Anti-fibrosis potential of pirarubicin via inducing apoptotic and autophagic cell death in rabbit conjunctiva

Xu, Lijuan; Rong, Shi Song; Xu, Yifeng; Zheng, Lei; Qiu, Weiqiang; Zhang, Xia; Jiang, Lou-jing; Duan, Runping; Tian, Tian; Yao, Yufeng

doi:10.1016/j.exer.2020.108215

Cited by 6 publications

(8 citation statements)

References 68 publications

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“…Intracellular ROS levels were determined using a 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe (Beyotime Biotechnology, Shanghai, China) by measuring the oxidative conversion of cell-permeable DCFH-DA to fluorescent dichlorofluorescein according to the manufacturer’s instructions and a previous study ( Xu et al, 2020 ). Briefly, 10 μM DCFH-DA solution diluted in serum-free DMEM/F12 medium was added and incubated with the cells, then they were kept in the dark for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals

Zhang

Zhao

et al. 2023

eLife

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This study aimed to investigate the protective effect of metformin on trabecular meshwork (TM) and explore its molecular mechanisms in vivo and in vitro. Ocular hypertension (OHT) mouse models were induced with dexamethasone and further treated with metformin to determine the intraocular pressure (IOP)-lowering effect. Cultured human TM cells (HTMCs) were pre-stimulated with tert-butyl hydroperoxide (tBHP) to induce oxidative damage and then supplemented with metformin for another 24 hr. The expression of fibrotic markers and integrin/Rho-associated kinase (ROCK) signals, including α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), fibronectin, integrin beta 1, ROCK 1/2, AMP-activated protein kinase, myosin light chain 1, and F-actin were determined by western blotting and immunofluorescence. Reactive oxygen species (ROS) content was analysed using flow cytometry. Transmission electron microscopy was performed to observe microfilaments in HTMCs. It showed that metformin administration reduced the elevated IOP and alleviated the fibrotic activity of aqueous humour outflow in OHT models. Additionally, metformin rearranged the disordered cytoskeleton in the TM both in vivo and in vitro and significantly inhibited ROS production and activated integrin/ROCK signalling induced by tBHP in HTMCs. These results indicated that metformin reduced the elevated IOP in steroid-induced OHT mouse models and exerted its protective effects against oxidative injury by regulating cytoskeleton remodelling through the integrin/ROCK pathway. This study provides new insights into metformin use and preclinical evidence for the potential treatment of primary open-angle glaucoma.

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Section: Methodsmentioning

confidence: 99%

Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals

Zhang

Zhao

et al. 2023

eLife

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“…Cells were lysed 24 h after drug treatment as previously described ( Xu, et al,2020 ). After gel separation and membrane transfer, myocilin (Abcam), integrin 1 (Abcam), AMPK (CST), pAMPK (CST), ROCK1/2 (Abcam), MLC1 (Abcam), and F-actin (Abcam) were detected.…”

Section: Methodsmentioning

confidence: 99%

“…At the time of harvest, the mice were re-anesthetized, and the eyes were fixed in 4% paraformaldehyde overnight, embedded in paraffin or optimum cutting temperature compound in sagittal axis. The sections were incubated overnight with primary antibodies at 4°C according to the manufacturers’ instructions ( Xu, et al,2020 ).α-SMA (CST), TGF-β (Abcam), F-actin (Abcam), fibronectin(Abcam)were detected. The secondary antibodies were goat anti-rabbit or anti-mouse (CST) at a 1:500 dilution.…”

Section: Methodsmentioning

confidence: 99%

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Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals

Zhang

Yin

et al. 2022

Preprint

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Background: This study aimed to investigate the protective effect of metformin on the trabecular meshwork (TM) and explore its molecular mechanisms in vivo and in vitro. Methods: Ocular hypertension (OHT) mouse models were induced with dexamethasone (DEX) and further treated with metformin to determine its IOP lowering effect. Cultured human TM cells (HTMC) were pre-stimulated with tert-butyl hydroperoxide (tBHP) to induce oxidative damage and then supplemented with metformin for another 24 h. The expression of fibrotic markers and integrin/ROCK signals, including α-SMA, TGF-β, fibronectin, F-actin, integrin beta 1, Rho-associated kinase (ROCK)1/2, AMP-activated protein kinase (AMPK), myosin light chain 1 (MLC 1), and F-actin were determined by western blotting (WB) and immunofluorescence (IF). Reactive oxygen species (ROS) content was analysed using flow cytometry (FCM). Results: Administration of metformin reduced the elevated IOP and alleviated the fibrotic activity of aqueous humour outflow in OHT models. Additionally, metformin rearranged the disordered cytoskeleton in the TM both in vivo and in vitro. Furthermore, metformin significantly inhibited ROS production and activated integrin/ROCK signalling induced by tBHP in HTMC. Conclusion: Metformin reduced the elevated IOP in steroid-induced OHT mouse models and exerted its protective effects against oxidative injury by regulating cytoskeleton remodelling through the integrin/ROCK pathway. This study provides new insights into metformin use and preclinical evidence for the potential treatment of primary open-angle glaucoma. Funding: This work was supported by the Key R&D Program of Zhejiang (2022C03112), Leading Scientific and Technological Innovation Talents in Zhejiang Province (2021R52012), National Key R&D Program of China (2020YFC2008200), Zhejiang Provincial National Science Foundation of China (LQ18H120010), Key Innovation and Guidance Program of the Eye Hospital, School of Ophthalmology & Optometry,Wenzhou Medical University (YNZD 2201903), and the Wenzhou Municipal Technological Innovation Program of High-level Talents (No. 604090352/577).

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“…The expression of FN1 was increased in blood samples from pterygium patients (Engelsvold et al 2013). In our study, we also detected the increased expression of FN1 in PCE samples.In addition EMT-related genes, other pathway genes closely related to the development of pterygium were also enriched in this study; the related pathways included in ammation, oxidative stress (including DNA repair, excision repair and DNA damage), death (including autophagy, apoptosis and ferroptosis), angiogenesis, and lymph angiogenesis (Chui et al 2011;Liu et al 2013;Notara et al 2015;Xu et al 2020a…”

mentioning

confidence: 99%

Identification of pterygium-related conjunctival epithelium microRNA and mRNA expression profiles by high-throughput screening analysis

Chen

et al. 2022

Preprint

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Purpose. To detect the hub mRNAs and construct a microRNA (miRNA)-mRNA regulatory network by comparing pterygium-related conjunctival epithelium (PCE) and normal conjunctival epithelium (NCE) patient samples by high-throughput sequencing.Methods. Bulbar conjunctival epithelium samples were obtained from patients with primary pterygium and from donated normal eyeballs. Bioinformatics analysis, which included differential gene expression, gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis, was applied for the functional enrichment analysis of differentially expressed mRNAs (DEmRNAs). Protein-protein interaction (PPI) analysis and gene set enrichment analysis (GSEA) were performed on DEmRNAs whose pathways were related to the development of pterygium. Potential miRNAs binding to these hub DEmRNAs were predicted using software, and a hub mRNA-miRNA regulatory network was constructed. MiRNAs were verified through RT-qPCR.Results. Overall, the results showed 280 upregulated and 256 downregulated DEmRNAs. To explore the potential functions of DEmRNAs, we performed bioinformatics analysis to detect the potential functions of DEmRNAs and the relevant pathways related to the pathogenesis of pterygium in PCE. In addition, we enriched six pathways that were involved in the development of pterygium using a heatmap. Then, the GSEA results showed that galactose metabolism, epithelial mesenchymal transition and so on were significantly enriched among upregulated mRNAs (P＜0.001, and NES ≥ 1). Cytokine-cytokine receptor interaction, primary immunodeficiency, MAPK signalling pathway and so on were significantly enriched among downregulated mRNAs (P＜0.001, and NES ≤ -1). Then, a miRNA/mRNA regulatory network was constructed with 20 miRNAs and 48 differentially expressed genes (DEGs). The RT-qPCR validation study confirmed nine downregulated miRNAs (miR-653-3p, miR-3617-5p, miR-4681, miR-4802-3p, miR-888-3p, miR-7106-3p, miR-543, miR-200b-3p and miR-203a-3p) and two upregulated miRNAs (miR-5579b-3p and miR-6844).Conclusion. Our study shows the mRNA expression profile and miRNA/mRNA regulatory network of the PCE and provides a new perspective on the pathogenesis of pterygium.

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Anti-fibrosis potential of pirarubicin via inducing apoptotic and autophagic cell death in rabbit conjunctiva

Cited by 6 publications

References 68 publications

Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals

Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals

Metformin protects trabecular meshwork against oxidative injury via activating integrin/ROCK signals

Identification of pterygium-related conjunctival epithelium microRNA and mRNA expression profiles by high-throughput screening analysis

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