CX3CL1 缺乏通过其受体 CX3CR1 减轻 LPS 处理的 MC3T3-E1 细胞中的炎症、凋亡并加速成骨分化和矿化。

CX3CL1 deficiency ameliorates inflammation, apoptosis and accelerates osteogenic differentiation, mineralization in LPS-treated MC3T3-E1 cells via its receptor CX3CR1.

机构信息

Medical school, Yangzhou Polytechnic College, Yangzhou, Jiangsu 225009, China.

Department of Rehabilitation, Northern Jiangsu People's Hospital, Yangzhou, Jiangsu 225001, China; Clinical Medical College of Yangzhou University, Yangzhou, Jiangsu 225009, China.

出版信息

Ann Anat. 2023 Feb;246:152036. doi: 10.1016/j.aanat.2022.152036. Epub 2022 Nov 24.

DOI:10.1016/j.aanat.2022.152036

PMID:36436718

Abstract

BACKGROUND

Osteoporosis is a devastating skeletal disease responsible for bone fragility and fracture. CX3C chemokine ligand 1 (CX3CL1) is an inflammatory chemokine which has been identified to possess increased expression in the serum of postmenopausal osteoporotic patients. This paper was to illuminate the impacts of CX3CL1 on inflammation, apoptosis and osteogenic differentiation, mineralization in LPS-treated osteoblasts and investigate the regulatory mechanism.

METHODS

The viability of MC3T3-E1 cells exposed to elevating doses of LPS was detected by CCK-8 assay. CX3CL1 and C-X3-C motif chemokine receptor 1 (CX3CR1) expression were detected by RT-qPCR and western blot. CX3CR1 expression was examined again following CX3CL1 depletion. The binding of CX3CL1 with CX3CR1 was testified through Co-IP assay. In MC3T3-E1 cells co-transduced with CX3CL1 interference and CX3CR1 overexpression plasmids following LPS exposure, cell activity and inflammation were separately estimated via CCK-8 assay and RT-qPCR. Apoptosis was measured by TUNEL assay and western blot. Osteoblast differentiation was evaluated by ALP activity assay, RT-qPCR and western blot. Osteoblast mineralization was assessed by ARS staining, RT-qPCR and western blot. Results The experimental data presented that LPS attenuated the viability and enhanced CX3CL1 and CX3CR1 expression in MC3T3-E1 cells in a dose-dependent manner. CX3CR1 interacted with CX3CL1 and was positively modulated by CX3CL1. The suppressive role of CX3CL1 absence in LPS-evoked viability decrease, inflammation and apoptosis in MC3T3-E1 cells was reversed by CX3CR1 elevation. Besides, CX3CR1 reversed the promoted osteoblast differentiation and mineralization imposed by CX3CL1 interference.

CONCLUSIONS

CX3CL1 knockdown eased inflammation, apoptosis and promoted osteogenic differentiation, mineralization in MC3T3-E1 cells upon LPS exposure through down-regulating CX3CR1.

摘要

背景

骨质疏松症是一种破坏性骨骼疾病，可导致骨骼脆弱和骨折。趋化因子（C-X3-C 基序）配体 1（CX3CL1）是一种炎症趋化因子，其在绝经后骨质疏松症患者的血清中表达增加。本文旨在阐明 CX3CL1 对 LPS 处理的成骨细胞中炎症、细胞凋亡和成骨分化、矿化的影响，并探讨其调节机制。

方法

通过 CCK-8 检测升高剂量 LPS 暴露下 MC3T3-E1 细胞的活力。通过 RT-qPCR 和 Western blot 检测 CX3CL1 和 C-X3-C 基序趋化因子受体 1（CX3CR1）的表达。通过 CX3CL1 耗竭再次检测 CX3CR1 的表达。通过 Co-IP 检测 CX3CL1 与 CX3CR1 的结合。在 LPS 暴露后共转染 CX3CL1 干扰和 CX3CR1 过表达质粒的 MC3T3-E1 细胞中，通过 CCK-8 检测和 RT-qPCR 分别评估细胞活性和炎症。通过 TUNEL 检测和 Western blot 检测细胞凋亡。通过 ALP 活性测定、RT-qPCR 和 Western blot 评估成骨细胞分化。通过 ARS 染色、RT-qPCR 和 Western blot 评估成骨细胞矿化。结果：实验数据表明，LPS 呈剂量依赖性减弱 MC3T3-E1 细胞活力并增强 CX3CL1 和 CX3CR1 的表达。CX3CR1 与 CX3CL1 相互作用，并受 CX3CL1 正向调节。CX3CR1 升高逆转了 CX3CL1 缺失对 LPS 诱导的 MC3T3-E1 细胞活力下降、炎症和细胞凋亡的抑制作用。此外，CX3CR1 逆转了 CX3CL1 干扰引起的成骨细胞分化和矿化的促进作用。