Purpose: To investigate expression and role of nucleotide-binding oligomerization domain 2 (NOD2) in the ocular surface of experimental dry eye (EDE), which is a nod-like receptor member and is involved in innate immune response. Methods: C57BL/6 female mice were divided into the groups: untreated (UT), EDE, and NOD2 knockout (KO) mice exposed to desiccating stress for 14 days. Clinical parameters were measured at 7 and 14 days. Immunofluorescent staining for NOD2 and Western blot for RIP2 and nuclear factor kappa B (NF-κB) were performed at 14 days. Levels of inflammatory cytokines were measured using multiplex immunobead assay. Flow cytometry, PAS staining, and TUNEL staining were performed. Results: After desiccating stress, NOD2 was expressed in the corneal epithelium of the EDE group. The EDE group showed a significantly increased RIP2 expression compared to the UT and NOD2 KO groups. A significantly lower expression of NF-κB and lower levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ, and IL-6 were noted in the NOD2 KO group than in the EDE group. The NOD2 KO group had lower CD11b+ and CD4+CCR5+ T cells, TUNEL positive cells and corneal staining score and higher density of conjunctival goblet cell density, tear volume, and tear film break-up time than the EDE group. The UT group showed significant differences in inflammatory and clinical parameters compared to the EDE and NOD2 KO groups.Conclusions: The NOD2 receptor pathway induced inflammation and apoptosis by activation of RIP2 and NF-κB on the ocular surface of EDE, thereby reducing tear secretion. Therefore, NOD2 pathway may be involved in the pathogenesis of dry eye.
목적: 건성안의 안구 표면에서 nod-like 수용체 구성원이며 선천 면역 반응에 관여하는 nucleotide-binding Oligomerization Domain 2 (NOD2)의 발현과 역할을 알아보고자 하였다.방법: C57BL/6 생쥐를 정상군(1군), 대조군(2군)과 NOD2 knockout군(3군)으로 나누었고, 2군과 3군에서 14일간 건성안을 유발하였다. 유발 7일과 14일째 눈물분비량과 눈물막파괴시간을 측정하고 형광염색 정도를 점수화하였다. 유발 14일째 Immunofluorescent staining을 통해 NOD2D의 발현을 평가하였고, Western blot를 통해 RIP2와 NF-κB의 발현을 평가하였다. Multiplex immunobead assay를 이용하여 염증인자 IL-1β, IL-6, TNF-a, IFN-γ를 측정하였고, flow cytometry를 이용하여 CD11b+ 및 CD4+ CCR5+ T 세포를 측정하였다. PAS 염색을 이용하여 결막 술잔세포수를 측정하였고, TUNEL를 이용하여 각, 결막 세포의 사멸을 관찰하였다.결과: 건성안 유발 후, 2군 각막상피에서 NOD2의 발현을 보였다. 유발 7일과 14일째 3군은 2군에 비해 눈물분비량과 눈물막파괴시간 및 형광염색 점수가 유의하게 높았다 (P