Human Corneal Epithelial Cell Migration Research Articles

Deoxynivalenol Damages Corneal Epithelial Cells and Exacerbates Inflammatory Response in Fungal Keratitis.

Fungal keratitis (FK) is a kind of infectious keratopathy with a high rate of blindness worldwide. Deoxynivalenol (DON) has been proven to have multiple toxic effects on humans and animals. The aim of this study was to explore a possible pathogenic role of DON in FK. We first made an animal model of FK in New Zealand white rabbits, and then attempted to detect DON in a culture medium in which Fusarium solani had been grown and also in the corneal tissue of the animal model of Fusarium solani keratitis. Next, a model of DON damage in human corneal epithelial cells (HCECs) was constructed to evaluate effects of DON on the activity, migration ability, cell cycle, and apoptosis in the HCECs. Then, putative the toxic damaging effects of DON on rabbit corneal epithelial cells and the impact of the repair cycle were studied. The expression levels of inflammatory factors in the corneas of the animal model and in the model of DON-damaged HCECs were measured. The Fusarium solani strain used in this study appeared to have the potential to produce DON, since DON was detected in the corneal tissue of rabbits which had been inoculated with this Fusarium solani strain. DON was found to alter the morphology of HCECs, to reduce the activity and to inhibit the proliferation and migration of HCECs. DON also induced the apoptosis and S-phase arrest of HCECs. In addition, DON was found to damage rabbit corneal epithelial cells, to prolong the corneal epithelial regeneration cycle, and to be associated with the upregulated expression of inflammatory factors in HCECs and rabbit corneas. DON appears to have a toxic damaging effect on HCECs in FK, and to induce the expression of inflammatory factors, leading to the exacerbation of keratitis and the formation of new blood vessels. Future studies will explore the possibility of developing a test to detect DON in ophthalmic settings to aid the rapid diagnosis of FK, and to develop DON neutralizers and adsorbents which have the potential to improve keratocyte status, inhibit apoptosis, and alleviate inflammation, therein providing new thinking for therapy of clinical FK.

Long Noncoding RNA MIATNB Regulates Hyperosmotic Stress-induced Corneal Epithelial Cell Injury by Inhibiting Autophagy in Dry Eye Disease

Purpose Dry eye disease (DED) has a complex etiology and the roles of long noncoding RNAs (lncRNAs) in its pathophysiology are not completely understood. Autophagy is a self-eating process important for cell survival and homeostasis. The present study explored the role of myocardial infarction-associated transcript neighbor (MIATNB) long non-coding RNA in hyperosmolarity-induced autophagy and apoptosis in human corneal epithelial cell (HCEC)-based model of dry eye disease. Methods In vitro assays were performed with a human SV40 immortalized corneal epithelial cell line. Different concentrations of NaCl were used to create hyperosmolarity. HCECs were cultured in presence of 70–120 mM NaCl for 24 h to create an in vitro model of dry eye. RT-qPCR was performed to assess the expression of dry eye related LC3B, ATG16L, BECN1, ATG1, ATG7, ATG13, ATG5, ATG10, and ATG101 mRNAs and western blot analysis of LC3B and P62 and RFP -GFP-tagged LC3. Flow cytometry and western blot analysis of caspase 3, BCL2 and BAX were performed to detect apoptosis. Chloroquine (CQ) was used to inhibit autophagy pharmacologically. Results Autophagy flux was activated in HCECs subjected to hyperosmotic stress. Hyperosmolarity activated apoptosis and inhibited HCEC migration and autophagy. Hyperosmolarity upregulated MIATNB expression, while MIATNB knockdown inhibited autophagosome degradation and promoted HCEC apoptosis. Under hyperosmolar conditions, MIATNB knockdown also inhibited the degradation of autophagolysosomes and stimulated HCEC apoptosis. Conclusion MIATNB plays a vital role in dry eye pathogenesis and serves as a bridge between autophagy and apoptosis. Targeting MIATNB for DED treatment should be further evaluated.

The RNA m5C Methylase NSUN2 Modulates Corneal Epithelial Wound Healing.

The emerging epitranscriptomics offers insights into the physiopathological roles of various RNA modifications. The RNA methylase NOP2/Sun domain family member 2 (NSUN2) catalyzes 5-methylcytosine (m5C) modification of mRNAs. However, the role of NSUN2 in corneal epithelial wound healing (CEWH) remains unknown. Here we describe the functional mechanisms of NSUN2 in mediating CEWH. RT-qPCR, Western blot, dot blot, and ELISA were used to determine the NSUN2 expression and overall RNA m5C level during CEWH. NSUN2 silencing or overexpression was performed to explore its involvement in CEWH both in vivo and in vitro. Multi-omics was integrated to reveal the downstream target of NSUN2. MeRIP-qPCR, RIP-qPCR, and luciferase assay, as well as in vivo and in vitro functional assays, clarified the molecular mechanism of NSUN2 in CEWH. The NSUN2 expression and RNA m5C level increased significantly during CEWH. NSUN2 knockdown significantly delayed CEWH in vivo and inhibited human corneal epithelial cells (HCEC) proliferation and migration in vitro, whereas NSUN2 overexpression prominently enhanced HCEC proliferation and migration. Mechanistically, we found that NSUN2 increased ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) translation through the binding of RNA m5C reader Aly/REF export factor. Accordingly, UHRF1 knockdown significantly delayed CEWH in vivo and inhibited HCEC proliferation and migration in vitro. Furthermore, UHRF1 overexpression effectively rescued the inhibitory effect of NSUN2 silencing on HCEC proliferation and migration. NSUN2-mediated m5C modification of UHRF1 mRNA modulates CEWH. This finding highlights the critical importance of this novel epitranscriptomic mechanism in control of CEWH.

Exosomes from bone marrow-derived mesenchymal stem cells facilitate corneal wound healing via regulating the p44/42 MAPK pathway.

This study was aimed at exploring the function of Exosomes isolated from bone marrow-derived mesenchymal stem cells (BMSC-Exos) in corneal wound healing and at revealing the underlying mechanisms involving the p44/42 mitogen-activated protein kinase (MAPK) pathway. The isolated BMSC-Exos were identified by transmission electron microscopy, Western blot, and nanoparticle tracking analysis. After coculture with BMSC-Exos, the proliferation and migration of human corneal epithelial cells (HCEs) were evaluated. The protein expression of p-MEK/MEK and p44/42 MAPK was detected by Western blot. A mouse model of alkali-burned cornea was established via NaOH exposure. After injection with BMSC-Exos, the pathological changes and expression of α-SMA (a fibrosis marker) and CD31 (a vascularization marker) in corneal tissues were detected. BMSC-Exos enhanced the proliferation and migration of HCEs in a dose-dependent manner. The p44/42 MAPK pathway was activated by the treatment of BMSC-Exos, and its blocking using U0126 partially abrogated the effects of BMSC-Exos on promoting the proliferation and migration of HCEs. In vivo, the injection of BMSC-Exos facilitated the remission of the pathological changes (inflammation) and weakened the upregulation of α-SMA (fibrosis) and CD31 (vascularization) in corneal tissues of mice with alkali-burn injury. BMSC-Exos promoted the proliferation and migration of HCEs via activating the p44/42 MAPK pathway in vitro and also inhibited alkali burn-induced inflammation, fibrosis, and vascularization in corneal tissues in vivo. BMSC-Exos may be promising resources for promoting corneal wound healing.

Dexamethasone attenuates dry eye-induced pyroptosis by regulating the KCNQ1OT1/miR-214 cascade

Dry eye disease (DED) is an inflammatory disorder of the ocular surface seriously affecting the quality of life of patients. Topical dexamethasone (Dex) administration protects the cornea from the hyperosmotic stress (HS) induced by tears. Pyroptosis participates in the activation of epithelial inflammation during DED. However, it remains unclear whether Dex attenuates the progression of DED through pyroptosis. In this study, we aimed to investigate the effect of Dex on DED using both cell and animal models and its underlying mechanism. The inflammatory factors contained in tears were detected using a cytokine assay. The pyroptosis in DED mice and human corneal epithelial cells (HCECs) treated with hyperosmotic medium under various treatments was evaluated by immunohistochemical assays (IHC) or western blotting (WB). RNA expression was manipulated with siRNA or agomir microRNAs and measured using a polymerase chain reaction. The scratch assay was used to assess the migration rate of HCECs. Remaining corneal defects were evaluated using fluorescein staining and photographed using a digital camera. Dex could suppress the release of inflammatory factors and notably attenuate pyroptosis, KCNQ1OT1 expression, and NF-κB activation induced by HS injury in vivo and in vitro. KCNQ1OT1 upregulation could activate pyroptosis by sponging miR-214. Furthermore, KCNQ1OT1 knockdown and miR-214 overexpression reversed the effect of HS, promoted the migration of HCECs, and accelerated corneal wound healing. Dex effectively suppressed HS-induced pyroptosis through the KCNQ1OT1/miR-214/caspase-1 signaling axis by inhibiting the NF-κB activation. Our results provide a novel understanding of the mechanism of Dex as an anti-inflammatory drug in DED.

The therapeutic potential of chondroitin sulfate in Aspergillus fumigatus keratitis

PurposeTo explore the therapeutic effect of chondroitin sulfate (CS) on Aspergillus fumigatus (A. fumigatus) keratitis. MethodsThe nontoxic concentration of CS was determined using the Draize eye test and cell counting kit-8. Cell scratch test and cell proliferation test were evaluated the impact of CS on the proliferation and migration of human corneal epithelial cells (HCECs). Adherence assay and plate counting were used to detect fungal load in vivo and in vitro, respectively. Clinical score and hematoxylin-eosin (HE) staining were applied to assess the therapeutic effects of CS in an A. fumigatus keratitis mice model. The neutrophil infiltration and activity were detected by flow cytometry (FCM), immunofluorescence staining, and myeloperoxidase (MPO) assay. Toll-like receptor 4 (TLR-4) expression in RAW 264.7 cells and mouse cornea was detected by immunofluorescence staining. Real-time PCR (RT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were applied to examine the expression of inflammatory mediators. ResultsCS at 400 μg/mL (non-cytotoxic) significantly promoted proliferation and migration of HCECs. In an A. fumigatus keratitis mice model, CS treatment alleviated fungal keratitis (FK) severity by decreasing corneal fungal load and inhibiting neutrophil infiltration. In RAW 264.7 cells, the mRNA and protein levels of TLR-4, phosphorylated nuclear factor (NF)-κB (p-NF-κB), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-a (TNF-α), cyclooxygenase 2 (COX-2), and macrophage inflammatory protein-2 (MIP-2) were remarkably lower in the siTLR-4 treated group, while higher in rTLR-4 treated group than in the corresponding control group. CS treatment suppressed rTLR-4 induced the mRNA and protein expression of TLR-4, p-NF-κB, IL-1β, IL-6, COX-2, TNF-α, and MIP-2 in RAW cells. ConclusionCS may ameliorate the prognosis of A. fumigatus keratitis by promoting corneal epithelial proliferation, inhibiting the recruitment and activity of neutrophils, and inhibiting the inflammatory response by down-regulation of the TLR-4/NF-κB signaling.

Extracellular Vesicles Secreted by Corneal Myofibroblasts Promote Corneal Epithelial Cell Migration.

Corneal epithelial wound healing is a multifaceted process that encompasses cell proliferation, migration, and communication from the corneal stroma. Upon corneal injury, bidirectional crosstalk between the epithelium and stroma via extracellular vesicles (EVs) has been reported. However, the mechanisms by which the EVs from human corneal keratocytes (HCKs), fibroblasts (HCFs), and/or myofibroblasts (HCMs) exert their effects on the corneal epithelium remain unclear. In this study, HCK-, HCF-, and HCM-EVs were isolated and characterized, and human corneal epithelial (HCE) cell migration was assessed in a scratch assay following PKH26-labeled HCK-, HCF-, or HCM-EV treatment. HCE cells proliferative and apoptotic activity following EV treatment was assessed. HCF-/HCM-EVs were enriched for CD63, CD81, ITGAV, and THBS1 compared to HCK-EV. All EVs were negative for GM130 and showed minimal differences in biophysical properties. At the proteomic level, we showed HCM-EV with a log >two-fold change in CXCL6, CXCL12, MMP1, and MMP2 expression compared to HCK-/HCF-EVs; these proteins are associated with cellular movement pathways. Upon HCM-EV treatment, HCE cell migration, velocity, and proliferation were significantly increased compared to HCK-/HCF-EVs. This study concludes that the HCM-EV protein cargo influences HCE cell migration and proliferation, and understanding these elements may provide a novel therapeutic avenue for corneal wound healing.

Pathogenic Role of Diabetes-Induced Overexpression of Kallistatin in Corneal Wound Healing Deficiency Through Inhibition of Canonical Wnt Signaling.

It was reported previously that circulation levels of kallistatin, an endogenous Wnt signaling inhibitor, are increased in patients with diabetes. The current study was conducted to determine the role of kallistatin in delayed wound healing in diabetic corneas. Immunostaining and Western blot analysis showed kallistatin levels were upregulated in corneas from humans and rodents with diabetes. In murine corneal wound healing models, the canonical Wnt signaling was activated in nondiabetic corneas and suppressed in diabetic corneas, correlating with delayed wound healing. Transgenic expression of kallistatin suppressed the activation of Wnt signaling in the cornea and delayed wound healing. Local inhibition of Wnt signaling in the cornea by kallistatin, an LRP6-blocking antibody, or the soluble VLDL receptor ectodomain (an endogenous Wnt signaling inhibitor) delayed wound healing. In contrast, ablation of the VLDL receptor resulted in overactivation of Wnt/β-catenin signaling and accelerated corneal wound healing. Activation of Wnt signaling in the cornea accelerated wound healing. Activation of Wnt signaling promoted human corneal epithelial cell migration and proliferation, which was attenuated by kallistatin. Our findings suggested that diabetes-induced overexpression of kallistatin contributes to delayed corneal wound healing by inhibiting the canonical Wnt signaling. Thus, kallistatin and Wnt/β-catenin signaling in the cornea could be potential therapeutic targets for diabetic corneal complications.

Peucedanum japonicum Thunberg and Its Active Components Mitigate Oxidative Stress, Inflammation and Apoptosis after Urban Particulate Matter-Induced Ocular Surface Damage.

We previously demonstrated that urban particulate matter (UPM) exposure decreases the migration activity and survival of human corneal epithelial cells (HCECs). Herein, we investigated the potential to improve the corneal wound-healing ability of Peucedanum japonicum Thunb. leaf extract (PJE) and its active components on UPM-induced ocular surface damage in vitro and in vivo. PJE effectively assisted wound healing without altering HCEC survival and enhanced catalase (CAT), heme oxygenase 1 (HO1) and glutathione peroxidase 1 (GPX1) antioxidant gene expression. A corneal wound was uniformly induced on the right eye in all experimental animals and divided into eight groups such as two control groups (wounded right eye group—NR and non-wounded left eye group—NL), UPM treated group and PJEs (25, 50, 100, 200, 400 mg/kg) treated groups. Corneal abrasion model rats exposed to UPM showed delayed wound healing compared to unexposed rats, but wound healing was dose-dependently enhanced by PJE oral administration. Seventy-two hours after wound generation, inflammatory cells, apoptotic cells and interleukin-6 (IL-6) expression were increased substantially after UPM exposure, but PJE treatment significantly reduced the wound to an almost normal level while enhancing re-epithelialization without changing corneal thickness. Next, we tried to identify the key molecules for enhancing wound healing through fractionation. The major compounds in the fraction, confirmed by high-performance liquid chromatography (HPLC), were chlorogenic acid (CA), neochlorogenic acid (NCA) and cryptochlorogenic acid (CCA). Each type of CA isomers showed slightly different half maximal effective (EC50) and maximal effective (ECmax) concentrations, and their mixtures synergistically enhanced HCEC migration. Thus, corneal abrasion wound recovery after UPM exposure improved after PJE treatment, and the active PJE components were identified, providing an important basis to develop therapeutics for ocular surface damage using PJE.

Thrombospondin 1‐induced exosomal proteins attenuate hypoxia‐induced paraptosis in corneal epithelial cells and promote wound healing

Thrombospondin-1 (TSP1) is involved in corneal wound healing caused by chemical injury. Herein, we examined the effects of TSP1 on hypoxia-induced damages and wound-healing activity in human corneal epithelial (HCE) cells. Exosomal protein expression was determined using liquid chromatography-tandem mass spectrometry, and HCE cell migration and motility were examined through wound-healing assay and time-lapse microscopy. Reestablishment of cell junctions by TSP1 was assessed through confocal microscopy and 3D image reconstruction. Our results show that CoCl2 -induced hypoxia promoted HCE cell death by paraptosis. TSP1 protected these cells against paraptosis by attenuating mitochondrial membrane potential depletion, swelling and dilation of endoplasmic reticulum and mitochondria, and mitochondrial fission. Exosomes isolated from HCE cells treated with TSP1 contained wound healing-associated proteins that were taken up by HCE cells to promote tissue remodeling and repair. TSP1 protected HCE cells against hypoxia-induced damages and inhibited paraptosis progression by promoting cell migration, cell-cell adhesion, and extracellular matrix remodeling. These findings indicate that TSP1 ameliorates hypoxia-induced paraptosis in HCE cells and promotes wound healing and remodeling by regulating exosomal protein expression. TSP1 may, therefore, play important roles in the treatment of hypoxia-associated corneal diseases.

Acetylsalicylic Acid Promotes Corneal Epithelium Migration by Regulating Neutrophil Extracellular Traps in Alkali Burn.

Neutrophils are the first cells to migrate into the cornea in response to alkali burns, and excessive neutrophil infiltration is associated with inflammatory injury and a poorer prognosis. In an effort to understand the mechanisms underlying the inflammation mediated by neutrophils after alkali burns, we examined the role of alkali-activated neutrophils on human corneal epithelial cells (HCEs) proliferation and migration, as well as the effects of acetylsalicylic acid (ASA) and dexamethasone (DXM) on NETosis. We stimulated human neutrophils with sodium hydroxide (NaOH) and observed dose- and time-dependent neutrophil extracellular traps (NETs) formation. We also observed that ASA, but not DXM, significantly inhibited NaOH-induced NETosis. Furthermore, the activation of nuclear factor (NF)-κB, but not the production of reactive oxygen species, was involved in ASA-regulated NETosis. Moreover, NETs were found to be involved in alkali-activated neutrophils (ANs) induced neutrophil-HCE adhesion. ANs enhanced HCEs proliferation via phagocytosis. Meanwhile, ANs inhibited HCEs migration through the release of NETs, which was partially rescued by 5 mM ASA. In conclusion, ANs may interfere with HCEs proliferation and migration by phagocytosis and NETs formation, respectively. ASA may enhance HCEs migration by decreasing NETs formation through inhibition of NF-κB activation and could be a promising strategy for improving the prognosis of corneal alkali burns.

MicroRNA-184 negatively regulates corneal epithelial wound healing via targeting CDC25A, CARM1, and LASP1

BackgroundMicroRNAs (miRNAs) play critical roles in corneal development and functional homeostasis. Our previous study identified miR-184 as one of the most highly expressed miRNAs in the corneal epithelium. Even though its expression level plummeted dramatically during corneal epithelial wound healing (CEWH), its precise role in mediating corneal epithelial renewal was unresolved. The present study aimed to reveal the function and mechanism of miR-184 in regulating CEWH.MethodsQuantitative RT-PCR analysis characterized the miR-184 expression pattern during CEWH in mice. Ectopic miR-184 injection determined its effect on this process in vivo. We evaluated the effects of miR-184 and its target genes on the proliferation, cell cycle, and migration of human corneal epithelial cells (HCECs) using MTS, flow cytometry, and wound-healing assay, respectively. Bioinformatic analysis, in conjunction with gene microarray analysis and cell-based luciferase assays, pinpointed gene targets of miR-184 contributing to CEWH.ResultsMiR-184 underwent marked downregulation during mouse CEWH. Ectopic miR-184 overexpression delayed this process in mice. Furthermore, miR-184 transfection into HCECs significantly inhibited cell proliferation, cell cycle progression, and cell migration. MiR-184 directly targeted CDC25A, CARM1, and LASP1, and downregulated their expression in HCECs. CARM1 downregulation inhibited both HCEC proliferation and migration, whereas a decrease in LASP1 gene expression only inhibited migration.ConclusionsOur results demonstrate that miR-184 inhibits corneal epithelial cell proliferation and migration via targeting CDC25A, CARM1, and LASP1, suggesting it acts as a negative modulator during CEWH. Therefore, identifying strategies to suppress miR-184 expression levels has the potential to promote CEWH.

Long-Term Exposure to Urban Particulate Matter on the Ocular Surface and the Incidence of Deleterious Changes in the Cornea, Conjunctiva and Retina in Rats.

We investigated the time-dependent deleterious ocular changes induced by urban particulate matter (UPM) in vitro and in vivo. UPM treatment decreased human corneal epithelial cell migration and survival. Fluorescein scores were consistently increased by UPM application for 16 weeks. One week of rest at 2 or 4 weeks led to a recovery trend, whereas two weeks of rest at 8 weeks induced no change. UPM treatment decreased the tear film break-up time at 2 weeks, which was thereafter maintained until 16 weeks. No changes were found after periods of rest. UPM-treated eyes exhibited greater corneal epithelium thickness than normal eyes at 2 weeks, which recovered to normal at 4 and 8 weeks and was significantly decreased at 16 weeks. Apoptotic cell number in the epithelium was increased at 2 weeks, which remained constant except at 8 weeks. IL-6 expression in the cornea of the right eye continually increased for 16 weeks, and significant recovery was only observed at 8 weeks after 2 weeks of rest. Ocular pressure was significantly increased in the right eye at 12 and 16 weeks. Topical UPM application to the eye induced deleterious changes to various closely related parts of the eye.

Purinergic Signaling Involvement in Thymosin β4-mediated Corneal Epithelial Cell Migration

ABSTRACT Purpose: This study aimed to determine the effect of thymosin beta 4 (Tβ4) on human corneal epithelial cell migration and the downstream signaling pathways. Tβ4 has a role in tissue development, cell migration, inflammation, and wound healing. A previous study showed that Tβ4 directly binds to F0-F1 ATP synthase. Other studies reported the role of extracellular ATP and purinergic receptors in cell migration with several cell types. Despite advancing to the clinical stage for treatment of eye disorders, the effect of Tβ4 on human corneal epithelial cell (HCEC) migration and proliferation and the precise downstream signaling pathway(s) have not been identified. Methods: Various concentrations of Tβ4 were tested in vitro on human corneal epithelial cell proliferation using the CCK-8 Kit and on cell migration using the gap closure migration assay. Additionally, ATP levels at various time points were determined using the ATP Lite One-Step Kit. The Fluo 8 NO Wash Calcium Assay Kit was used to measure the intracellular Ca2+ concentration after treatment with various concentrations of Tβ4. P2X7 inhibitors were tested on ATP signaling and migration. Total- and phospho-ERK1/2 levels were determined in western blot. Results: Tβ4 enhanced HCEC proliferation and migration in a dose- and time-dependent manner. Moreover, these functions were related to increased extracellular ATP levels, intracellular Ca2+ influx, and ERK1/2 phosphorylation. Tβ4-mediated HCEC migration was inhibited by specific P2X7 purinergic receptor antagonists suggesting the role of this receptor in Tβ4-mediated human corneal epithelial cell migration. Conclusions: These results suggest that Tβ4-mediated HCEC proliferation and migration are associated with increased ATP levels, P2X7 R-mediated Ca2+ influx, and the ERK1/2 signaling pathway. This study begins to describe the mechanisms for Tβ4-mediated corneal healing and regeneration.

Effects of epidermal growth factor on transforming growth factor-beta1-induced epithelial-mesenchymal transition and potential mechanism in human corneal epithelial cells.

To evaluate the effects of epidermal growth factor (EGF) on transforming growth factor-beta1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in human corneal epithelial cells (HCECs). HCECs were cultured and treated with TGF-β1 for establishing the model of EMT in vitro. Biological effect of EGF on TGF-β1-induced EMT was evaluated. Proteins and mRNAs expression changes of E-cadherin, N-cadherin and Fibronectin (EMT-relative markers) after TGF-β1 or TGF-β1 combined EGF treatment were detected by Western blot and RT-PCR, respectively. Viability and migration of HCECs were measured by CCK-8, transwell cell migration assay and cell scratch wound healing assay. Activation of Smad2, ERK, p38, JNK and Akt signaling pathways were evaluated by Western blot. Inhibitors of relevant signaling pathways were added to the HCECs to explore the key signal mechanism. With treatment of TGF-β1 only, three EMT-relative proteins and mRNA expression showed that EMT up-regulated in a concentration-dependent and time-dependent manner, with significantly decreasing cell viability (TGF-β1≥5 ng/mL, P<0.05) and increasing cell migration (TGF-β1≥5 ng/mL, P<0.01). The phosphorylation of Smad2 and p38 was a key process of TGF-β1-induced EMT. Meanwhile, EMT-relative proteins and mRNA expression showed that EGF inhibited TGF-β1-indued EMT, with significantly increasing cell viability (EGF≥10 ng/mL, P<0.01). It was noteworthy that EGF significantly enhanced cell migration although EMT was inhibited (EGF≥10 ng/mL, P<0.01), and the blockage of p38 (by SB202190, a p38 inhibitor) was a potential mechanism of this phenomenon. EGF inhibits TGF-β1-induced EMT via suppressive p38, and promotes cells proliferation and migration in a non-EMT process by inhibiting p38 pathway.

The role of netrin-1 in the mouse cornea during Aspergillus fumigatus infection

PurposeTo explore the effects of netrin-1 on inflammation in Aspergillus fumigatus-infected mouse corneas and on proliferation and migration in human corneal epithelial cells (HCECs). MethodsNetrin-1 and the receptor A2BAR were detected in normal and infected corneas from C57BL/6 mice and RAW 264.7 cells. The mice were injected subconjunctivally with recombinant netrin-1. The severity of the disease was determined by clinical scores, photography with a slit lamp, RT-PCR, western blotting, myeloperoxidase (MPO) assays and immunofluorescence staining of polymorphonuclear neutrophilic leukocytes (PMNs). The effects of netrin-1 on RAW 264.7 cells in vitro were determined by RT-PCR. The role of A2BAR was demonstrated in vivo by detecting the expression of IL-1β, TNF-α, and IL-10 in corneas pretreated subconjunctivally with an A2BAR antagonist (PSB1115). RAW 264.7 cells were stimulated with Aspergillus fumigatus (A. fumigates) and netrin-1 with or without PSB1115 pretreatment. A cell counting kit-8 (CCK-8) assay was used to evaluate cell proliferation ability, and cell migration ability was determined by cell scratch experiments with HCECs. ResultsNetrin-1 expression decreased slightly after A. fumigatus infection and then increased to its peak. A2BAR expression increased at 1 day post infection (p.i.), with a subsequent decline. Compared to the PBS control, exogenous netrin-1 attenuated the inflammatory response, PMN infiltration, and expression of the proinflammatory factors IL-1β and TNF-α, while IL-10 expression was up-regulated. In RAW 264.7 cells, recombinant netrin-1 obviously inhibited the mRNA expression of IL-1β and TNF-α and promoted the mRNA expression of the anti-inflammatory cytokine IL-10. Pretreatment with PSB1115 resulted in disease aggravation and higher levels of the proinflammatory factors IL-1β and TNF-α both in vivo and in vitro. And the effect of netrin-1 on inflammatory factors was abolished by PSB1115. Moreover, compared to the control treatment, exogenous netrin-1 significantly facilitated the proliferation and migration of HCECs. ConclusionsNetrin-1 attenuates inflammation in C57BL/6 mice infected with A. fumigatus, and it may play this role via the receptor A2BAR. Additionally, netrin-1 can promote the proliferation and migration of HCECs.

DNA Methylation Regulates Corneal Epithelial Wound Healing by Targeting miR-200a and CDKN2B

DNA methylation is a key epigenetic modification involved in various biological processes and diseases. Corneal epithelial wound healing (CEWH) is essential for restoring corneal integrity and transparency after injury. However, the role of DNA methylation in CEWH remains elusive. Here, we investigate the function and underlying mechanism of DNA methylation in regulating CEWH. Dot blots and global methylation assays determined DNA methylation levels during CEWH. Quantitative RT-PCR and Western blot analysis examined the expression of DNA methyltransferases (DNMTs), cyclin-dependent kinase inhibitor 2B (CDKN2B), and miR-200a during CEWH, respectively. MTS assays and flow cytometry were used to analyze human corneal epithelial cell (HCEC) proliferation and cell cycle, respectively. The in vitro scratch wound assay assessed HCEC migration and an in vivo murine corneal epithelial debridement model evaluated wound healing. Using bisulfite sequencing PCR, we determined the DNA methylation status of the candidate genes. Transfection of miR-200a mimic or inhibitor assessed the function of miR-200a in HCECs. Rescue experiments were performed to clarify the correlation between DNMT1 and miR-200a/CDKN2B during CEWH. DNMT1 and DNMT3B expression was significantly upregulated during CEWH, resulting in a significant global DNA hypermethylation. DNMT1 downregulation dramatically delayed CEWH in vivo, and suppressed HCEC proliferation and migration. MiR-200a inhibited HCEC migration. Furthermore, miR-200a and CDKN2B were identified as molecular targets of DNA methylation and as having a causal connection with DNMT1. DNMT1-mediated DNA hypermethylation can enhance the process of CEWH by directly targeting miR-200a and CDKN2B. This insight pinpoints novel potential drug targets for promoting CEWH.

VITEX NEGUNDO EXTRACTS PROTECT HUMAN CORNEAL EPITHELIAL CELLS FROM ULTRAVIOLET C-INDUCED PHOTOTOXICITY

Objective: The objective of the study was to investigate the protective effects of Vitex negundo (VN) extracts against ultraviolet (UV) light C-induced oxidative stress in human corneal epithelial cells (HCEC) in vitro.&#x0D; Methods: The VN extracts were checked for their antioxidant activities by biochemical tests. The preliminary phytochemical analysis was done to check the presence of secondary metabolites. To trigger oxidative stress, the HCEC were exposed to UVC irradiation (200 J/m2). Then, different concentration of VN extracts was administered after 1 h of UVC irradiation. The effects of UVC irradiation and the extracts on the HCEC viability, morphology, cell migration by scratch assay, and reactive oxygen species (ROS) production by dichlorofluorescein diacetate method were measured.&#x0D; Results: VN ethanol (VNE) extract showed the highest antioxidant activity than the aqueous extract. Phytochemical screening shows the presence of alkaloids, carbohydrates, proteins, phenolics, and tannins. The extracts showed no cytotoxicity on the HCEC and also the VNE extract did not alter the morphology of the cells. Results suggest that the VNE extract enhanced the proliferation and migration of HCEC. The VNE extract significantly decreased UVC-induced oxidative toxicity by inhibiting the intracellular ROS production and maintained cell viability.&#x0D; Conclusion: VN extracts with its strong antioxidant potential protects HCEC from UVC-induced oxidative stress. The results suggest that these extracts can be used as prophylactic agents or as a therapeutic agent in overcoming UV light-induced damage in various ocular surface diseases such as pterygium and photokeratitis.

Effect of Nitric Oxide on Human Corneal Epithelial Cell Viability and Corneal Wound Healing

Although the wound healing effects of nitric oxide (NO) are known, the mechanism by which NO modulates corneal wound healing remains unclear. In this study, we investigated the effect of exogenous NO donor (NaNO2) on corneal wound healing. We found that NaNO2 (0.1 μM to 100 μM) increased human corneal epithelial cell (HCEC) viability and migration. It also modulated the phosphorylation of mitogen-activated protein kinases (MAPKs) in a time- dependent manner in those HCECs. Further, p38 MAPK phosphorylation increased at 6 h and normalized at 24 h, while the phosphorylation of extracellular signal regulated kinase (ERK) was increased both at 6 h and 24 h. Topical treatment with NaNO2 (10 μM) enhanced corneal epithelial healing and decreased corneal opacity in murine corneal alkali burn model by modulating inflammatory cytokines. Our findings suggest that NO increased HCEC proliferation and migration via time-dependent MAPK activation and eventually enhanced corneal recovery from the alkali burn.

Effect of human autologous serum and fetal bovine serum on human corneal epithelial cell viability, migration and proliferation in vitro.

To analyze the concentration-dependent effects of autologous serum (AS) and fetal bovine serum (FBS) on human corneal epithelial cell (HCEC) viability, migration and proliferation. AS was prepared from 13 patients with non-healing epithelial defects Dulbecco's modified eagle medium/Ham's F12 (DMEM/F12) with 5% FBS, 0.5% dimethyl sulphoxide (DMSO), 10 ng/mL human epidermal growth factor, 1% insulin-transferrin-selenium, then were incubated in serum media: DMEM/F12 supplemented by 5%, 10%, 15% or 30% AS or FBS. HCEC viability was analyzed using cell proliferation kit XTT, migration using a wound healing assay, proliferation by the cell proliferation enzyme-linked immunosorbent assay (ELISA) BrdU kit. Statistical analysis was performed using the generalized linear model, the values at 30% AS or 30% FBS were used as the baselines. HCEC viability was the highest at 30% AS or 15% FBS and the lowest at 10% AS or 30% FBS application. HCEC migration was the quickest through 30% AS or 30% FBS and the slowest through 5% AS or 5% FBS concentrations. Proliferation was the most increased through 15% AS or 5% FBS and the least increased through 30% AS or 30% FBS concentrations. HCEC viability at 10% and 15% AS was significantly worse (P=0.001, P=0.023) compared to baseline and significantly better at 15% FBS (P=0.003) concentrations. HCEC migration was significantly worse (P≤0.007) and HCEC proliferation significantly better (P<0.001) in all concentration groups compared to baseline. For the best viability of HCEC 30% AS or 15% FBS, for HCEC migration 30% AS or 30% FBS, for proliferation 15% AS or 5% FBS should be used. Therefore, we suggest the use of 30% AS in clinical practice.