell adhesion molecules in human dental pulp after tooth preparation and application of temporary luting cements. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 107, 137-144.

Bian, Z. M., Elner, S. G., Yoshida, A., Kunkel, S. L., Su, J. & Elner, V. M. (2001) Activation of p38, ERK1/2 and NIK pathways is required for IL-1beta and TNF-alpha-induced chemokine expression in human retinal pigment epithelial cells. Exp Eye Res 73, 111-121.

Bletsa A, Fristad I, Berggreen E. (2009) Sensory pulpal nerve fibres and trigeminal ganglion neurons express IL-1RI: a potential mechanism for development of inflammatory hyperalgesia. Int Endod J 42, 978-86.