Corneal strain influences keratocyte proliferation and migration through upregulation of ALDH3A1 expression

Abstract

Keratocytes are the primary resident cells in the corneal stroma. They play an essential role in maintaining corneal physiological function. Studying the factors that affect the phenotype and behavior of keratocytes offers meaningful perspectives for improving the understanding and treatment of corneal injuries. In this study, 3% strain was applied to human keratocytes using the Flexcell® Tension Systems. Real-time quantitative PCR (RT-qPCR) and western blot were used to investigate the influence of strain on the expression of intracellular aldehyde dehydrogenase 3A1 (ALDH3A1). ALDH3A1 knockdown was achieved using double-stranded RNA-mediated interference (RNAi). Immunofluorescence (IF) staining was employed to observe the impact of changes in ALDH3A1 expression on nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear translocation. Keratocyte proliferation and migration were assessed by bromodeoxyuridine (BrdU) assay and scratch wound healing assay, respectively. Mouse injury models and single-cell RNA sequencing of keratocytes from keratoconus patients were used to assess how strain influenced ALDH3A1 in vivo. Our results demonstrate that 3% strain suppresses keratocyte proliferation and increases ALDH3A1. Increased ALDH3A1 inhibits NF-κB nuclear translocation, a key step in the activation of the NF-κB signaling pathway. Conversely, ALDH3A1 knockdown promotes NF-κB nuclear translocation, ultimately enhancing keratocyte proliferation and migration. Elevated ALDH3A1 levels were also observed in mouse injury models with increased corneal strain and keratoconus patients. These findings provide valuable insights for further research into the role of corneal strain and its connection to corneal injury repair.

Keywords: ALDH3A1; NF‐κB; biomechanics; corneal injuries; corneal strain; keratocytes; migration; proliferation.

FIGURE 1

IL‐1β promotes keratocytes proliferation. (A) Keratocytes were exposed to 1 ng/mL IL‐1β for 0 (ctrl), 12, or 24 h, respectively. Proliferation rate was assessed using BrdU assay. n = 3. Statistical analyses were conducted by One‐way ANOVA with Tukey's multiple comparisons test. (B) IL‐1𝛽 was administrated when keratocytes were scratched using a 1 mL pipette tip, followed by a 48‐hour incubation. 0, 12, 24, 36 and 48 h post scratching, photographs of the wounded area were captured. The means of the remaining wound area were calculated. Keratocytes without IL‐1β treatment served as the control (ctrl) group. n = 3. For each timepoint, statistical analyses were conducted by unpaired t‐test. Data is expressed as the mean ± SD. ****p < .0001.

FIGURE 2

Strain upregulates ALDH3A1 and downregulates IL‐8 expression. Keratocytes were subjected to 3% strain for 24, 48, or 72 h, respectively. Unstrained keratocytes served as control (ctrl) group. (A) The mRNA expression levels of ALDH3A1 and IL‐8 were assessed by RT‐qPCR. n = 3. For each timepoint, statistical analyses were conducted by unpaired t‐test. (B) Time‐dependent mRNA expression pattern of ALDH3A1 and IL‐8 in 3% strained keratocytes. n = 3. Statistical analyses were conducted by One‐way ANOVA with Tukey's multiple comparisons test. Data were presented as mean ± SD and as target gene expression/GAPDH, normalized to the ctrl group. (C) Time‐dependent protein expression levels of ALDH3A1 in 3% strained keratocytes. β‐actin served as a loading control. Representative data from one of three independent western blot experiments conducted using samples from three different donors. (D) The densitometry analysis of the western blot bands of ALDH3A1. n = 3. The densitometry of β‐actin bands served as the normalization reference. Statistical analyses were conducted by One‐way ANOVA with Tukey's multiple comparisons test. Data were presented as mean ± SD. *p < .05, **p < .01, ***p < .001, ****p < .0001.

FIGURE 3

Strain reverses the IL‐1β effects on ALDH3A1 and IL‐8 expression. We administered IL‐1β treatment to keratocytes, which were concurrently subjected to 3% strain over periods of 24, 48, and 72 h. (A) The mRNA expression levels of ALDH3A1 and IL‐8 were assessed by RT‐qPCR. n = 3. Statistical analyses were conducted by One‐way ANOVA with Tukey's multiple comparisons test. (B, C) Time‐dependent mRNA expression pattern of ALDH3A1 and IL‐8 in keratocytes under different treatment conditions. Data were presented as mean ± SD and as target gene expression/GAPDH, normalized to the ctrl group. n = 3. Statistical analyses were conducted by One‐way ANOVA with Tukey's multiple comparisons test. (D) Protein expression levels of ALDH3A1 in keratocytes under different treatment conditions. β‐actin served as a loading control. Representative data from one of three independent western blot experiments conducted using samples from three different donors. (E) The densitometry analysis of the western blot bands of ALDH3A1. n = 3. The densitometry of β‐actin bands served as the normalization reference. Statistical analyses were conducted by One‐way ANOVA with Tukey's multiple comparisons test within each timepoint. Data were presented as mean ± SD. *p < .05, **p < .01, ***p < .001, ****p < .0001.

FIGURE 4

Strain prevents NF‐κB nuclear translocation. Keratocytes were subjected to concurrent treatment with IL‐1β and 3% strain for 24 h. (A) Immunofluorescent staining images of keratocytes. NF‐κB were stained in red, and nuclei were stained in blue. (400‐fold magnification; scale bar, 50 μm) Arrows indicate the nuclear translocation of NF‐κB. Representative data from one of five independent immunofluorescence staining experiments conducted using samples from three different donors. (B) Protein expression levels of NF‐κB in keratocytes nuclear extract. β‐actin served as a loading control for total protein. Histone H3 served as a loading control for nuclear protein. Representative data from one of three independent western blot experiments conducted using samples from three different donors. (C) The densitometry analysis of the western blot bands of NF‐κB. n = 3. The densitometry of Histone H3 bands served as the normalization reference. Statistical analyses were conducted by One‐way ANOVA with Tukey's multiple comparisons test within each timepoint. Data were presented as mean ± SD. *p < .05, **p < .01.

FIGURE 5

ALDH3A1 inhibits NF‐κB nuclear translocation and further downregulates IL‐8. (A) The mRNA expression levels of ALDH3A1 were assessed by RT‐qPCR. n = 3. Statistical analyses were conducted by unpaired t‐test. Data were presented as mean ± SD and as target gene expression/GAPDH, normalized to the NT siRNA group. (B) Protein expression level of ALDH3A1 in keratocytes 48 h post transfected with NT siRNA or siRNA targeting ALDH3A1 (ALDH3A1 siRNA). β‐actin served as a loading control. Representative data from one of three independent western blot experiments conducted using samples from three different donors. (C) The densitometry analysis of the western blot bands of ALDH3A1. n = 3. The densitometry of β‐actin bands served as normalization. Statistical analyses were conducted by unpaired t‐test. Data were presented as mean ± SD. (D) Keratocytes were treated with NT siRNA or ALDH3A1 siRNA for 48 h, following which nuclear extract protein samples were collected, and subjected to western blot analysis. Protein expression level of NF‐κB within the nuclear extract was assessed by western blot β‐actin served as a loading control for total protein. Histone H3 served as a loading control for nuclear protein. Representative data from one of four independent western blot experiments conducted using samples from three different donors. (E) The densitometry analysis of the western blot bands of NF‐κB. n = 3. The densitometry of Histone H3 bands served as the normalization reference. Statistical analyses were conducted by unpaired t‐test. Data were presented as mean ± SD. (F) The mRNA expression levels of IL‐8 were assessed by RT‐qPCR. n = 3. Statistical analyses were conducted by unpaired t‐test. Data were presented as mean ± SD and as target gene expression/GAPDH, normalized to the NT siRNA group. **p < .01, ***p < .001.

FIGURE 6

Downregulation of ALDH3A1 promotes keratocyte migration and proliferation. (A) Impact of ALDH3A1 on keratocytes migration. Keratocytes were cultured as a monolayer and treated separately with non‐targeting siRNA (NT siRNA) or siRNA targeting ALDH3A1 (ALDH3A1 siRNA) for 48 h, then scratch‐wounded using a 1 mL pipette tip. Images of the scratched area were captured at 0‐, 24‐, and 48‐h post‐scratch (4‐fold magnification). Representative data from one of three independent experiments conducted using samples from three different donors. (B) The means of the remaining wound area were calculated. n = 3. For each timepoint, statistical analyses were conducted by unpaired t‐test. (C) Keratocytes were treated separately with non‐targeting siRNA (NT siRNA) or siRNA targeting ALDH3A1 (ALDH3A1 siRNA) for 48 h. The proliferation rate was assessed using BrdU assay. n = 3. Statistical analyses were conducted by unpaired t‐test. Data is expressed as the mean ± SD. *p < .05, **p < .01, ***p < .001.

FIGURE 7

Strain suppresses keratocytes proliferation. (A) EdU staining images of keratocytes. EdU positive cells were stained in red, and nuclei were stained in blue. (200‐fold magnification; scale bar, 100 μm). Representative data from one of three independent EdU staining experiments conducted using samples from three different donors. (B) The statistical analysis of the EdU positive cells. n = 9. Statistical analyses were conducted by One‐way ANOVA with Tukey's multiple comparisons test. Data is presented as mean ± SD. **p < .01, ****p < .0001.

FIGURE 8

Strain upregulates ALDH3A1 and downregulates IL‐8 in vivo. (A) The corneal thickness of the Cut injury model. n = 6. Statistical analyses were conducted by unpaired t‐test. Data were presented as mean ± SD. (B) The corneal thickness of the Scratch injury model. n = 6. Statistical analyses were conducted by unpaired t‐test. Data were presented as mean ± SD. (C) IOP of the Cut injury model. n = 5. Statistical analyses were conducted by unpaired t‐test. (D) IOP of the Scratch injury model. n = 5. Statistical analyses were conducted by unpaired t‐test. (E) Immunofluorescent staining images of the cross‐section of the cornea injury site in the Cut injury model. ALDH3A1 were stained in green, and nuclei were stained in blue. (scale bar, 100 μm). Representative data from one of four independent experiments conducted using samples from four different mice. (F) Immunofluorescent staining images of the cross‐section of the cornea injury site in the Scratch injury model. ALDH3A1 were stained in green, and nuclei were stained in blue. (scale bar, 100 μm). Representative data from one of four independent experiments conducted using samples from four different mice. (G) The scRNA‐Seq data of the expression levels of ALDH3A1 and IL‐8 in keratocytes from keratoconus patients. Keratocytes were isolated from cornea tissues of 3 keratoconus patients and 4 healthy donors (served as ctrl group). One‐sided Wilcoxon rank‐sum test was performed to assess ALDH3A1 and IL‐8 specificity within keratocytes. ns, no significant difference, ***p < .001, ****p < .0001.