Porphyromonas gingivalis 자극된 RAW 264.7 cells에서 Weissella cibaria CMU의 항염 효과
Anti-inflammatory effect of Weissella cibaria CMU in Porphyromonas gingivalis-stimulated RAW 264.7 cells
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초록
본 연구는 건강한 어린이의 타액에서 분리한 프로바이오틱스 균주인 Weissella cibaria CMU (oraCMU)가 Porphyromonas gingivalis (P. gingivalis)로 자극된 RAW 264.7 세포에 미치는 항염증 효과를 확인하고자 하였다. RAW 264.7 세포에 oraCMU를 multiplicity of infection (MOI) 0.1, 1, 10, 100으로 처리하여 MTS assay를 진행하였고, 이를 통해 세포 생존율을 측정하였다. Nitric oxide (NO) 생성량을 측정하기 위해 RAW 264.7 세포에 oraCMU를 MOI 0.1, 1, 10으로 처리한 후 P. gingivalis로 자극하여 NO assay를 진행하였다. Interleukin (IL)-1β및 IL-6 pro-inflammatory cytokines의 발현은 qRT-PCR을 시행하여 mRNA 양을 측정하였다. 신호전달계에서 염증성 전사인자의 발현 경로를 확인하기 위하여 western blot을 통해 핵 내로의 NF-κB 이동을 확인하여 oraCMU의 항염증 기전을 연구하였다. MTS assay 결과 MOI 0.1, 1, 10, 100에서 세포 생존율은 유의한 변화가 없음을 확인하였다. 또한 NO assay를 통해 P. gingivalis 유도된 RAW 264.7 세포에서 oraCMU 처리에 의해 NO 생성이 억제됨을 확인하였다. qRT-PCR을 통해 IL-1β와 IL-6의 mRNA 양을 측정한 결과 P. gingivalis에 의해 염증 반응이 유도된 RAW 264.7 세포에서 oraCMU 처리에 의해 pro-inflammatory cytokines의 발현이 감소하였음을 확인할 수 있었고, western blot 결과 핵 내의 NF-κB의 단백질 발현 또한 oraCMU 처리에 의해 억제됨을 확인하였다. 이러한 결과를 통해 P. gingivalis에 의해 염증반응이 유도된 RAW 264.7 세포에서 oraCMU는 항염증 효과를 보였으며, 이를 토대로 향후 식‧의약품 소재 개발에서 치주염 개선 가능성이 있는 후보물질이 될 수 있을 것으로 사료된다.
Abstract
This study aimed to investigate the anti-inflammatory effects of Weissella cibaria CMU (oraCMU), a probiotic strain isolated from the saliva of healthy children, on RAW 264.7 cells stimulated by Porphyromonas gingivalis (P. gingivalis). RAW 264.7 cells were treated with oraCMU at multiplicities of infection (MOI) of 0.1, 1, 10, and 100, and cell viability was assessed using an MTS assay. Nitric oxide (NO) production was measured using NO assay after treating RAW 264.7 cells with oraCMU at MOIs of 0.1, 1, and 10, followed by stimulation with P. gingivalis. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure mRNA levels of the pro-inflammatory cytokines interleukin (IL)-1β and IL-6. To investigate the signaling pathway of inflammatory transcription factor expression, Western blotting was performed to analyze the nuclear translocation of NF-κB and clarify the anti-inflammatory mechanism of oraCMU. The MTS assay showed no significant changes in cell viability at MOIs of 0.1, 1, 10, and 100. NO assay demonstrated that oraCMU treatment inhibited NO production in P. gingivalis-stimulated RAW 264.7 cells. Additionally, qRT-PCR analysis revealed reduced mRNA expression of IL-1β and IL-6 in P. gingivalis-stimulated RAW 264.7 cells treated with oraCMU. Western blot analysis further confirmed that oraCMU treatment inhibited NF-κB nuclear translocation. These findings suggest that oraCMU exerts anti-inflammatory effects on P. gingivalis-stimulated RAW 264.7 cells and may be a potential candidate for the development of food and pharmaceutical products for periodontitis treatment.
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