Erhöhte NF-κB- und iNOS-Expression in Keratozyten von Keratokonuspatienten – Hinweise auf eine entzündliche Komponente?

Zusammenfassung

Hintergrund In den letzten Jahren mehren sich Hinweise auf eine entzündliche Komponente beim Keratokonus (KC). Ein Schlüsselgen bei entzündlichen Prozessen ist der Nuclear Factor Kappa B (NF-κB). NF-κB ist ein Transkriptionsfaktor, der unter anderem das Enzym Nitric Oxide Synthase (NOS), das mit dem konkurrierenden Enzym Arginase (Arg) bei entzündlichen Prozessen involviert ist, aktiviert. Ziel dieser Studie war es, die Isotypen von NOS und Arginase zu analysieren, die Expression NF-κB, NOS und Arginase sowie den regulativen Mechanismus von NOS und Arginase in Keratozyten von Keratokonuspatienten mithilfe des Inhibitors 1400W in vitro zu untersuchen.

Methoden Primäre humane Keratozyten wurden durch enzymatische Behandlung mit Kollagenase A aus humanen Korneoskleralscheiben (n = 8) und von Explantaten von geplanten perforierenden Keratoplastiken (KC-Patienten) isoliert (n = 8) und in DMEM/F12-Kulturmedium, versetzt mit 5% fetalem Kälberserum, kultiviert. Die Expression von NF-κB, NOS und Arginase wurden mit quantitativer PCR (qPCR) und Westernblot-Analyse (WB) untersucht. Nitrit- und Ureakonzentrationen im Zellkulturüberstand wurden nach Zugabe des NOS-Inhibitors 1400W (0 – 40 µM) analysiert.

Ergebnisse In den Keratozyten wurden ausschließlich die Isotypen iNOS (induzierbare NO-Synthase) und Arg-II nachgewiesen. Die mRNA-Expression von NF-κB und iNOS waren in KC-Keratozyten höher als in normalen Zellen (p = 0,0135 und p = 0,0001), während in der Arg-II-Expression keine Unterschiede messbar waren. Im WB war bei NF-κB eine höhere Bandenintensität messbar (p = 0,0012), bei iNOS konnten keine Unterschiede in der Bandenintensität nachgewiesen werden. Im Überstand der KC-Keratozyten wurden geringere Konzentrationen von Nitrit und Urea nach Zugabe des Inhibitors 1400W gemessen (p = ≤ 0,014), nicht jedoch bei normalen Zellen (p ≥ 0,178).

Schlussfolgerung Aufgrund der erhöhten Expression von NF-κB und iNOS muss von einer inflammatorischen Komponente beim Keratokonus ausgegangen werden. Die unterschiedliche Regulation der KC-Keratozyten durch den iNOS-Inhibitor 1400W legt eine veränderte metabolische Aktivität nahe, die durch entzündliche Prozesse hervorgerufen werden kann.

Abstract

Purpose In recent years, there has been increasing evidence of an inflammatory component in keratoconus. A key gene in inflammatory processes is the nuclear factor kappa B (NF-κB). NF-κB is a transcription factor for the enzyme nitric oxide synthase (NOS), which is involved with the competing enzyme arginase (Arg) in inflammatory processes. The aim of this study was to analyze the isotypes of NOS and arginase, the expression of NF-κB, NOS and arginase, and the regulatory mechanism of NOS and arginase in keratocytes of keratoconus patients using the inhibitor 1400W in vitro.

Methods Human keratocytes were isolated from surgically removed corneas of 8 KC patients and 8 normal human corneal buttons and were cultured to confluence, in vitro. Quantitative PCR and Western blot analysis were performed to examine NF-κB, NOS and arginase expression in keratocytes. Nitrite and urea concentrations in the supernatant of the cells were analyzed using 0 – 40 µM 1400W iNOS inhibitor concentrations.

Results Only the isotypes iNOS and Arg-II were detected in the keratocytes. The mRNA expression of NF-κB and iNOS were higher in KC keratocytes than in normal cells (p = 0.0135 and p = 0.0001), whereas no differences were measurable in Arg-II expression. In the WB, a higher band intensity was measurable in NF-κB (p = 0.0012), and in iNOS, no differences in band intensity could be detected. In the supernatant of the KC keratocytes, lower concentrations of nitrite and urea were measured after the addition of the inhibitor 1400W (p ≤ 0.014), but not in normal cells (p ≥ 0.178).

Conclusion Due to the increased expression of NF-κB and iNOS, an inflammatory component in keratoconus must be assumed. The different regulation of the KC keratocytes by the iNOS inhibitor 1400W suggests an altered metabolic activity which can be caused by inflammatory processes.

Schlüsselwörter

Keratokonus - NF-κB - iNOS - Arginase II - Entzündung

Key words

keratoconus - NF-κB - iNOS - arginase II - inflammation

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