Senescence and Stress Signaling Pathways in Corneal Cells After Nitrogen Mustard Injury

Abstract

Mustard gas keratopathy (MGK), a complication of exposure to sulfur mustard, is a blinding ocular surface disease involving key cellular pathways, including apoptosis, oxidative stress, and inflammation. Recent studies indicate that cellular senescence contributes to the pathophysiology of mustard gas toxicity. This study aimed to assess senescence and stress-related pathways-particularly mitogen-activated protein kinase (MAPK) signaling-in nitrogen mustard (NM)-induced corneal injury. In vitro, primary human corneal epithelial (P-HCECs), primary human corneal mesenchymal stromal cells (hcMSCs), and human corneal-limbal epithelial cell (HCLE) lines were exposed to varying concentrations of NM. The results demonstrated a dose-dependent increase in cellular senescence, characterized by reduced Ki67 expression, elevated p16, and p21 mRNA levels, as well as activation of the MAPK pathway activation. Treatment with a selective p38-MAPK inhibitor significantly reduced senescence markers and improved cell proliferation following exposure to NM. Overall, these studies indicate that NM exposure triggers cellular senescence and stress-related MAPK signaling, while p38-MAPK inhibition mitigates these effects, suggesting a potential therapeutic strategy.

Keywords: BIRB796; MAPK; MAPK inhibitor; cornea; mustard; mustard keratopathy; nitrogen mustard; ocular surface; senescence.

Figure 1

Phase-contrast microscopy images showing significant morphological changes in human corneal mesenchymal stromal cells (hcMSCs) treated with nitrogen mustard at concentrations of 0.1, 0.03, and 0.01 mM after three days (A). Notable morphological alterations in primary human corneal epithelial cells (HCECs) treated with nitrogen mustard at 0.1 and 0.015 mM are observed after two days (B). Giemsa staining further illustrates significant morphological changes in hcMSCs treated with nitrogen mustard at 0.1, 0.03, and 0.01 mM after one week (C) (scale bar (red): 100 μm).

Figure 2

Immunofluorescence staining demonstrates a dose-dependent reduction in Ki-67 expression in human corneal mesenchymal stem cell (hcMSC) cultures, observed 2.5 weeks post-exposure to nitrogen mustard (NM) (scale bar (red): 50 μm) (A). Quantitative analysis reveals a significant decline in Ki-67levels in NM-treated cells at varying concentrations compared to untreated controls, with 95% confidence intervals (* p < 0.01; ** p < 0.001) (B). Ki-67 (green), DAPI (blue).

Figure 3

Expression of senescence markers in human corneal mesenchymal stem cells (hcMSCs) and primary human corneal epithelial cells (P-HCECs) following exposure to 0.05 and 0.1 mM nitrogen mustard (NM). Fluorescence staining illustrates the significant increase in p21-positive cells in hcMSCs two and a half weeks post-exposure to varying concentrations of NM, accompanied by 95% confidence intervals (scale bar: 50 μm) (* p < 0.01; ** p < 0.001) (A). Western blot analysis shows the detection of p16 and p21 levels in P-HCECs after one week of NM treatment, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) used as a housekeeping gene for normalizing protein expression levels, with standard deviation (P21 (green): * p < 0.001; P16: * p < 0.01, ** p < 0.001) (B).

Figure 4

mRNA expression of p16 and p21 in human corneal mesenchymal stem cells (hcMSCs) and primary human corneal epithelial cells (P-HCECs) after exposure to nitrogen mustard (NM). The relative fold increase was assessed in hcMSCs three days after NM exposure, with standard deviation (P16: * p < 0.05, ** p < 0.001; P21: * p < 0.01, ** p < 0.001) (A). The relative fold increase was also assessed in P-HCECs five days after NM exposure, with standard deviation (* p < 0.01; ** p < 0.001) (B).

Figure 5

Representative images of SA-β-gal staining showing the effects of nitrogen mustard (NM) in different groups, including unexposed cells and 0.03 mM and 0.1 mM NM-exposed cells after 48 h of treatment in primary human corneal epithelial cells (P-HCECs), and 0.1 mM NM-exposed in human corneal mesenchymal stromal cells (hcMSCs) Blue arrows indicate epithelial cells stained with SA-β-gal (scale bar: 50 μm) (A). Quantitative analysis of SA-β-gal-positive cells revealed that the untreated control group had a significantly lower area density of β-galactosidase compared to both the 0.1 mM NM treatment group and the 0.03 mM NM treatment group in P-HCECs and to the 0.1 mM NM treatment group in hcMSCs, with 95% confidence intervals shown (* p < 0.001) (B).

Figure 6

Western blot analysis of MAPK protein expression in human corneal mesenchymal stem cells (hcMSCs) and human corneal–limbal epithelial (HCLE) cells following exposure to varying concentrations of nitrogen mustard (NM). The results demonstrate a significant dose-dependent increase in the phosphorylation levels of MAPK proteins, including p-p44/42, p-p38, and p-SAPK/JNK (A). Quantitative analysis of the protein expression was conducted using one-way ANOVA and Tukey’s post hoc test, with standard deviation shown (P-P44/42: * p < 0.01, ** p < 0.001; P-P38 and SAPK/JNK: * p < 0.001) (B). Arrow (blue) showing senescent cells (green), scale bar (red).

Figure 7

BIRB796, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), significantly reduced nitrogen mustard (NM)-induced phosphorylation of p38 in a dose-dependent manner in human corneal limbal epithelial (HCLE) cells after 20 h. Phosphorylated p38 (P-p38) protein expression was detected via Western blotting (A) and followed by a quantitative analysis of the protein expression with standard deviation shown (* p < 0.01; ** p < 0.001) (B). Arrow (black) showing Markers band.

Figure 8

Phase-contrast microscopy images of human corneal limbal epithelial (HCLE) cells captured 2 days post-exposure to nitrogen mustard (NM), either alone or in combination with the MAPK inhibitor BIRB796. The images illustrate the characteristic features of cellular senescence. NM-treated cells exhibited flattened morphology and increased cell size compared to untreated and BIRB796-treated cells, which maintained a morphology comparable to that of the untreated cells (scale bar: 100 μm) (A). Cell counts of HCLE cells following various treatment exposures are presented as the mean number of cells per high-power field (HPF) across seven sections, with 95% confidence intervals (* p < 0.001) (B).

Figure 9

Immunofluorescence detection of p16 and p21 in human corneal mesenchymal stem cells (hcMSCs) 5 days after exposure to 0.1 mM nitrogen mustard (NM) and a combination of NM and BIRB796 (0.1 µM) (scale bar: 50 μm) P16 (Red), DAPI (Blue). P21 (Green) (A). Corresponding quantitative analysis, with standard deviation (* p < 0.01; ** p < 0.001) (B).

Figure 10

Representative images of β-galactosidase staining in three experimental groups: untreated cells, cells exposed to 0.1 mM nitrogen mustard (NM), and cells exposed to a combination of 0.1 mM NM and BIRB796 after 72 h (scale bar: 50 μm) (A). Quantitative analysis of SA-β-gal-positive cells shows that the control group exhibited a significantly lower area density of β-galactosidase compared to both the 0.1 mM NM-treated group and the NM + BIRB group (* p < 0.001 for each comparison), with 95% confidence intervals shown. Additionally, the area density of β-galactosidase was significantly higher in the NM-treated group than in the NM + BIRB group (* p < 0.001) (B).