Indocyanine green (ICG) and brilliant blue G (BBG) are commonly used vital dyes to remove internal limiting membrane (ILM) in vitreoretinal surgery. The vital dyes have shown cytotoxic effects in ocular cells. Autophagy is a stress responsive pathway for either protecting cells or promoting cell death. However, the role of autophagy in ocular cells in response to the vital dyes remains unknown. In this study, we found that ICG and BBG reduced cell viability in both human retinal pigment epithelial ARPE-19 and mouse photoreceptor 661W cells. ICG and BBG induced lipidated GFP-LC3-II and LC3-II in ARPE-19 and 661W cells. Combination treatment with the autophagy inhibitor chloroquine indicated that ICG and BBG reduced autophagic flux in ARPE-19 cells, whereas the vital dyes induced autophagic flux in 661W cells. Moreover, genetic and pharmacological ablation of autophagy enhanced vital dyes-induced cytotoxicity in ocular cells. Dietary supplements, including resveratrol, lutein, and CoQ10, induced autophagy and diminished the cytotoxic effects of ICG and BBG in ocular cells. These results suggest that autophagy may protect ARPE-19 and 661W cells from vital dyes-induced damage.

Age-related macular degeneration (AMD) is an ocular disease with retinal degeneration. Retinal pigment epithelium (RPE) degeneration is mainly caused by long-term oxidative stress. Kinase activity could be either protective or detrimental to cells during oxidative stress; however, few reports have described the role of kinases in oxidative stress. In this study, high-throughput screening of kinome siRNA library revealed that erb-b2 receptor tyrosine-protein kinase 2 (ERBB2) knockdown reduced reactive oxygen species (ROS) production in ARPE-19 cells during oxidative stress. Silencing ERBB2 caused an elevation in microtubule associated protein light chain C3-II (MAP1LC3B-II/I) conversion and sequesterone (SQSTM)1 protein level. ERBB2 deprivation largely caused an increase in autophagy-regulating protease (ATG4B) expression, a protease that negatively recycles MAP1LC3-II at the fusion step between the autophagosome and lysosome, suggesting ERBB2 might modulate ATG4B for autophagy induction in oxidative stress-stimulated ARPE-19 cells. ERBB2 knockdown also caused an accumulation of nuclear factor erythroid 2-related factor 2 (NRF2) and enhanced its transcriptional activity. In addition, ERBB2 ablation or treatment with autophagy inhibitors reduced oxidative-induced cytotoxic effects in ARPE-19 cells. Furthermore, ERBB2 silencing had little or no additive effects in ATG5/7-deficient cells. Taken together, our results suggest that ERBB2 may play an important role in modulating autophagic RPE cell death during oxidative stress, and ERBB2 may be a potential target in AMD prevention.

Dental pulp stem cells (DPSCs) and stem cells from the apical papilla (SCAPs) are oral mesenchymal stem cells capable of self‐renewal and have a potential for multilineage differentiation. Increasing evidence shows that microRNAs (miRNAs) play important roles in stem cell biology. Here, we focused on exploring miR‐146a‐5p and its relationship to the undifferentiated status of STRO‐1+ SCAPs and STRO‐1+ DPSCs, as well as its role during STRO‐1+ DPSC differentiation and proliferation. Our data indicated that baseline miR‐146a‐5p expression is significantly lower in STRO‐1+ SCAPs than in STRO‐1+ DPSCs and increased in the latter during osteogenic induction. Moreover, we identified miR‐146a‐5p as a key miRNA that promotes osteo/odontogenic differentiation of STRO‐1+ DPSCs and attenuates cell proliferation. Additionally, it was observed that STRO‐1+ DPSC mineralization results in the downregulation of notch receptor 1 (NOTCH1) and hes family bHLH transcription factor 1 (HES1). Interference with neurogenic locus notch homolog protein 1 (Notch 1) signaling was verified to enhance differentiation and suppress STRO‐1+ DPSC proliferation. It was further observed that miR‐146a‐5p directly targets the 3′‐untranslated region (3′‐UTR) of NOTCH1 and inhibits expression of both NOTCH1 and HES1mRNAs and Notch 1 and transcription factor HES‐1 (HES‐1) proteins in STRO‐1+ DPSCs. We conclude that miR‐146a‐5p exerts its regulatory effect on STRO‐1+ DPSC differentiation and proliferation partially by suppressing Notch signaling.

Introduction. Pulp regeneration, as a treatment for pulp necrosis, has significant advantages over root canal therapy for the preservation of living pulp. To date, research on pulp regeneration has mainly focused on the transplantation of pulp stem cells into the root canal, but there is still a lack of research on the migration of pulp cells into the root canal via cell homing. Stem cells from the apical tooth papilla (SCAP) are recognized as multidirectional stem cells, but these cells are difficult to obtain. MicroRNAs are small noncoding RNAs that play crucial roles in regulating normal and pathologic functions. We hypothesized that some types of microRNAs might improve the migration and proliferation function of dental pulp stem cells (DPSCs), which are easily obtained in clinical practice, and as a result, DPSCs might replace SCAP and provide valuable information for regenerative endodontics. Methods. Magnetic activated cell sorting of DPSCs and SCAP was performed. Next-generation sequencing was performed to examine DPSCs and SCAP miRNAs expression and to identify the most significant differentially expressed miRNA. CCK-8 and transwell assays were used to determine the impact of this miRNA on DPSCs proliferation and migration. Results. The most significant differentially expressed miRNA between DPSCs and SCAP was miR-224-5p. Downregulating miR-224-5p promoted DPSCs proliferation and migration; the opposite results were observed when miR-224-5p was upregulated. Conclusion. MiR-224-5p promotes proliferation and migration in DPSCs, a finding that is of great significance for further exploring the role of dental pulp stem cells in regenerative endodontics.

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