Regulation of ghrelin receptor by microbial and inflammatory signals in human osteoblasts

Abstract: Recently, it has been suggested that the anti-inflammatory hormone ghrelin (GHRL) and its receptor GHS-R may play a pivotal role in periodontal health and diseases. However, their exact regulation and effects in periodontitis are not known. The aim of this in-vitro study was to investigate the effect of microbial and inflammatory insults on the GHS-R1a expression in human osteoblast-like cells. MG-63 cells were exposed to interleukin (IL)-1β and Fusobacterium nucleatum in the presence and absence of GHRL for up to 2 d. Subsequently, gene expressions of GHS-R1a, inflammatory mediators and matrix metalloproteinase were analyzed by real-time PCR. GHS-R protein synthesis and NF-κB p65 nuclear translocation were assessed by immunocytochemistry and immunofluorescence microscopy, respectively. IL-1β and F. nucleatum caused a significant upregulation of GHS-R1a expression and an increase in GHS-R1a protein. Pre-incubation with a MEK1/2 inhibitor diminished the IL-1β-induced GHS-R1a upregulation. IL-1β and F. nucleatum also enhanced the expressions of cyclooxygenase 2, CC-chemokine ligand 2, IL-6, IL-8, and matrix metalloproteinase 1, but these stimulatory effects were counteracted by GHRL. By contrast, the stimulatory actions of IL-1β and F. nucleatum on the GHS-R1a expression were further enhanced by GHRL. Our study provides original evidence that IL-1β and F. nucleatum regulate the GHS-R/GHRL system in osteoblast-like cells. Furthermore, we demonstrate for the first time that the proinflammatory and proteolytic actions of IL-1β and F. nucleatum on osteoblast-like cells are inhibited by GHRL. Our study suggests that microbial and inflammatory insults upregulate GHS-R1a, which may represent a protective negative feedback mechanism in human bone.

摘要：近年来，有研究指出抗炎激素饥饿素（ghrelin, GHRL）及其受体生长激素促分泌素受体（GHS-R）可能在牙周健康与疾病中发挥关键作用。然而，二者在牙周炎中的具体调控机制与效应仍未明确。本项体外研究旨在探究微生物与炎性刺激对人成骨样细胞中GHS-R1a表达的影响。将MG-63细胞置于添加或不添加GHRL的培养环境中，分别用白细胞介素（IL）-1β与具核梭杆菌处理，处理时长最长达2天。随后通过实时荧光定量PCR分析GHS-R1a、炎性介质及基质金属蛋白酶的基因表达水平；分别采用免疫细胞化学与免疫荧光显微镜术检测GHS-R蛋白合成情况及NF-κB p65核转位水平。实验结果显示，IL-1β与具核梭杆菌可显著上调GHS-R1a的基因表达，并提升GHS-R1a的蛋白水平。MEK1/2抑制剂预孵育可削弱IL-1β诱导的GHS-R1a上调效应。IL-1β与具核梭杆菌还可增强环氧合酶2、CC趋化因子配体2、IL-6、IL-8及基质金属蛋白酶1的表达，但上述促炎效应可被GHRL拮抗。与之相反，GHRL可进一步增强IL-1β与具核梭杆菌对GHS-R1a表达的促上调作用。本研究提供了原创性证据，证明IL-1β与具核梭杆菌可调控成骨样细胞中的GHS-R/GHRL系统。此外，本研究首次证实，GHRL可抑制IL-1β与具核梭杆菌对成骨样细胞的促炎及蛋白水解效应。本研究提示，微生物与炎性刺激可上调GHS-R1a的表达，这可能代表了人类骨骼中的一种保护性负反馈调控机制。