Cerium oxide nanoparticles attenuates fibrosis and inflammation in thyroid-associated ophthalmopathy via JNK pathway

Abstract

Objective: Thyroid-associated ophthalmopathy (TAO) is an autoimmune orbital disorder characterized by pathological alterations including extraocular muscle fibrosis and orbital inflammation. Cerium oxide nanoparticles (CeO2-NPs, CNPs) are gaining popularity in ophthalmology due to their potential antifibrotic and anti-inflammatory properties. This study aims to investigate the inhibitory effects of CNPs on fibrosis and inflammation in TAO orbital fibroblasts (OFs) derived from TAO patients.

Methods: OFs obtained by primary culture of orbital adipose tissue from 8 TAO patients. Probing the safe action concentration of CNPs and Anisomycin using CCK8 and detecting intracellular reactive oxygen species (ROS) generation using ROS Assay kit. Wound-Healing Assay was used to examine the degree of fibrosis of OFs. The expression of Fibronectin, COL1A1, α-Smooth muscle action, Hyaluronan Synthase 2, c-Jun N-terminal Kinase (JNK) and pJNK were detected by the RT-PCR and WB, and Hyaluronic Acid secretion was detected by ELISA. Inflammatory factors Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNFα) expression were detected by RT-PCR and ELISA.

Results: CNPs below 100 μg/mL and Anisomycin below 5 μM did not affect OFs proliferation. CNPs inhibit intracellular ROS generation. CNPs inhibit OFs fibrosis and suppress the expression of fibrosis indicators. These antifibrotic effects were mediated by inhibition of JNK phosphorylation, and were reversible by a JNK agonist. Furthermore, CNPs reduce both mRNA levels and secretion of inflammatory factors, IL-6 and TNF-α.

Conclusion: CNPs demonstrated the ability to inhibit fibrosis in TAO OFs by reducing JNK phosphorylation, as well as dose-dependently suppressed ROS generation and inflammatory response in TAO OFs.

Keywords: JNK phosphorylation; cerium oxide nanoparticles; fibrosis; inflammation; orbital fibroblasts; thyroid-associated ophthalmopathy.

FIGURE 1

Action concentration of drugs and ROS generation in OFs. (A,B) Cell viability measurement using the CCK-8 assay after treatment with CNPs or Anisomycin for 2 days. (C) ROS generation of OFs after treatment with 0, 10, 50 and 100 μg/mL CNPs and 5 ng/mL TGF-β. (D) ROS generation of OFs in control, TGF-β, CNPs + TGF-β and CNPs + Anisomycin + TGF-β groups. (E,F) Fluorescence intensity analysis in Figures (C,D). Data are presented as mean ± SEM (n ≥ 3). ns:P > 0.05, *P < 0.05, ***P < 0.001, ****P < 0.0001.

FIGURE 2

CNPs exert an inhibitory effect on OFs fibrosis. (A,B) Wound-Healing assay and cell migration analysis after treatment with 0, 10, 50 and 100 μg/mL CNPs and 5 ng/mL TGF-β, at 24 h and 48 h. (C) Secretion of HA after treatment with different concentrations of CNPs. (D) mRNA levels of Fibronectin, COL1A1, α-SMA and HAS2 after treatment with CNPs. (E) The WB results for Fibronectin, COL1A1 and α-SMA in each group, and the protein levels were quantified and normalized to the level of GAPDH for each sample. Data are presented as mean ± SEM (n ≥ 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 3

CNPs regulates JNK phosphorylation during OFs fibrosis inhibition. (A) Wound-Healing assay and cell migration analysis in control, TGF-β, CNPs + TGF-β and CNPs + Anisomycin + TGF-β groups. (B) mRNA levels of Fibronectin, COL1A1, α-SMA and HAS2 in each group. (C) The WB results for COL1A1, α-SMA and pJNK/JNK in each group, and the protein levels were quantified and normalized to the level of GAPDH for each sample. Data are presented as mean ± SEM (n ≥ 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

FIGURE 4

CNPs exert an inhibitory effect on OFs inflammation. (A) mRNA levels of IL-6 and TNF-α after treatment with 0, 10, 50 and 100 μg/mL CNPs and 5 ng/mL IL-1β. (B) Secretion of IL-6 and TNF-α after treatment with different concentrations of CNPs. Data are presented as mean ± SEM (n ≥ 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.