Previous studies have revealed that high levels of serum homocysteine (Hcy) are closely associated with the development of juvenile and age-related cataracts. An increased concentration of Hcy is likely to induce gene specific demethylation in DNA promoter regions. The aim of the present study was to prevent this demethylation by administering acetyl-l-carnitine (ALCAR) to human lens epithelial cells (HLECs). Different concentrations of Hcy were used to treat HLECs for 3, 6, 12 and 24 h and the findings were used to determine the optimum dose to induce endoplasmic reticulum (ER) stress. Similarly, the concentration of ALCAR was standardized. The production of reactive oxygen species (ROS) and the percentage of cells undergoing cell death were measured. The levels of antioxidants, ER stress-associated proteins, mRNA levels of nuclear factor erythroid-2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1) and promoter DNA methylation of the Keap1 gene were also assessed. Hcy was observed to induce ER stress, produce ROS and lead to cell death. However, administration of ALCAR prevented these effects to a significant degree. Additionally, western blot analysis revealed that ALCAR increased the levels of antioxidant proteins, including catalase, superoxide dismutase, glutathione peroxidase, Nrf2, Keap1 and glutathione. Similarly, the reverse transcription-quantitative polymerase chain reaction experiments on Nrf2 and Keap1, as well as the bisulfite genomic DNA sequencing analysis revealed a preventive effect of ALCAR against Hcy-induced ER stress. The ER stress-induced activation of the unfolded protein response is responsible for demethylation of Keap1 promoter DNA to activate the expression of the Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. This decrease in Nrf2 activity represses the transcription of numerous antioxidant enzyme genes and alters the redox-balance towards lens oxidation. However, treatment with ALCAR led to significant protection from these effects. The present results suggested that ALCAR either prevents or ameliorates the actions of the antioxidant system in HLECs at the level of the protein and the gene. Further advanced studies are required for the development of ALCAR as an anti-cataract agent.

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ABSTRACT. In this study, we aimed to investigate the feasibility of directed differentiation of human amniotic epithelial cells into conjunctival epithelium under specific conditions as well as of constructing tissueengineered conjunctiva for ocular surface reconstruction. Human amniotic epithelial cells were cultured with induced denuded conjunctival matrix and conjunctival homogenate. Immunohistochemistry of cytokeratin-4, cytokeratin-13, and muc5ac as well as PAS staining were performed. The concentration of muc5ac at different times was measured using ELISA. The differentiated cells with quantum dots were transferred onto a denuded amniotic membrane to establish tissue-engineered conjunctiva and transplanted into a rabbit model with a conjunctival defect. After induction of human amniotic epithelial cells, differentiated cells showed conjunctival epithelium phenotype, while trace amounts of mu5ac in the culture medium measured by ELISA increased gradually within 1 to 7 days. Successfully tissue-engineered conjunctiva had similar structure as normal conjunctiva and was transplanted into a rabbit model with conjunctiva defect. After 2 weeks post-surgery, conjunctiva grafts 13824 S.P.Yang et al.©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 14 (4): 13823-13834 (2015) survived and were integrated. Immunohistochemistry showed conjunctival epithelium phenotype, positive cells were found in PAS staining. Thus, human amniotic epithelial cells could differentiate into conjunctival epithelium-like cells and goblet cells with partially physiological function, and we successfully restored ocular surface integrity in the rabbit model using tissue-engineered conjunctiva.

Diabetic retinopathy (DR) is a common complication of diabetes mellitus (DM), which results in vision loss. This study explored the role of miR-126 in high-glucose-induced human retinal endothelial cells (HRECs) and its underlying molecular mechanisms. The results showed that the expression levels of miR-126 and interleukin-17A (IL-17A) in high-glucose-induced HRECs were downregulated and upregulated, respectively. Functionally, overexpression of miR-126 promoted proliferation and suppressed apoptosis in high-glucose-induced HRECs, while IL-17A reversed the effects induced by miR-126. However, overexpression of IL-17A inhibited the proliferation and induced apoptosis, while knockdown of IL-17A accelerated the proliferation and repressed apoptosis. In addition, miR-126 repressed the expression of IL-17A, Bax, and caspase-3, while promoting the expression of survivin and phosphorylation of PI3K and AKT; restoration of IL-17A rescued these effects. Furthermore, IL-17A was identified as a target of miR-126. This indicates that miR-126 enhances proliferation and inhibits apoptosis in high-glucose-induced HRECs by activating the PI3K–AKT pathway, increasing survivin levels, and decreasing Bax and caspase-3 expression by targeting IL-17A, suggesting that miR-126 could be a novel target for preventing DR.

Dan-zhi-xiao-yao-san is a Traditional Chinese Medicinal formulation widely used for the treatment of neuropsychological disorders. The present study examined the anxiolytic and neuroprotective effects of Dan-zhi-xiao-yao-san in a rat model of chronic stress. The results of an elevated plus maze test showed that Dan‑zhi‑xiao‑yao‑san significantly attenuated the levels of anxiety-induced stress as evidenced by increases in the time spent in the open arm region, as well as the percentage of entries into this area. In addition, Dan-zhi-xiao-yao-san alleviated stress‑induced neuronal death, as indicated by histological examination. Furthermore, mechanistic studies suggested that the anxiolytic and neuroprotective effects of Dan-zhi-xiao-yao-san may be mediated via attenuation of chronic stress‑induced upregulation of α‑synuclein and corticosterone, and downregulation of protein phosphatase 2A (PP2A) in the hippocampal region of the brain at the mRNA and protein level. In addition, Dan‑zhi‑xiao‑yao‑san decreased the serum levels of stress‑induced corticosterone in the model animals. In conclusion, the present study demonstrated that Dan‑zhi‑xiao‑yao‑san exerted anxiolytic and neuroprotective effects in a rat model of chronic stress via attenuation of stress‑induced upregulation of α‑synuclein and corticosterone, and downregulation of PP2A in the hippocampus.

Previous studies have revealed that uncontrollable stress can impair the synaptic plasticity and firing property of hippocampal neurons, which influenced various hippocampal-dependent tasks including memory, cognition, behavior, and mood. In this work, we had investigated the effects and mechanisms of the Chinese herbal medicine Xiao Yao San (XYS) against corticosterone-induced stress injury in primary hippocampal neurons (PHN) cells. We found that XYS and RU38486 could increase cell viabilities and decrease cell apoptosis by MTT, immunofluorescence, and flow cytometry assays. In addition, we observed that XYS notably inhibited the nuclear translocation of GR and upregulated the mRNA and protein expressions levels of Caveolin-1, GR, BDNF, TrkB, and FKBP4. However, XYS downregulated the FKBP51 expressions. Furthermore, the results of the electrophoretic mobility shift assay (EMSA) and double luciferase reporter gene detection indicated that FKBP4 promotes the transcriptional activity of GR reaction element (GRE) by binding with GR, and FKBP51 processed the opposite action. The in vivo experiment also proved the functions of XYS. These results suggested that XYS showed an efficient neuroprotection against corticosterone-induced stress injury in PHN cells by upregulating GRE transcriptional activity, which should be developed as a potential candidate for treating stress injury in the future.

Diabetic retinopathy (DR) is one of the most severe and common complications caused by diabetic mellites. Inhibiting NLRP3 inflammasome activation displays a crucial therapeutic value in DR. Studies have shown that KCNQ1OT1 plays a critical role in regulating NLRP3 inflammasome activation and participates in the pathogenesis of diabetic complications. The present study aims to explore the role, and the potential mechanism of KCNQ1OT1 in regulating the activation of NLRP3 inflammasome in DR. The expression of KCNQ1OT1 and the activation of NLRP3 inflammasome were increased in experimental DR models. KCNQ1OT1 knockdown alleviated NLRP3 inflammasome-associated molecules expression. In addition, KCNQ1OT1 was found to be localized mainly in the cytoplasm of Müller cells and facilitated TXNIP expression by acting as a miR-17-5p sponge. KCNQ1OT1 promoted the activation of NLRP3 inflammasome through miR-17-5p/TXNIP axis. Moreover, the clinical samples of patients with DR showed that the expression of KCNQ1OT1 and the activation of NLRP3 inflammasome were all increased, further supporting the hypothesis that the KCNQ1OT1 dysregulation may be the molecular mechanism of the pathogenesis of DR. Therefore, KCNQ1OT1 may serve as a new therapeutic target for DR.