Inflammatory Genes Associated with Pristine Multi-Walled Carbon Nanotubes-Induced Toxicity in Ocular Cells

Background: The wide application of multi-walled carbon nanotubes (MWCNTs) in various fields has raised enormous concerns regarding their safety for humans. However, studies on the toxicity of MWCNTs to the eye are rare and potential molecular mechanisms are completely lacking. This study was to evaluate the adverse effects and toxic mechanisms of MWCNTs on human ocular cells. Methods: Human retinal pigment epithelial cells (ARPE-19) were treated with pristine MWCNTs (7– 11 nm) (0, 25, 50, 100 or 200 μg/mL) for 24 hours. MWCNTs uptake into ARPE-19 cells was examined using transmission electron microscopy (TEM). The cytotoxicity was evaluated by CCK-8 assay. The death cells were detected by Annexin V-FITC/PI assay. RNA profiles in MWCNT-exposed and non-exposed cells (n = 3) were analyzed using RNA-sequencing. The differentially expressed genes (DEGs) were identified through the DESeq2 method and hub of which were filtered by weighted gene co-expression, protein–protein interaction (PPI) and lncRNA-mRNA co-expression network analyses. The mRNA and protein expression levels of crucial genes were verified using quantitative polymerase chain reaction (qPCR), colorimetric analysis, ELISA and Western blotting. The toxicity and mechanisms of MWCNTs were also validated in human corneal epithelial cells (HCE-T). Results: TEM analysis indicated the internalization of MWCNTs into ARPE-19 cells to cause cell damage. Compared with untreated ARPE-19 cells, those exposed to MWCNTs exhibited significantly decreased cell viabilities in a dose-dependent manner. The percentages of apoptotic (early, Annexin V positive; late, Annexin V and PI positive) and necrotic (PI positive) cells were significantly increased after exposure to IC50 concentration (100 μg/mL). A total of 703 genes were identified as DEGs; 254 and 56 of them were, respectively, included in darkorange2 and brown1 modules that were significantly associated with MWCNT exposure. Inflammation-related genes (including CXCL8, MMP1, CASP3, FOS, CXCL2 and IL11 ) were screened as hub genes by calculating the topological characteristics of genes in the PPI network. Two dysregulated long non-coding RNAs ( LUCAT1 and SCAT8 ) were shown to regulate these inflammation-related genes in the co-expression network. The mRNA levels of all eight genes were confirmed to be upregulated, while caspase-3 activity and the release of CXCL8, MMP1, CXCL2, IL11 and FOS proteins were demonstrated to be increased in MWCNT-treated ARPE-19 cells. MWCNTs exposure also can induce cytotoxicity and increase the caspase-3 activity and the expression of LUCAT1, MMP1, CXCL2, and IL11 mRNA and protein in HCE-T cells. Conclusion: Our study provides promising biomarkers for monitoring MWCNT-induced eye disorders and targets for developing preventive and therapeutic strategies. Keywords: multi-walled carbon nanotubes, ocular toxicity, transcriptome, inflammation, apoptosis, long non-coding RNAs