Kojic acid enhances the proliferation of human corneal epithelial cells via p38 and p21 signaling pathways.

PURPOSE: Treatment of corneal injury depends on the self-proliferation ability of human corneal epithelial cells (HCEp). Our previous study revealed kojic acid had the anti-senescence function on human corneal endothelial cells. In this study, we researched the enhancive proliferation effect of kojic acid in HCEp. METHODS: Cell viability was evaluated by MTT assay. The expression of proliferation-related protein was detected by western blotting and immunofluorescence assay. RESULTS: Kojic acid could enhance HCEp proliferation, characterized by promoting cell proliferation rate, decreasing the expression levels of p21, galectin 8 and ki67, and increasing that of p-p38. The p38 signaling pathway inhibitor, SB203580, could reverse the enhancive proliferation function of kojic acid. Furthermore, knockdown of p21 had similar enhancive proliferation effect to kojic acid. CONCLUSION: Kojic acid might enhance HCEp proliferation through p38 and p21 signaling pathways, potentially via reduced expression levels of galectin 8 and ki67. Hence, kojic acid might be a potential drug to accelerate the healing of corneal epithelial injury.

The novel mutation P36R in LRP5L contributes to congenital membranous cataract via inhibition of laminin γ1 and c-MAF.

PURPOSE: The present study investigated a pathogenic mutation and its mechanism on membranous cataract in a congenital membranous cataract family. METHODS: An autosomal dominant four-generation Chinese congenital membranous cataract family was recruited and whole-exome sequencing was performed to screen for sequence variants. Candidate variants were validated using polymerase chain reaction and Sanger sequencing. Wild-type and mutant low-density lipoprotein receptor-related protein 5-like (LRP5L) plasmids were constructed and transfected into human lens epithelial cells (HLE B-3) and human anterior lens capsules. The cell lysates, nuclear and cytoplasmic proteins, and basement membrane components of HLE B-3 cells were harvested. LRP5L and laminin γ1 were knocked down in HLE B-3 cells using specific small-interfering RNA. The protein expression levels of LRP5L, laminin γ1, and c-MAF were detected using immunoblotting and immunofluorescence. RESULTS: We identified a novel suspected pathogenic mutation in LRP5L (c.107C > G, p.P36R) in the congenital membranous cataract family. This mutation was absent in 300 normal controls and 300 age-related cataract patients. Bioinformatics analysis with PolyPhen-2 and SIFT suggested that LRP5L-P36R was pathogenic. LRP5L upregulated laminin γ1 expression in the cytoplasmic proteins of HLE B-3 cells and human anterior lens capsules, and LRP5L-P36R inhibited the effects of LRP5L. LRP5L upregulated c-MAF expression in the nucleus and cytoplasm of HLE B-3 cells, and LRP5L-P36R inhibited c-MAF expression via inhibition of laminin γ1. CONCLUSION: Our study identified a novel gene, LRP5L, associated with congenital membranous cataract, and its mutant LRP5L-P36R contributed to membranous cataract development via inhibition of laminin γ1 and c-MAF.

Laminin α4 overexpression in the anterior lens capsule may contribute to the senescence of human lens epithelial cells in age-related cataract.

Senescence is a leading cause of age-related cataract (ARC). The current study indicated that the senescence-associated protein, p53, total laminin (LM), LMα4, and transforming growth factor-beta1 (TGF-ß1) in the cataractous anterior lens capsules (ALCs) increase with the grades of ARC. In cataractous ALCs, patient age, total LM, LMα4, TGF-ß1, were all positively correlated with p53. In lens epithelial cell (HLE B-3) senescence models, matrix metalloproteinase-9 (MMP-9) alleviated senescence by decreasing the expression of total LM and LMα4; TGF-ß1 induced senescence by increasing the expression of total LM and LMα4. Furthermore, MMP-9 silencing increased p-p38 and LMα4 expression; anti-LMα4 globular domain antibody alleviated senescence by decreasing the expression of p-p38 and LMα4; pharmacological inhibition of p38 MAPK signaling alleviated senescence by decreasing the expression of LMα4. Finally, in cataractous ALCs, positive correlations were found between LMα4 and total LM, as well as between LMα4 and TGF-ß1. Taken together, our results implied that the elevated LMα4, which was possibly caused by the decreased MMP-9, increased TGF-ß1 and activated p38 MAPK signaling during senescence, leading to the development of ARC. LMα4 and its regulatory factors show potential as targets for drug development for prevention and treatment of ARC.

Three kinds of corneal host cells contribute differently to corneal neovascularization.

BACKGROUND: Corneal neovascularization (angiogenesis and lymphangiogenesis) compromises corneal transparency and transplant survival, however, the molecular mechanisms of corneal host epithelial and stromal cells in neovascularization have not yet been fully elucidated. Furthermore, the contribution and mechanism of corneal host endothelial cells involved in neovascularization are largely unexplored. METHODS: Liquid chromatography-mass spectrometry, immunoblotting, and ELISA were used to screen and identify potential neovascularization-related factors in human full-thickness vascularized corneal tissues. Lipopolysaccharide was used to induce inflammation in three kinds of corneal host cells in vitro, including corneal epithelial, stromal, and endothelial cells. Fungus was used to establish an animal model of corneal neovascularization in vivo. Tube formation and spheroid sprouting assays were used to evaluate the contribution of three kinds of corneal host cells to the degree of neovascularization under various stimuli. Matrix metalloproteinase (MMP)-2, alpha-crystallin A chain (CRYAA), galectin-8, Bcl-2, neuropilin-2, MMP-9 plasmids, and recombinant human fibronectin were used to identify the key proteins of corneal host cells involved in corneal inflammatory neovascularization. FINDINGS: All three kinds of corneal host cells influenced corneal neovascularization to varying degrees. MMP-9 in human corneal epithelial cells, MMP-2, and CRYAA in human corneal stromal cells, and MMP-2 and galectin-8 in human corneal endothelial cells are potential key proteins that participate in corneal inflammatory neovascularization. INTERPRETATION: Our data indicated that both the effects of key proteins and corneal host cells involved should be considered for the treatment of corneal inflammatory neovascularization.

Kojic acid inhibits senescence of human corneal endothelial cells via NF-κB and p21 signaling pathways.

Fuchs endothelial dystrophy (FED) and late cornea allograft failure of cornea transplantation are associated with human corneal endothelial cells (HCEC) senescence. Kojic acid has various functions, however, its anti-senescence effect has never been identified. In this study, we investigated the anti-senescence effect of kojic acid on HCEC. Cell viability, migration ability and senescence were evaluated by MTT assay, migration assay, and senescence-associated beta-galactosidase (SA-ß-Gal) staining, respectively. Senescence-related protein expression was analyzed by western blotting and immunofluorescence assay. Angiogenesis of human umbilical vein endothelial cells (HUVEC) was examined by tube formation assay and spheroid sprouting assay. The results showed that kojic acid could inhibit HCEC senescence, characterized by enhancing migration, decreasing the levels of SA-ß-Gal staining, galectin 8, laminin α1, laminin α2, laminin γ1 and p21, and increasing that of p-NF-κB of senescent HCEC. The p-NF-κB inhibitor could reverse the anti-senescent effect of kojic acid, and p21 siRNA showed similar anti-senescence effect with kojic acid. In addition, kojic acid could alleviate HUVEC tube formation induced by senescent HCEC, which could be reversed by p-NF-κB inhibitor. The p21 siRNA could alleviate HUVEC spheroid sprouting induced by senescent HCEC. These results indicated that kojic acid might inhibit HCEC senescence and following resulted angiogenesis via NF-κB and p21 signaling pathways, possibly through downregulation of galectin 8 and laminins. Therefore, kojic acid is a promising drug for HCEC senescence-related diseases such as FED and late cornea allograft failure.

Corneal integrity and thickness of central fovea after phacoemulsification surgery in diabetic and nondiabetic cataract patients.

INTRODUCTION: Here we intended to investigate the changes in corneal endothelial cells and foveal thickness after phacoemulsification surgery on the eyes of diabetic and non-diabetic cataract patients. MATERIAL AND METHODS: A total of 120 cataract patients who were scheduled for phacoemulsification surgery and intraocular lens implantation were recruited and divided into 2 categories according to the diagnosis of diabetes mellitus. Changes in integrity, endothelial cell density (ECD), coefficient of variation (CV), percentage of hexagonal cells (PHC), central corneal thickness (CCT), and central foveal thickness (CFT) were all recorded at preoperative day 1 and postoperative day 3, 1 week, 1 month, 3 months and 6 months. RESULTS: None of the recorded variables showed any difference between the nondiabetic and diabetic groups before surgery (p > 0.05). During the postoperative 6 months, ECD and PHC decreased and CV increased in both groups (all ptime < 0.05), whereas CCT and CFT fluctuated in both groups significantly (both ptime < 0.05), with their individual peaks at postoperative 1 week in the diabetic group. The groups differed significantly in ECD, PHC, and CV at each time point postoperatively (all pgroup < 0.05). Furthermore, the diabetic group had improved CFT during the postoperative 1 month and higher CCT during the 6 months postoperatively than the nondiabetic group (all pgroup < 0.05). The time and group interactions were significant for ECD, CV, PHC, CCT and CFT (all pgroup × time < 0.05). CONCLUSIONS: The diabetic group had more changes in corneal endothelial cells and foveal thickness than the nondiabetic group postoperatively.