Blockade of extracellular high-mobility group box 1 attenuates inflammation-mediated damage and haze grade in mice with corneal wounds.

PURPOSE: To investigate the expression of extracellular high mobility group box 1 (HMGB1) and the effect of its inhibitor glycyrrhizin (GL) in corneal wound healing. METHODS: We treated C57BL/6J mice with GL or PBS before and after establishing a corneal injury model. Fluorescein staining, Ki-67 expression, haze grade, and haematoxylin/eosin (H&E) staining were used to assess treatment efficacy. The expression of HMGB1, NF-κB-p65, the NLRP3 inflammasome, IL-1ß, CCL2, CXCL2, TGF-ß1, α-SMA, fibronectin, and collagen III and neutrophil influx were examined by immunohistochemical staining, western blot, and RT-qPCR at various time points after corneal injury. RESULTS: After corneal injury, HMGB1 transferred from the nucleus to the cytoplasm and was passively released or actively secreted into the corneal stroma from epithelial cells and inflammatory cells; however, this increase was attenuated by GL treatment. Furthermore, GL indirectly attenuated the expression of IL-1ß by directly inhibiting extracellular HMGB1 functions, which activated the NF-κB-p65/NLRP3/IL-1ß signalling pathway. Moreover, application of GL alleviated the neutrophil infiltration that delays wound healing, accompanied by the downregulation of expression of the chemokines CCL2 and CXCL2. More interestingly, application of GL reduced the degree of haze grade through inactivating extracellular HMGB1 functions that induced TGF-ß1 release and myofibroblast differentiation. In addition, fluorescein and H&E staining and Ki-67 levels suggest that GL promotes regeneration of corneal epithelium. CONCLUSIONS: After corneal injury, extracellular HMGB1 can be an essential driver to trigger a neutrophil- and cytokine-mediated inflammatory injury amplification loop. The application of GL promotes the cornea to restore transparency and integrity, which may be related to the attenuation of extracellular HMGB1 levels and function.

L­carnitine alleviates oxidative stress­related damage via MAPK signaling in human lens epithelial cells exposed to H2O2.

L­carnitine (LC) is well known for its antioxidative properties. The present study aimed to evaluate the effects of LC on human lens epithelial cells (HLECs) and to analyze its regulatory mechanism in cataractogenesis. HLE B­3 cells were cultured with hydrogen peroxide (H2O2) and were pretreated with or without LC. The Cell Counting kit­8 assay was used to determine cell viability. Reactive oxygen species (ROS) assay kit was used to measure the cellular ROS production induced by H2O2 and LC. In addition, reverse transcription­quantitative PCR and western blot analysis were performed to detect the expression levels of oxidative damage markers and antioxidant enzymes. Notably, ROS overproduction was observed upon exposure to H2O2, whereas LC supplementation markedly decreased ROS levels through activation of the antioxidant enzymes forkhead box O1, peroxiredoxin 4 and catalase. Furthermore, LC suppressed the expression of apoptosis­associated genes (caspase-3) and inflammation­associated genes [interleukin (IL)1, IL6, IL8 and cyclooxygenase­2]. Conversely, LC promoted proliferating cell nuclear antigen, cyclin­dependent kinase (CDK)2 and CDK4 expression, which may increase proliferation of HLECs that were incubated with H2O2. In addition, epithelial­mesenchymal transition occurred upon ROS accumulation, whereas the effects of H2O2 on AQP1 and vimentin expression were reversed upon LC supplementation. Notably, this study revealed that LC restored the oxidant/antioxidant balance and protected against cell damage through the mitogen­activated protein kinase signaling pathway. In conclusion, LC may serve a protective role in curbing oxidative damage and therefore may be considered a potential therapeutic agent for the treatment of cataracts.

Role of Smad3 signaling in the epithelial­mesenchymal transition of the lens epithelium following injury.

Transforming growth factor­ß (TGF­ß) is important in the development of posterior capsule opacification (PCO), and inhibition of the TGF­ß pathway may represent a novel method of treating PCO. Drosophila protein, mothers against decapentaplegic homolog 3 (Smad3) is a phosphorylated receptor­activated Smad required for the transmission of TGF­ß signals. Smad3 knockout (KO) disturbs the activation of TGF­ß signaling, thus inhibiting the onset of PCO. In the present study, lens epithelial cell (LEC) damage induced by extracapsular cataract extraction was simulated by puncture of the anterior capsule using a 26­gauge hypodermic needle. The effect of Smad3 in the trauma­induced epithelial­mesenchymal transition (EMT) of the lens epithelium in Smad3­KO and wild­type (WT) mice was then observed. The expression levels of EMT markers and extracellular matrix components were measured in the two groups by reverse transcription­quantitative polymerase chain reaction analysis, western blot analysis and immunofluorescence staining. Apoptosis was also detected in the punctured anterior capsule. The Smad3­KO mice exhibited lower expression levels of α­smooth muscle actin, lumican, osteopontin, fibronectin and collagen, compared with the WT mice. Additionally, the Smad3­KO mice exhibited a higher percentage of apoptotic cells than the WT mice. Smad3 signaling was associated with the induction of trauma­induced EMT, and Smad3 KO interfered with TGF­ß signaling pathway activation, but did not completely inhibit the trauma­induced EMT in LECs. Therefore, Smad3 may be a target in the treatment of PCO and other fibrosis­related diseases.

Paracrine effects of mesenchymal stem cells on the activation of keratocytes.

AIMS: The purpose of this study is to investigate the impact of mesenchymal stem cell (MSC)-derived soluble factors on the function of keratocytes, with a particular focus on the processes involved in wound healing, including keratocyte activation, migration and proliferation as well as extracellular matrix (ECM) synthesis. METHODS: Primary cultured rabbit keratocytes were treated with MSC-conditioned medium (MSC-CM). The paracrine factors released by bone marrow MSCs were examined by ELISA. Time-lapse microscope was used to examine wound closure in vitro. Mouse model of corneal injury was made by epithelial scraping after ethanol injury. RESULTS: MSC-CM significantly increased the wound closure rate of corneal stromal cells in vitro. This enhancement of wound closure by MSC-CM was due to the promotion of cell migration. MSC-CM enhanced keratocyte survival following ethanol injury via inhibiting apoptosis. The expression of ECM component genes in keratocytes was upregulated by MSC-CM. In addition, MSC-CM promoted corneal epithelial wound healing following chemical injury. A number of wound healing mediators were detected in MSC-CM, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), transforming growth factor beta 1 (TGFß1), interleukin 8 (IL8), interleukin 6 (IL6) and monocyte chemoattractant protein 1 (MCP1). CONCLUSION: MSC secretes certain factors that accelerate corneal re-epithelialisation. The paracrine effects of MSC on corneal wound healing including improvements in cell viability, migration and ECM formation.