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Purpose: Despite being crucial to neuronal survival, the role Müller cells play in geographic atrophy (GA) has only recently been considered. We investigated whether Müller cells retain their normal functional profile or form a fibrotic scar when remodeling in human GA eyes and our subretinal sodium iodate (NaIO3) model., Methods: Sprague Dawley rats given subretinal injections of NaIO3 (5 mg/mL) were sacrificed at 3 and 12 weeks. Cryosections and retinal flatmounts from rats and cryosections from human GA eyes were stained with antibodies against the Müller cell proteins glutamine synthetase (GS), inwardly rectifying potassium channel 4.1 (Kir4.1), aquaporin 4 (AQP4), cellular retinaldehyde-binding protein 1 (CRALBP), and glial fibrillary acidic protein (GFAP), as well as alpha smooth muscle actin (α-SMA), fibronectin, and collagens I and IV. The immunofluorescence intensity of AQP4 and Kir4.1 was quantified using Image J, and Kir4.1 protein levels were verified by western blot., Results: In both human GA eyes and NaIO3-injected rats, Müller cell processes at the external limiting membrane (ELM) descent and in the subretinal membrane exhibited increased GS expression. GFAP was elevated throughout the Müller cells. AQP4 staining at the ELM descent was particularly pronounced throughout the radial processes, including those extending into the subretinal space. In NaIO3-injected rats, perivascular Kir4.1 expression significantly decreased in the atrophic retina, but expression increased in the subretinal glial membrane. α-SMA and extracellular matrix proteins were not detected in the subretinal membrane., Conclusions: Our findings underscore the persistence of homeostatic proteins, albeit altered, in Müller cells as they remodel and extend into the subretinal space.

It has been confirmed that the expression of miR-501-3p is closely related to the behavior of several cancers. This study aimed to elucidate the effects of miR-501-3p/SPC24 axis on the behavior of renal cancer cells and to identify its prognostic value in renal cancer. First, the expression of miR-501-3p in the renal cell carcinoma (RCC) cell line was detected using real-time quantitative polymerase chain reaction (RT-qPCR). Second, cell function identification experiments were performed, including CCK-8, scratch, transwell invasion, and flow cytometry assays. Several databases were applied to explore the possible mechanism of miR-501-3p tumor suppressor effect in RCC. To explore the value of miR-501-3p/SPC24 axis in predicting renal cancer patient overall survival (OS), GEPIA (http://gepia.cancer-pku.cn/index.html) was used. Finally, western blot was performed to detect the expression level of SPC24 in renal cancer cells predicted by bioinformatics analysis. Dual-Luciferase Reporter Assay was used to verify if SPC24 is a target of mir-501-3p. MiR-501-3p was found to be down-regulated in cancer cells and tissues and to play a role in suppressing tumor cell proliferation, cell viability, cell migration, and cell invasion, while promoting apoptosis. We also found that high expression levels of SPC24 were associated with shorter OS time in patients diagnosed with renal cell carcinoma. In addition, the results of TCGA data analysis and western blot showed that the tumor suppressor effect of miR-501-3p may be achieved by targeting SPC24. The MiR-501-3p/SPC24 axis affects cell proliferation, migration, invasion, apoptosis, and prognosis in renal cell carcinoma.

Purpose: The purpose of this study was to investigate the role of LRRK2 in the inflammatory response to fungal keratitis (FK) and elucidate the underlying mechanisms., Methods: The protein levels of leucine-rich repeat kinase 2 (LRRK2), p-LRRK2, and stimulator of interferon genes (STING)-related proteins were assessed by western blot analysis. ELISA and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to evaluate the inflammatory response induced by Aspergillus fumigatus. Mass spectrometry was performed to identify the interaction partners of LRRK2. The glutathione S-transferase (GST) pull-down assay and co-immunoprecipitation (co-IP) were used to verify the interaction between LRRK2 and STING. Additionally, fungal load determinations and clinical score assessments were conducted to determine corneal infection in a mouse model., Results: A. fumigatus stimulation promoted the phosphorylation of LRRK2 through Toll-like receptor 2 (TLR2) in human corneal epithelial cells (HCECs) and mouse corneas. LRRK2 overexpression enhanced the A. fumigatus-induced inflammatory response, and LRRK2 knockdown alleviated A. fumigatus keratitis both in vitro and in vivo. Mass spectrometry identified STING as a novel interaction partner of LRRK2. Moreover, A. fumigatus treatment enhanced the interaction between LRRK2 and STING, resulting in the phosphorylation and activation of STING. The phosphorylated STING then triggered its downstream signaling pathways, exacerbating the severity of A. fumigatus keratitis. LRRK2 inhibitor (LRRK2-IN-1) significantly mitigated the inflammatory response and corneal damage caused by A. fumigatus stimulation., Conclusions: LRRK2 inhibition ameliorates A. fumigatus-induced inflammation through modulating STING signaling pathways in both HCECs and mouse models. Our results suggest that targeted inhibition of LRRK2 could be a promising strategy for FK treatment.

Purpose: Allograft rejection remains a major cause of failure in high-risk corneal transplants, but the underlying mechanisms are not fully understood. This study aimed to investigate the contribution of transplantation stress-induced cellular senescence to corneal allograft rejection and to elucidate the associated molecular mechanisms., Methods: Age-matched murine corneal transplantation models were established. Cellular senescence was evaluated using senescence-associated β-galactosidase (SA-β-Gal) staining, western blot, and immunofluorescence staining. The role of cellular senescence in corneal allograft rejection was analyzed using p16 knockout mice and adoptive transfer experiments. Senolytic treatment with ABT-263 was administered intraperitoneally to evaluate its effects on corneal allograft rejection. RNA sequencing and pharmacological approaches were employed to identify the underlying mechanisms., Results: Surgical injury induced a senescence-like phenotype in both donor corneas and recipient corneal beds, characterized by an increased accumulation of SA-β-Gal-positive cells in the corneal endothelium and stroma and elevated expression of senescence markers p16 and p21. Using genetic and adoptive transfer models, transplantation stress-induced senescence was shown to exacerbate corneal allograft rejection. Importantly, clearance of senescent cells by ABT-263 significantly suppressed ocular alloresponses and immune rejection. Mechanistically, RNA sequencing and loss-of-function experiments demonstrated that the anti-rejection effects of senolytic treatment were closely dependent on angiotensin-converting enzyme 2 (ACE2)., Conclusions: These findings highlight transplantation stress-induced senescence as a pivotal pathogenic factor in corneal allograft rejection. Senolytic therapy emerges as a potential novel strategy to mitigate transplant rejection and improve corneal allograft survival.

Nasopharyngeal carcinoma (NPC) is a malignant tumor predominantly influenced by Epstein-Barr virus infection and genetic factors. The transforming growth factor-beta (TGF-β) superfamily is implicated in various cellular processes, including tumorigenesis. This study aimed to detect the role of one TGF-β superfamily member activin receptor type IIB (ACTRIIB) in NPC. This study analyzed NPC datasets, including GSE12452, GSE102349, and GSE53819. ACTRIIB expression and N-glycosylation levels were assessed by western blot, real-time PCR, immunofluorescence, and immunohistochemistry in NPC cells and tissues. As indicated by the datasets, ACTRIIB was significantly upregulated in NPC tissues, and the up-regulation was associated with poor prognosis. This study confirmed the N-glycosylation of ACTRIIB primarily at the forty-second amino acid, an asparagine. The N-glycosylation of ACTRIIB promoted the localization of ACTRIIB to the cell membrane and prevented the degradation of the protein by lysosomes, through which ACTRIIB activated the downstream Smard1/2 to promote tumor cell proliferation and invasion. Inhibition of N-glycosylation or knockdown of ACTRIIB resulted in reduced cell proliferation and invasion and increased the cell sensitivity to docetaxel. In conclusion, N-glycosylation of ACTRIIB was a critical post-translational modification that enhanced protein stability and induced membrane localization, which facilitates the functions of ACTRIIB in cell proliferation and invasion in NPC. Inhibition of ACTRIIB N-glycosylation could potentially serve as a therapeutic strategy to improve the efficacy of chemotherapy in NPC.

Purpose: Scleral extracellular matrix (ECM) remodeling and weakened scleral stiffness are characteristic of myopia. The purpose of this study was to investigate the precise underlying mechanisms of scleral remodeling regulated by mechanical signals emanating from the ECM., Methods: The expression and regulation of YES-associated protein (YAP) were confirmed in human samples or guinea pig myopia models by Western blot (WB) or ELISA. To mimic the biomechanical microenvironment associated with myopia, stiff (50 kPa) and soft (8 kPa) substrates were established. The underlying mechanisms were further investigated by quantitative real-time RT-PCR, WB, and fluorescence staining in cells treated with siRNAs, plasmids or inhibitors. In vivo, a YAP activator, inhibitor and F-actin polymerization facilitator were applied to evaluate their therapeutic significance for myopia., Results: Our findings revealed that YAP expression is decreased in the sclera of guinea pigs and humans with myopia. Under mechanical stimuli, YAP functions as a mediator, transducing mechanical signals and modulating collagen expression. Furthermore, integrin α1β1 acts as a regulator of YAP and operates through modification of the F-actin cytoskeleton. Specifically, in response to mechanical forces, integrin α1β1 modulates F-actin restructuring. This modified actin cytoskeletal architecture subsequently facilitates the nuclear translocation of YAP, ultimately leading to the suppression of COL1A1 expression., Conclusions: Our results suggest that the integrin α1β1-F-actin-YAP-COL1A1 axis constitutes a vital regulatory mechanism intrinsically associated with the pathogenesis of myopia.

Purpose: The purpose of this study was to investigate the role and mechanism of caspase-8 in the development of choroidal neovascularization induced by age-related macular degeneration, with the aim of identifying a potential therapeutic target for neovascular age-related macular degeneration., Methods: Mouse models of laser photocoagulation-induced choroidal neovascularization and hypoxic human choroidal endothelial cells were utilized to examine the involvement of caspase-8 in choroidal neovascularization development. The toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8 pathway was explored in hypoxic human choroidal endothelial cells to elucidate its contribution to pathological angiogenesis. Various experimental techniques, including inhibition assays and immunoblotting analysis, were employed to assess the effects and mechanisms of caspase-8 activation., Results: Inhibition of caspase-8 demonstrated attenuated choroidal neovascularization development in mice subjected to laser photocoagulation. Activation of the toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8 pathway was observed in hypoxic human choroidal endothelial cells. Upon activation by the toll-like receptor 4/TIR domain-containing adaptor molecule 1 axis, caspase-8 directly cleaved caspase-1, leading to the cleavage of interleukin-1β and interleukin-18 by caspase-1. Consequently, activation of interleukin-1β and interleukin-18 through the toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8/caspase-1 pathway promoted the proliferative, migratory, and tube-forming abilities of hypoxic human choroidal endothelial cells., Conclusion: The findings of this study indicate that caspase-8 plays a crucial role in promoting choroidal neovascularization by activating interleukin-1β and interleukin-18 through the toll-like receptor 4/TIR domain-containing adaptor molecule 1/caspase-8/caspase-1 pathway in choroidal endothelial cells. Therefore, targeting caspase-8 may hold promise as a therapeutic approach for neovascular age-related macular degeneration.

Hypothesis: Western blot analysis of human prestin in the blood reveals multiple bands, rather than a single band., Background: Previously, using the ELISA method, prestin was shown to be a good biomarker of outer hair cell (OHC) health and sensorineural hearing loss that could be measured in the blood. Recently, we found that a Western blot approach in an experimental model demonstrated three prestin bands providing greater insights into prestin in the blood and its origins. This approach has not yet been explored in humans., Methods: Serum samples from 25 healthy human subjects were analyzed. An automated Western blot for each sample was generated, and bands were analyzed and compared with transient evoked otoacoustic emission levels (TEOAE)., Results: There were five bands at ~32, ~50, ~94, ~139, and ~171 kDa, respectively. Notably, the ~50-kDa band consistently was the most prominent. When the subjects were divided based on TEOAE level, those with high emission levels had a significantly larger ~94-kDa band than those with low emission levels., Conclusions: Western blot characterization of OHC biomarker prestin in humans shows that the band closest to the previously estimated molecular weight of prestin (81 kDa) is related to a functional measure of OHCs. This finding increases confidence in the value of serum prestin as a biomarker. The Western blot method appears to offer higher-resolution information on serum prestin. Future work will be carried out under pathological conditions to inform on the application of this quantitative method in clinical settings., Competing Interests: The authors disclose no conflicts of interest., (Copyright © 2024, Otology & Neurotology, Inc.)

Purpose: Bufalin (BU) is a bioactive ingredient extracted from the skin and parotid venom glands of Bufo raddei, which can effectively inhibit angiogenesis. The aim of this study was to investigate whether BU could affect corneal neovascularization (CoNV)., Methods: A rat CoNV model (right eye) was constructed by administration of NaOH, and the left eye served as a control. Corneal damage scores of rats were detected. Hematoxylin & eosin, TUNEL, and Masson staining examined pathological changes, apoptosis, and fibrosis of corneal tissues. Immunohistochemistry and western blotting assessed the expression of proteins., Results: BU intervention resulted in a significant reduction in corneal inflammatory cells, repair of corneal epithelial hyperplasia, significant reduction in stromal edema, and reduction in vascular proliferation. BU can inhibit corneal neovascularization., Conclusion: This study demonstrated that BU inhibits CoNV, fibrosis, and inflammation by modulating the STAT3 signaling pathway, elucidating the intrinsic mechanism of its protective effect. BU has great potential in the treatment of CoNV caused by corneal alkali burns.

Retinal damage accounts for irreversible vision loss following ocular alkali burn (OAB), but the underlying mechanisms remain largely unexplored. Herein, using an OAB mouse model, we examined the impact of oxidative stress (OS) in retinal damage and its molecular mechanism. Results revealed that OS in the retina was enhanced soon after alkali injury. Antioxidant therapy with N-acetylcysteine (NAC) preserved the retinal structure, suppressed cell apoptosis and decreased retinal inflammation, confirming the role of OS. Moreover, enhanced OS was linked to mitochondrial dysfunction, mtDNA leakage and initiation of the cytosolic DNA-sensing signaling. The activation of the major DNA sensors cyclic GMP-AMP Synthase (cGas) and cGAS-Stimulator of Interferon Genes (cGAS/STING) pathway was then identified. Notably, inhibiting cGAS/STING signaling with C-176 markedly reduced inflammation and cell apoptosis and ultimately protected the retina against OAB. Overall, our study reveals the vital function of OS in the occurrence of OAB-induced retinal damage and the involvement of cGAS/STING activation. Furthermore, our provides preclinical validation of the use of an antioxidant or a STING inhibitor as a potential therapeutic approach to protect the retina after OAB., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Senescent retinal pigment epithelial cells play a key role in neovascular age-related macular degeneration (nAMD); however, the mechanisms underlying the angiogenic ability of these cells remain unclear. Herein, we investigated the effects of the senescent adult retinal pigment epithelial cell line-19 (ARPE-19) on wound healing, cell migration and survival, and tube formation abilities of human umbilical vein endothelial cells (HUVECs). Additionally, we used Brown Norway rats to establish a laser-induced choroidal neovascularization (CNV) model for further nAMD-related studies. We found that the wound healing, cell migration, and tube formation abilities of HUVECs were significantly enhanced following culture in conditioned media from senescent ARPE-19 cells; this was attributed to the activation of the transforming growth factor β-activated kinase 1 (TAK1)/p38 MAPK pathway. Consistently, we found that the TAK1 inhibitors 5Z-7-oxozeaenol and takinib reversed the effects of conditioned media from senescent ARPE-19 cells on the wound healing, migration, survival, and tube formation abilities of HUVECs. We further investigated the therapeutic effects of 5Z-7-oxozeaenol on the laser-induced CNV rat model. We found that TAK1 was activated in IB4+ areas in laser-induced CNV lesions; inhibiting the activity of TAK1 using 5Z-7-oxozeaenol significantly alleviated CNV lesion formation and fluorescein leakage in fundus fluorescein angiography and greatly improved a-waves, b-waves, and OP values, as recorded by electroretinography. Thus, senescent RPE cells may promote angiogenesis via the TAK1/p38 MAPK pathway. Further, inhibiting TAK1 expression alleviates pathological neovascularization and improves retinal function in a laser-induced CNV rat model, highlighting the therapeutic potential of this approach for treating nAMD., Competing Interests: Declaration of interest The authors declare that they have no competing interests., (Copyright © 2025 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

OPA1 is a dynamin-related GTPase that modulates mitochondrial dynamics and cristae integrity. Humans carry eight different isoforms of OPA1 and mice carry five, all of which are expressed as short- or long-form isoforms. These isoforms contribute to OPA1's ability to control mitochondrial energetics and DNA maintenance. However, western blot isolation of all long and short isoforms of OPA1 can be difficult. To address this issue, we developed an optimized western blot protocol based on improving running time to isolate five different isoforms of OPA1 in mouse cells and tissues. This protocol can be applied to study changes in mitochondrial structure and function. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Western Blot Protocol for Isolating OPA1 Isoforms in Mouse Primary Skeletal Muscle Cells., (© 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

Diabetes mellitus (DM) is always accompanied by various complications, where diabetic retinopathy was a serious microvascular complications threatening the visual function of patients. This study evaluated the significance of miR-151a-5p and its effect on DR progression aiming to explore a novel biomarker for disease screening and monitoring. Study enrolled 137 patients with DM and 103 diabetes patients with DR. Serum miR-151a-5p was compared with PCR, and its clinical significance was evaluated from the perspectives of diagnosis and severity prediction. High-glucose-treated human retinal cell model was established, the effect of miR-151a-5p on high-glucose-induced cell injury was assessed based on cell growth, inflammation, oxidative stress, and endoplasmic reticulum stress. In mechanism, the downstream targets of miR-151a-5p were predicted, based on the function enrichment, the involvement of DKK3 and the MAPK signaling was estimated. Increasing miR-151a-5p was identified as a risk factor for DR in DM patients diagnosing DR patients and was positively correlated with disease severity predicting severe development of DR. Silencing miR-151a-5p alleviated high-glucose-induced reducing proliferation, activated inflammation, oxidative stress, and endoplasmic reticulum stress in human retinal cells. Negative regulation of DKK3 by miR-151a-5p was observed, and the knockdown of DKK3 could reversed the protective effect of miR-151a-5p. High-glucose activate the MAPK signaling, which was suppressed by the miR-151a-5p/DKK3 axis, and MAPK signaling was demonstrated to mediate the functional role of the miR-151a-5p/DKK3 axis. miR-151a-5p can be considered as a biomarker for the onset and progression of DR. miR-151a-5p potentially modulates the progression of DR through regulating inflammation, oxidative stress, and endoplasmic reticulum stress via the MAPK signaling., Competing Interests: Competing interests The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study aimed to investigate the effects of O-linked N-acetylglucosamine modification (O-GlcNAcylation) on astroglial-mesenchymal transition through connexin43 (Cx43) pathway under high-glucose conditions. The primary rat astrocytes were cultured under normal and high-glucose conditions, and level of GFAP, α-SMA and Cx43 was investigated. To explore the influence of O-GlcNAcylation on astroglial-mesenchymal transition, Thiamet G treatment was employed to enhance O-GlcNAcylation, while Alloxan was used to decrease it. Cx43 knockdown was acquired through lentivirus constructs to explore its role in astrocyte transition. The levels of GFAP and α-SMA expressions were examined, while astrocyte proliferation was evaluated using the CCK-8 assay, and migration was assessed through wound healing assays. The results showed that primary rat astrocytes were identified by GFAP antibody staining. Under high-glucose conditions, the levels of GFAP, α-SMA, and Cx43 increased, as confirmed by Western blot and immunofluorescence. O-GlcNAcylation augmentation induced by Thiamet G treatment significantly increased the expression of GFAP, α-SMA, and Cx43 compared to both normal and high-glucose conditions. Conversely, the inhibition of O-GlcNAcylation reversed the high-glucose-induced increase in GFAP and α-SMA. Cx43 knockout led to the downregulation of GFAP and α-SMA compared to high-glucose and O-GlcNAcylation-augmented conditions. Additionally, levels of O-GlcNAcylation and VEGF were reduced in Cx43 knockout group. Consistently, CCK8 and wound healing assays demonstrated that Cx43 knockout could inhibit astrocyte proliferation and migration compared to the high-glucose and O-GlcNAcylation augmented groups. These findings demonstrate that astroglial-mesenchymal transition occurs under high-glucose conditions, and can be promoted by O-GlcNAcylation augmentation, but suppressed by Cx43 knockout. The study underscores the significant role of Cx43 in this transition and its potential involvement in diabetic complications., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Posterior capsule opacification (PCO) is the most common complication after cataract surgery. In this study, we used transforming growth factor beta-2 (TGF-β2)-induced SRA01/04 cells to mimic PCO cell model and explored the functions and underlying mechanisms of specific protein 1 (SP1) in TGF-β2-induced SRA01/04 cell development. MTT assay and EdU assay were carried out to explore the proliferation of SRA01/04 cells. Transwell assay and wound-healing assay were performed to investigate SRA01/04 cell migration and invasion. Chromatin Immunoprecipitation (ChIP) assay, dual-luciferase reporter assay and Co-immunoprecipitation (Co-IP) assay were used to analyze the relations of SP1, COL1A2 and ZEB1. TGF-β2 treatment led to the promotion of SRA01/04 cell proliferation, migration, invasion and EMT process. COL1A2 level was induced by TGF-β2 treatment and COL1A2 knockdown inhibited TGF-β2-induced SRA01/04 cell proliferation, migration, invasion and EMT. SP1 could activate the transcription of COL1A2. SP1 overexpression promoted TGF-β2-induced SRA01/04 cell injury by regulating COL1A2 expression. Moreover, COL1A2 interacted with ZEB1 and COL1A2 knockdown-mediated effects on the proliferation, migration, invasion and EMT of TGF-β2-induced SRA01/04 cells were abrogated by elevating ZEB1. SP1 regulated COL1A2 and then mediated ZEB1 to affect the proliferation, migration, invasion and EMT of TGF-β2-induced SRA01/04 cells., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objective: We aimed to explore the role of connexin26 (Cx26) and connexin30 (Cx30) in the cochlea in noise-induced permanent threshold shifts (PTS) and temporary threshold shift (TTS)., Study Design: Prospective, controlled., Setting: Laboratory., Methods: A mouse model of noise-induced PTS and TTS was constructed. Western blots were used to detect the expression of Cx26 and Cx30 in the cochlea. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to assess the potential biological pathways., Results: Both the expression of Cx26 and Cx30 showed a trend of first rising and then falling in noise-induced PTS. The expression of Cx26 increased greatly in the 24 hours noise exposure (P < .05) and reached the highest level in the 4 hours after noise exposure (P < .05), then decreased gradually and returned to the control level on the seventh day after the noise exposure, when compared with the control group. The expression of Cx30 showed a similar trend in noise-induced PTS. However, both the expression of Cx26 and Cx30 showed a trend of first falling and then rising in noise induced TTS. The expression of Cx26/Cx30 reached its lowest level in the 4 hours after noise exposure (P < .05), and then increased to the control level on the second day after noise exposure (P > .05), compared with the control group. The first KEGG and GO pathway may be related with oxidative phosphorylation., Conclusion: Cx26 and Cx30 may have an effect in noise induced PTS and TTS. Future studies are needed to confirm the results., (© 2024 American Academy of Otolaryngology–Head and Neck Surgery Foundation.)

Sulfur mustard gas (SM), an alkylating and vesicating agent, has been used frequently in many wars and conflicts. SM exposure to the eye results in several corneal abnormalities including scar/fibrosis formation. However, molecular mechanism for SM induced corneal fibrosis development is poorly understood. After SM insult to the eye, excessive synthesis/secretion of extracellular matrix components (ECM) including collagen (COL) I, COL III, and lysyl oxidase (LOX) by corneal myofibroblasts causes corneal fibrosis, however, precise mechanism remains elusive. This study tested the hypothesis that Phosphoinositide 3-kinase (PI3K) signaling alterations post SM in cornea enhances stromal ECM synthesis and corneal fibrosis. New Zealand White Rabbits were used. The right eyes were exposed to SM (200 mg-min/m 3 ) and left eye to the air for 8min at MRI Global. Rabbit corneas were collected on day-3, day-7, and day-14 for molecular analysis. SM exposure caused a significant increase in mRNA expression of PI3K, AKT, COL I, COL III, and LOX and protein levels of LOX in a time-dependent manner in rabbit corneas. The in vitro studies were performed with human corneal stromal fibroblasts (hCSFs) by growing cultures in -/+ nitrogen mustard (NM) and LY294002, a PI3K specific inhibitor, for 30min, 8h, 24h, 48h, and 72h. NM significantly increased mRNA and protein levels of PI3K, AKT, COL I, COL III, and LOX. On the contrary, LY294002 in NM hCSFs significantly reduced PI3K, AKT, COL I, COL III, and LOX protein expression. We concluded that PI3K signaling mediates stromal collagen synthesis and LOX production following SM injury., (Published by Elsevier Ltd.)

Intraocular pressure (IOP) is regulated through the balance of production and drainage of aqueous humor. The main route of aqueous-humor outflow comprises the trabecular meshwork (TM) and Schlemm's canal (SC). We reported that IL-6 trans-signaling can inhibit TGF-β signaling in TM cells and may affect regulation of IOP. However, the function of IL-6 trans-signaling in SC cells remains unclear. Therefore, we investigated the role of IL-6 trans-signaling in monkey SC cells. Simultaneous treatment with IL-6 and soluble IL-6 receptor (sIL-6R) significantly decreased the trans-endothelial electrical resistance (TER) of SC cells and reduced aqueous-humor outflow resistance. Moreover, activation of IL-6 trans-signaling significantly reduced expression of fibronectin, ZO-1 and claudin-5, and increased that of several matrix metalloproteinases. We also investigated the effect of IL-6 trans-signaling on TGF-β2-induced changes in SC cells. Simultaneous treatment with IL-6 and sIL-6R significantly suppressed the TGF-β2-induced increase in the TER of SC cells but did not affect the activity of the TGF-β2 signaling pathway. By contrast, the TGF-β2-induced increases in the expression of fibronectin and collagen type I were significantly decreased upon simultaneous treatment with IL-6 and sIL-6R. The results show that IL-6 trans-signaling suppressed TGF-β2-induced increase in outflow resistance., Competing Interests: Competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

We investigated the mechanism of action of atropine in myopia control by examining its effect on choroidal hemodynamics. Blood flow was evaluated using indocyanine green (ICG) fluorescence and molecular variation during the development of form-deprivation myopia (FDM) and atropine treatment in guinea pigs. Guinea pigs were divided randomly into the normal control (NC), FDM, and FDM + 1% atropine (ATR) groups, and evaluated by spherical equivalent refractive error (SE) and axial length (AL). Choroidal hemodynamic parameters were measured via ICG fluorescence imaging including the maximal ICG fluorescence intensity (I max ), rising time (T rising ), blood flow index (BFI), and mean transit time (MTT). Additionally, the expression in the choroid-RPE complex of choroidal vascular endothelial growth factor A (VEGFA) and HIF-1 α were assessed via Western blotting. Atropine inhibited the development of FDM, with effects of FD on both SE and AL being reduced. ICG fluorescence hemodynamic wide-field maps and time-series curves revealed that the atropine significantly accelerated choroidal blood flow, with reduced T rising and MTT, while increasing I max , BFI and the number of lobulated choriocapillaris structures compared with the FDM group. In terms of molecular markers, atropine inhibited the effect of FDM, increasing VEGFA levels and reducing HIF-1α expression. These findings suggest that atropine improved choroidal hemodynamics and changed vascular markers, potentially contributing to its role in inhibiting the progression of myopia in the FDM model., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the authors., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Burn injuries trigger a systemic hyperinflammatory response, leading to multiple organ dysfunction, including significant hepatic damage. The liver plays a crucial role in regulating immune responses and metabolism after burn injuries, making it critical to develop strategies to mitigate hepatic impairment. This study investigates the role of methylation-controlled J protein (MCJ), an inner mitochondrial protein that represses complex I in burn-induced oxidative stress and mitochondrial dysfunction, using an in vitro Alpha Mouse Liver 12 cell model., Methods: Alpha Mouse Liver 12 cells were treated with serum from burn-injured mice (SBIM) to simulate burn injury in vitro. Methylation-controlled J protein was silenced using shRNA. Cell viability, apoptosis markers, reactive oxygen species levels, antioxidant response elements, electron transport chain components, and mitochondrial respiration were assessed using various techniques, including Cell Counting Kit-8 assay, Western blotting, MitoSOX Red staining, and Seahorse XF analysis., Results: Serum from burn-injured mice treatment (10%) for 8 hours reduced Alpha Mouse Liver 12 cell viability to 50% of control levels and increased MCJ expression fivefold. It also significantly upregulated apoptosis markers: cleaved caspase-3 (4-fold), Bax (3.8-fold), and cytosolic cytochrome c (3.5-fold). Methylation-controlled J protein silencing improved cell viability to 85% of control levels and reduced apoptosis markers by 75% to 78%. Serum from burn-injured mice increased reactive oxygen species levels by 3-fold, while MCJ silencing reduced this by 2.5-fold. Antioxidant proteins (NRF2, HO-1, NQO-1, GCLC, catalase) were suppressed by SBIM but upregulated 3.2- to 3.8-fold with MCJ silencing. Serum from burn-injured mice reduced electron transport chain components (NDUFS1, SDHB, MTCO2) by 45% to 65%, which MCJ silencing restored 2.5- to 3-fold. Mitochondrial respiration improved significantly with MCJ silencing: basal respiration (+26%), maximal respiration (+66%), adenosine triphosphate production (+25%), and spare respiratory capacity (+63%)., Conclusion: Methylation-controlled J protein plays a critical role in burn-induced hepatocyte damage. Its silencing alleviates SBIM-induced cytotoxicity, oxidative stress, and mitochondrial dysfunction. These findings highlight MCJ as a potential therapeutic target for preserving liver function in burn patients, warranting further in vivo studies to explore its clinical potential., (Copyright © 2025 American Association for the Surgery of Trauma.)

The resistance of pathogenic bacteria to various clinical antibiotics is the major problem in treating bacterial keratitis. Dimethyl fumarate (DMF) has good anti-fungal and anti-inflammatory effects in fungal keratitis, but its effect on bacterial keratitis is unclear. This study aims to investigate DMF's anti-inflammatory and antibacterial effects. The pyroptosis model was constructed by intracellular infection of canine corneal epithelial cells (CCECs) with Staphylococcus pseudintermedius (S. pseudintermedius), and 200 μM DMF was added to explore its function. Western blot, ELISA, immunostaining, flow cytometry, qRT-PCR, and bacterial counts were used to examine the expression of the NLRP3-GSDMD signaling pathway, virulence genes, and oxidant mediators. 111 clinical keratitis isolates or S. pseudintermedius were treated with different concentrations of DMF to detect bacterial growth and biofilm formation. Adding DMF resulted in the inhibition of the NLRP3-GSDMD pathway while activating the NRF2 pathway. This led to a decrease in pyroptosis rate, intracellular bacteria count, and ROS content. Additionally, DMF blocked the mRNA expression of virulence genes ebpS, hlgB, siet, lukS-I, PVL, icaA, icaD, spsD, and spsL associated with S. pseudintermedius infection. Furthermore, DMF demonstrated concentration-dependent inhibition of the growth of clinical isolates and the formation of S. pseudintermedius biofilm. In conclusion, our results indicate that DMF can inhibit pyroptosis and the growth of various clinical isolates, making it a novel ophthalmic drug with anti-inflammatory and antibacterial properties., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Keratoconus (KC) is the most common ectatic corneal disease with unknown pathogenesis. This study aimed to investigate the role of methyltransferase-like enzyme 3 (METTL3) in KC pathogenesis. In the present study, we examined the levels of METTL3 and other N6-methyladenosine (m 6 A) modification-related proteins in KC samples and human stromal keratocyte (HTK) cells stimulated by mechanical stretch (MS) using Western blotting and immunohistochemistry. The level of m 6 A RNA methylation was quantified using the m 6 A RNA methylation assay kit. Genetic (Mettl3 knockdown mice) and pharmacological (STM2457) approaches were employed to investigate the effect of METTL3 on the expression of metalloproteinases (MMPs) in MS-treated corneal stromal cells (CSCs) via Western blotting and real-time polymerase chain reaction. Moreover, YAP signaling activity was assessed to explore the relationship between METTL3 and MMPs in MS-treated CSCs. Increased expression of METTL3 and decreased expression of METTL14, WTAP, and YTHDF2 were detected in KC samples and MS-stimulated HTK cells. Correspondingly, the m 6 A levels in KC specimens and MS-stimulated CSCs were significantly higher than those in controls but were significantly reduced when METTL3 activity was genetically and pharmacologically blocked. Inhibition of METTL3 significantly reduced the expression of MMP1 and MMP3 in mechanically stretched CSCs and reduced YAP activity. Furthermore, pharmacologically inhibiting YAP signaling in MS-stimulated HTK cells significantly reduced MMP1 and MMP3 expression. Our findings highlight the pathogenic role of METTL3 in KC. Further investigation is required to investigate the underlying mechanism., (Copyright © 2024. Published by Elsevier Ltd.)

We aimed to explore the protective effects and underlying mechanisms of taurine on retinal cells during acute ocular hypertension (AOH)-induced damage. Retinal morphology, apoptosis, mitochondrial structure, electroretinography, expression of GTP binding protein 3 (GTPBP3), and molecules in the unfolded protein response (UPR) were examined in an AOH mouse model and wild-type (WT) mice with or without intravitreal injection of taurine. For in vitro experiments, the GTPBP3 expression and endoplasmic reticulum (ER) stress were examined in R28 cell line under hydrogen peroxide (H 2 O 2 )-induced damage or hypoxia/reoxygenation (H/R)-induced damage, with or without taurine pretreatment. Taurine pretreatment alleviated retinal damage caused by AOH modeling. The GTPBP3 expression level decreased after AOH injury, and taurine pretreatment reversed this reduction. Retinas with decreased GTPBP3 expression showed reduced retinal ganglion cell (RGC) function, which could be reversed by intravitreal taurine injection. In H 2 O 2- , H/R-, and AOH-induced damage, UPR were activated and alleviated by taurine pretreatment. GTPBP3 knockdown in R28 cells also activated the UPR, which was alleviated by taurine. A UPR activator downregulated GTPBP3 levels in normal R28 cells, whereas a UPR inhibitor upregulated GTPBP3 levels in GTPBP3 knockdown R28 cells. In conclusion, this study provides important evidence that taurine prevents retinal cell damage in mice exposed to AOH and modulates GTPBP3 expression via the UPR pathway. Interventions targeting this mechanism can be used as potential therapeutic targets for AOH damage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The abrupt and substantial elevation of intraocular pressure (IOP) in acute glaucoma induces retinal ischemia/reperfusion (I/R) injury, resulting in progressive retinal ganglion cell (RGC) death and irreversible visual impairment. PANoptosis, a form of regulated cell death consisting of pyroptosis, apoptosis and necroptosis, is reported to be involved in high IOP-induced RGC death. However, the precise mechanisms of RGC death remain unclear, and neuroinflammation is considered to play a vital role. TAT-N24, a synthetic inhibitor targeting the p55 regulatory subunit of phosphatidylinositol 3-kinase (p55PIK) signaling, demonstrates anti-inflammatory effect in uveitis and may have certain neuroprotective effects. Therefore, we investigated whether TAT-N24 could shield RGCs from immunoinflammatory damage in an acute glaucoma mouse model and explored the potential mechanism associated with PANoptosis. A mouse model of acute ocular hypertension (AOH) was established. Intravitreal injection of TAT-N24 was conducted to evaluate its impact on RGC death. The expression levels of key components in PANoptosis were analyzed using RT-qPCR and Western blotting. Immunohistochemistry and immunofluorescence staining on eyeball sections were employed to assess the expression of p55PIK, Brn3a, and ionized calcium binding adaptor molecule 1 (Iba1). Retinal structure was examined by H&E staining, while cell apoptosis was evaluated by TdT-mediated dUTP nick end labeling (TUNEL). The results showed that intravitreal injection of TAT-N24 effectively alleviated RGC death and retinal damage induced by AOH injury. The key components in PANoptosis were markedly upregulated after AOH injury, while these components were significantly inhibited after TAT-N24 treatment. Moreover, the expression levels of Z-DNA-binding protein 1 (ZBP1)-PANoptosome (ZBP1, RIPK1, RIPK3, and Caspase-8), NLR family pyrin domain-containing protein 3 (NLRP3), and NLR family CARD domain-containing protein 4 (NLRC4) inflammasomes were notably elevated after AOH injury, which was significantly suppressed by TAT-N24. In conclusion, PANoptosis was involved in AOH-induced RGC death and retinal damage. TAT-N24 exhibited an anti-PANoptotic effect, protecting RGCs by inhibiting ZBP1-PANoptosome as well as NLRP3 and NLRC4 inflammasomes after AOH injury., Competing Interests: Declaration of competing interest None., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Posterior capsule opacification (PCO) due to the proliferation and migration of lens epithelial cells (LECs) is the main complication after surgery. Heat stress has demonstrated impressive results in halting cell proliferation and migration, while also facilitating cell death. This study aimed to investigate the role and mechanism of ferroptosis in the proliferation and migration of LECs under heat stress. CCK-8 assays, scratch assays, and transcriptome analysis were used to evaluate the impact of temperature on human lens epithelial cells (HLECs) and explore the potential mechanisms. The role of ferroptosis in the proliferation of HLECs induced by heat was investigated using the ferroptosis inhibitor Fer-1 and siRNA-mediated NCOA4 protein interference. Fluorescence staining and Western blot experiments were used to detect the expression of Fe 2+ , reactive oxygen species (ROS), and ferroptosis-related proteins NCOA4, FTH1, and SLC3A2. The results of CCK-8 assays, scratch assays, and transcriptome analysis demonstrated significant thermal effects on HLEC behavior. After heat treatment, there were significant changes in the fluorescence expression of Fe 2+ and ROS in the HLECs and lens explant. In addition, the expression of NCOA4, FTH1, and SLC3A2 also changed significantly. Using Fer-1 or NCOA4 siRNA-mediated interference restored cell viability decreased by thermal stress. Furthermore, interference with NCOA4 protein effectively restored the expression of Fe 2+ , ROS, and FTH1. In conclusion, heat stress has a significant effect on LECs by regulating ferroptosis and the interaction between NCOA4 and FTH1 proteins play an important role., Competing Interests: Conflict of interest The authors report no conflicts of interest in this study., (Copyright © 2024. Published by Elsevier Ltd.)

Currently, research on optic nerve injury predominantly focuses on the retina and optic nerve, but emerging evidence suggests that optic nerve injury also affects advanced visual structures like the superior colliculus (SC) and primary visual cortex (V1 region). However, the exact mechanisms have not been fully explored. This study aims to investigate the characteristics and mechanisms of pathology in the SC and V1 region after optic nerve crush (ONC) to deepen our understanding of the central mechanism of visual injury. After unilateral ONC, visual acuity in the injured eye declined, along with thinning of the retinal nerve fiber layer, and the latency and amplitude of FVEPs decreased. Furthermore, neuronal loss and degeneration were observed in the contralateral SC and V1 region, accompanied by astrocytic activation. Additionally, protein markers C3, and Serping1 for A1 astrocytes, which had neurotoxic effects and S100A10, and PTX3 for A2 astrocytes, which promoted tissue repair, were increased in the two regions. A1 astrocytes were mainly present in the early stages of observation, while A2 astrocytes were mainly increased later. Notably, NLRC4, GSDMD-N, cleaved caspase-1 expression, and IL-1β, IL-18 secretion increased in the contralateral SC and V1 region. Collectively, our findings reveal that A1 (neurotoxic) and A2 astrocytes (neuroprotective), NLRC4-mediated neuronal pyroptosis are enhanced in SC and V1 region contralateral to the ONC eye. The primary visual cortex responds to injury later than the superior colliculus after ONC, with less pronounced damage changes. Reactive astrocytes and NLRC4 inflammasome may act as promising targets for the prevention and treatment of optic nerve injury., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Microglia activation and autophagy changes are associated with the regulation of pain, but no study to date has been designed to address whether these features apply to trigeminal neuropathic pain. This study aimed to investigate how alterations in autophagy affect nociceptive behaviors may be associated with microglia activation in the caudal part of the spinal trigeminal nucleus (SpVC) in a rat model of trigeminal neuropathic pain. This model was established by chronic constriction injury of the infraorbital nerve. Autophagy inhibitors and agonists were injected into the lateral ventricle to regulate autophagy. The autophagy markers microtubule-associated protein light chain 3 I (LC3-I), LC3-II, sequestosome1 (p62), and LC-3 were examined by western blotting and/or immunofluorescence. The microglia marker ionized calcium binding adapter molecule 1 (Iba-1) was examined by immunohistochemistry. Nociceptive behavior changes were detected by measuring the mechanical thresholds and face-grooming duration. The results showed that microglia in SpVC were activated, and autophagy flux was blocked in the trigeminal neuropathic pain model. Autophagy agonists inhibited microglia activation and alleviated nociceptive behaviors. In contrast, autophagy inhibitors further activated microglia and exacerbated nociceptive behaviors. In a rat model of trigeminal neuropathic pain, autophagy blockage leads to microglia activation, which significantly influences nociceptive processes., (© 2024 Scandinavian Division of the International Association for Dental Research. Published by John Wiley & Sons Ltd.)

The study aimed to compare the effects of different types of excimer laser keratectomy on rabbit corneas and to identify the optimal disease model for corneal ectasia. Additionally, investigating the structural and molecular alterations in the novel disease model helped explore the mechanisms underlying biomechanical cues in corneal ectasia. 2.0-2.5 kg New Zealand white rabbits were treated with different types of excimer laser keratectomy, including comparisons between photorefractive keratectomy (PRK) and phototherapeutic keratectomy (PTK) surgeries, as well as comparisons of different ablation depths of PRK. Detailed tests on post-surgery corneas included pentacam analyzer, H&E staining and optical coherence tomography (OCT), transmission electron microscopy (TEM), raman spectroscopy and uniaxial tensile tests. Later, tandem mass tag-labeled proteomics and multiply statistic analysis were performed on post-PRK75 corneas. Western blot was used to validate protein expression. Herein, we found that tapered corneal thinning in post-PRK corneas predisposed to corneal ectasia. Greater ablation depth increased ectasia risk. PRK75 (ablation of 75% of corneal thickness using PRK mode) emerged as the optimal modeling approach, evidenced by significant and sustained corneal ectasia for 4 weeks. The 4-week post-PRK75 corneas were evaluated by changes in stromal cell microstructure, basement membrane, collagen lamellae, collagen covalent bonds and decreased corneal biomechanical strength. Additionally, PRK75 surgery induced 109 differentially expressed proteins (DEPs), with 51 previously linked to human corneal ectasia. The statistic analysis demonstrated the dysregulation of immue response was involved in the post-PRK75 corneas, and identified nine core proteins involved in corneal ectasia, including SERPINH1, ALDH1A1, MMP10, A2M, GSTM3, CD44, CLU, C3, and ITGB2. Therefore, we concluded that PRK75 was a novel and reliable modeling method for corneal ectasia, resemble human corneal ectasia. The intrinsic structural remodeling and molecular alteration in post-PRK75 corneas could shed lights on understanding the mechanism of biomechanical cues in corneal ectasia in the future., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Objective: This study aimed to investigate the role of transmembrane emp24 domain-containing protein 2 (TMED2) in oral squamous cell carcinoma (OSCC)., Methodology: A bioinformatics analysis was first conducted to explore TMED2 expression in OSCC and its relation with overall survival. The analysis results were further verified by assessing TMED2 expression levels in human normal oral keratinocyte cells and human OSCC cell lines using quantitative real-time polymerase chain reaction and the Western blot. Finally, the effects of TMED2 knockdown and overexpression on the expression levels of TMED2, ADP-ribosylation factor 1, extracellular signal-regulated kinase ½, and phospho-extracellular signal-regulated kinase ½ proteins were examined in cells using the Western blot., Results: The GEPIA2 database showed that OSCC tissues expressed more TMED2 than normal tissues. At the cellular level, TMED2 expression significantly increased in SCC-4, HSC-3, and CAL-27 cells than in human normal oral keratinocyte cells. TMED2 knockdown reduced cell proliferation, increased the apoptosis rate in SCC-4 cells, and led to a higher proportion of cells in the G0/G1 phase and a lower proportion in the S phase., Conclusion: TMED2 may promote OSCC cell proliferation and inhibit apoptosis, potentially by activation of the ADP-ribosylation factor 1/ extracellular signal-regulated kinase ½ signaling pathway.

Purpose: To investigate the role of S100A8/A9 in the pathogenesis of Sjögren's dry eye disease (SjDED) and explore its potential mechanism of action., Methods: S100A8/A9 expression was determined by western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Tear secretion, corneal fluorescein staining, and hematoxylin and eosin staining were used to evaluate the effect of paquinimod, a S100A8/A9 inhibitor, on dry eye disease in nonobese diabetic (NOD) mice. Immune cell infiltration and percentage were assessed by immunofluorescence and flow cytometry. Proinflammatory cytokine levels were examined by qRT-PCR or ELISA. The mechanism of action was analyzed using western blot, immunofluorescence, and chromatin immunoprecipitation., Results: S100A8/A9 was upregulated in peripheral blood mononuclear cells of patients with SjDED and lacrimal glands (LGs) of SjDED mice. The upregulation of S100A8/A9 was correlated with the dry eye severity and inflammatory infiltration levels in LGs. Administration of paquinimod ameliorated clinical and histopathological phenotypes of SjDED mice and reduced the proportion of Th17 cells in LGs, lymph nodes, and spleens. Further experiments revealed that S100A8/A9 did not directly affect Th17 generation and function but upregulated the expression of major histocompatibility complex Ⅱ (MHC Ⅱ) and Th17-polarizing cytokines in dendritic cells (DCs) to augment Th17 cell response. Mechanistically, S100A8/A9 induced the expression of Acod1 and thereby promoted the activation and nuclear translocation of signal transducer and activator of transcription 3 (STAT3), resulting in increased Il23a transcription. STAT3 activator reversed the therapeutic effect of paquinimod on SjDED mice., Conclusions: S100A8/A9 activated the Acod1/STAT3 pathway to promote DC-driven Th17 cell responses in SjDED. The S100A8/A9/Acod1/STAT3 pathway may represent a promising therapeutic target for SjDED.

Purpose: To investigate the presence of uridine-5'-triphosphate (UTP)-activated P2Y1-like nucleotide receptors (P2Y2R, P2Y4R, and P2Y6R) in conjunctival goblet cells (CGCs) and determine if they increase intracellular Ca2+ concentration ([Ca2+]i) and induce mucin secretion., Methods: Adult, male rat conjunctiva was used for culture of CGCs. To investigate the expression of P2YRs, mRNA was extracted from CGCs and used for reverse transcription PCR (RT-PCR) with commercially obtained primers specific to P2Y2R, P2Y4R, and P2Y6R. Immunofluorescence (IF) and western blot (WB) analyses were performed using first-passage CGCs and stained with antibodies specific to each P2YR. Furthermore, CGCs were incubated with fura-2/AM, and [Ca2+]i was measured after stimulation with the P2YR selective agonists UTP, uridine 5'-diphosphate (UDP), or UDP-glucose and agonists specific to P2Y2R (MRS 2768), P2Y4R (MRS 4062), and P2Y6R (MRS 2693). [Ca2+]i measurements after P2Y2R and P2Y6R siRNA treatment were performed. Mucin secretion was measured after stimulation of P2Y2R, P2Y4R, and P2Y6R., Results: mRNA for all pyrimidinergic P2Y1-like receptors was found as single bands of expected base pair number with RT-PCR. The presence of these P2YRs was confirmed with IF microscopy and WB analysis. UTP and UDP elicited concentration-dependent increases in [Ca2+]i. The receptor-specific agonists and UDP-glucose increased [Ca2+]i, although these responses were substantially lower than those elicited by UTP and UDP at 10-4 M and 10-3 M and did not show similar dose dependency. P2Y2R- and P2Y6R-depleted CGCs responded with reduced peak [Ca2+]i. UTP, MRS 2768 (P2Y2R), and UDP each stimulated mucin secretion from CGCs., Conclusions: P2Y2R, P2Y4R, and P2Y6R are present and functional in rat CGCs and may represent novel therapeutic targets for dry eye treatment and other types of ocular surface disease.

Purpose: Dry eye disease (DED) is a common ocular surface inflammatory disease with a complex pathogenesis. Herein, the role and effect of gasdermin E (GSDME) in DED pathogenesis were explored., Methods: In vitro, flow cytometry, Cell Counting Kit-8 (CCK-8) and lactate dehydrogenase (LDH) release assays were used to determine the effects of hyperosmotic stress on pyroptosis, apoptosis, and cell viability in human corneal epithelial cells (HCECs). Quantitative PCR (qPCR) and Western blot assays were used to detect GSDME expression in HCECs and in those transfected with si-GSDMD. In vivo, GSDMD-knockout (KO) mice were used to study the role of GSDME in DED pathogenesis. The qPCR, Western blotting, and immunofluorescence were used to explore the effects of GSDME on HCEC apoptosis, pyroptosis, and the expression of related genes and proteins in GSDMD-KO mice with scopolamine-induced dry eye., Results: Pyroptosis and cell membrane rupture occurred, and caspase-3 and GSDME protein expression increased after HCECs were treated with 312 to 500 mOsm sodium chloride. GSDME gene and protein expression levels were increased in HCECs from both si-GSDMD- and GSDMD-KO mice. Although caspase-3 expression was increased in the dry eye group of GSDMD-KO mice, HCEC apoptosis and the apoptosis-related factors PARP were not detected. The gene and protein expression levels of the pyroptosis-related factors ASC and IL-1β were greater than those in GSDMD-KO mice without dry eye., Conclusions: GSDME is involved in DED pathogenesis by mediating inflammation via the pyroptosis pathway, GSDME inhibition may be a therapeutic target for DED.

Purpose: This study aimed to evaluate early-phase safety of subretinal application of AAVanc80.CAG.USH1Ca1 (OT&#95;USH&#95;101) in wild-type (WT) pigs, examining the effects of a vehicle control, low dose, and high dose., Methods: Twelve WT pigs (24 eyes) were divided into three groups: four pigs each received bilateral subretinal injections of either vehicle, low dose (3.3 × 1010 vector genomes [vg] per eye), or high dose (1.0 × 1011 vg per eye). Total retinal thickness (TRT) was evaluated using optical coherence tomography and retinal function was assessed with full-field electroretinography (ff-ERG) at baseline and two months post-surgery. After necropsy, retinal changes were examined through histopathology, and human USH1C&#95;a1/harmonin expression was assessed by quantitative PCR (qPCR) and Western blotting., Results: OT&#95;USH&#95;101 led to high USH1C&#95;a1 expression in WT pig retinas without significant TRT changes two months after subretinal injection. The qPCR revealed expression of the human USH1C&#95;a1 transgene delivered by the adeno-associated virus vector. TRT changes were minimal across groups: vehicle (256 ± 21 to 243 ± 18 µm; P = 0.108), low dose (251 ± 32 to 258 ± 30 µm; P = 0.076), and high dose (242 ± 24 to 259 ± 28 µm; P = 0.590). The ff-ERG showed no significant changes in rod or cone responses. Histopathology indicated no severe retinal adverse effects in the vehicle and low dose groups., Conclusions: Early-phase clinical imaging, electrophysiology, and histopathological assessments indicated that subretinal administration of OT&#95;USH&#95;101 was well tolerated in the low-dose treatment arm. OT&#95;USH&#95;101 treatment resulted in high expression of human USH1C&#95;a1. Although histopathological changes were not severe, more frequent changes were observed in the high-dose group.

Purpose: Because chickens have excellent light perception properties, this study focused on investigating whether monochromatic light can cause photodamage in chicken retinal ganglion cells (RGCs)., Methods: Post-hatching day chickens were exposed to four different light-emitting diode light environments for five weeks, respectively, monochromatic blue light (480 nm), green light (560 nm), red light (660 nm), or white light (6000 K). The mechanisms through which monochromatic light influences the structure of the chicken retina were analyzed by detecting the morphological structure of the retina, gene and protein expression levels, and the ultrastructure of the optic nerve., Results: Blue light exposure for five weeks significantly impacted the thickness of the inner reticular layer, retinal synaptic function, and the number of RGCs in the chicken retina. Moreover, neurodegenerative disease characteristics, such as a reduction in the number of dendritic branches and the presence of myelin lesions, have also been observed in RGCs. The activation of the protein kinase RNA-like ER kinase-mediated unfolded protein response, along with the abnormal degradation of the autophagy substrate P62, accompanies these retinal pathological processes. Additionally, our findings indicate that the photosensitivity of OPN4 is linked to endoplasmic reticulum stress in RGCs, which may elucidate why chicken RGCs experience damage exclusively under blue light exposure., Conclusions: Blue light can damage the chicken retina through endoplasmic reticulum stress and autophagy, especially in RGCs. Our study enhances the understanding of the mechanisms underlying retinal photodamage and emphasizes the risk of retinal damage in low-illuminance blue light environments.

Purpose: To study the roles of tubulin acetylation and cyclic mechanical stretch (CMS) in trabecular meshwork (TM) cells and their impact on outflow pathway physiology and pathology., Methods: Primary TM cell cultures were subjected to CMS (8% elongation, 24 hours), and acetylated α-tubulin at lysine 40 (Ac-TUBA4) was assessed by western blotting and immunofluorescence. Enzymes regulating tubulin acetylation were identified via siRNA-mediated knockdowns of ATAT1, HDAC6, and SIRT2. Ac-TUBA4 levels were compared between glaucomatous (GTM) and non-glaucomatous (NTM) TM cells and in frozen sections of human cadaver eyes. The effect of tubulin acetylation on substrate stiffness and cell contractility was evaluated by culturing cells on substrates with varying stiffness and by collagen gel contraction assays, respectively. Microtubule stability was examined by monitoring resistance to nocodazole-induced depolymerization. The in vivo effect on intraocular pressure (IOP) was evaluated following intracameral injections of tubacin in mice., Results: CMS induced tubulin acetylation in human TM cells by downregulating the deacetylase HDAC6. Elevated Ac-TUBA4 levels were observed in GTM compared NTM cells and tissues. Tubulin acetylation was not affected by substrate stiffness and did not show a direct effect on TM cell contractility. Tubulin acetylation was found to provide protection against microtubule destabilization induced by nocodazole. Importantly, intracameral injection of tubacin, an HDAC6 inhibitor, significantly lowered IOP in mice., Conclusions: Our study highlights a critical role of tubulin acetylation in TM cell response to mechanical stress and its potential impact on IOP regulation. Tubulin acetylation could represent a therapeutic target for glaucoma.

Purpose: Progenitors for the corneal endothelium have been identified in the transition zone (TZ), but their cellular interactions remain undefined. Posterior limbal mesenchymal stromal cells (P-LMSCs) may support TZ cells in the posterior limbus. This study aims to characterize P-LMSCs and investigate their effects on TZ cells., Methods: Human P-LMSCs and TZ cells were isolated by explant culture. P-LMSCs were characterized by comparing with anterior limbal mesenchymal stromal cells (A-LMSCs) using immunocytochemistry. TZ cells were cocultured with P-LMSCs in a Transwell, with TZ cell and A-LMSC coculture and TZ cells only as the control groups. The proliferation and wound healing capacity of TZ cells were assessed by EdU assay and scratch wound assay. Colony forming assay, droplet digital PCR, Western blotting, and immunocytochemistry were used to compare the stemness of TZ cells. The effect of P-LMSC conditioned medium on endothelial wound healing was evaluated in organ-cultured mouse corneas. Endothelial regeneration was measured by trypan blue staining., Results: P-LMSCs expressed similar proteins (vimentin, Nestin, TRA-1-60, Oct3/4) as A-LMSCs. TZ cells cocultured with P-LMSCs had significantly higher proliferation, wound healing speed, and colony-forming efficiency than TZ cells only. TZ cells supported by P-LMSCs expressed higher levels of stem/progenitor markers (Nestin, Sox9, AP-2α, Pitx2) than the control groups. P-LMSC conditioned medium stimulated regeneration of mouse corneal endothelium from the TZ region., Conclusions: The proliferation and stemness of TZ cells were enhanced by P-LMSCs in both cell and organ culture models. Our study provides an innovative strategy for corneal endothelial rejuvenation.

Purpose: Protein arginine methyltransferase 1 (PRMT1) is an integral constituent of numerous cellular processes. However, its role in corneal epithelial wound healing (CEWH) remains unclear. This study investigates the impact of PRMT1 on cellular mechanisms underlying corneal epithelial repair and its potential to improve wound healing outcomes., Methods: The murine CEWH model was established using an Alger brush. Corneal epithelial-specific Prmt1 knockout mice were generated using the Cre-lox system. Quantitative reverse transcription polymerase chain reaction and Western blot analyses determined the expression of candidate genes at mRNA and protein expression levels. Human corneal epithelial cells (HCECs) were transfected with siRNA using Lipofectamine RNAiMAX or infected with lentivirus to precisely alter the expression of PRMT1 or Annexin A3 (ANXA3). EdU and a scratch wound-healing assay evaluated the effects of PRMT1 or ANXA3 on HCEC proliferation and migration, respectively. Rescue experiment and chromatin immunoprecipitation assay validate the correlation between PRMT1 and ANXA3., Results: Prmt1 is significantly upregulated during CEWH, accompanied by an elevated global arginine methylation level. Knockdown of PRMT1 in HCECs or in vivo knockout impairs cell proliferation, migration, and the CEWH process. Furthermore, ANXA3 was identified as a critical target of PRMT1, with PRMT1 enhancing ANXA3 expression through histone arginine methylation at its promoter region, establishing a causal correlation between them. Moreover, PRMT1 can modulate the NF-κB and JNK signaling pathways via ANXA3., Conclusions: PRMT1 is a critical epigenetic regulator in CEWH, promoting wound healing by upregulating ANXA3 via histone arginine methylation. These findings highlight the potential of targeting PRMT1 to enhance corneal epithelial repair.

Purpose: To investigate potential modes of programmed cell death in the lens epithelial cells (LECs) of patients with early age-related cortical cataract (ARCC) and to explore early-stage intervention strategies., Methods: Anterior lens capsules were collected from early ARCC patients for comprehensive analysis. Ultrastructural examination of LECs was performed using transmission electron microscopy. Cell death-associated protein markers were quantified via Western blot analysis, including those for paraptosis (ALIX, GRP78), apoptosis (cleaved caspase 3 and caspase 9), pyroptosis (N-GSDMD), and ferroptosis (GPX4). Intracellular vesicle-organelle colocalization was assessed through immunofluorescence. OGG1 protein expression and activity were evaluated through multiple methods, including Western blot, laser micro-irradiation, and immunofluorescence. The therapeutic potential of the OGG1 activator TH10785 on paraptosis was investigated using an ex vivo rat lens model., Results: Morphologic changes revealed significant endoplasmic reticulum (ER) swelling in ARCC patient LECs, with no characteristic apoptotic features. Paraptosis-related proteins exhibited significant alterations, while other cell death pathway markers (apoptosis, pyroptosis, and ferroptosis) remained unchanged. In the reactive oxygen species-induced paraptosis model, vesicular structures showed exclusive colocalization with ER-specific fluorescence. Elevated levels of the DNA damage marker 7,8-dihydro-8-oxoguanine were observed concurrent with decreased OGG1 activity. The OGG1 activator TH10785 showed efficacy in suppressing LECs paraptosis in ex vivo rat lens cultures., Conclusions: Paraptosis was identified in the LECs of patients with early ARCC. TH10785 activates OGG1 to suppress paraptosis in LECs, suggesting a novel therapeutic approach for early ARCC intervention.

Purpose: Ocular neovascularization is a major cause of blindness. Although fibroblast growth factor-2 (FGF2) has been implicated in the pathophysiology of angiogenesis, the underlying mechanisms remain incompletely understood. The purpose of this study was to investigate the role of FGF2 in retinal neovascularization and elucidate its underlying mechanisms., Methods: The oxygen-induced retinopathy mouse model was used to study the pathogenesis of retinal neovascularization. Immunofluorescence was used to quantify the neovascularization in retina. Data-independent acquisition proteomics were performed to quantify differentially expressed proteins in human retinal microvascular endothelial cells stimulated with FGF2 and associated pathways were analyzed. We carried out qRT-PCR and Western Blot assays to detect the expression of genes at mRNA and protein levels. The angiogenesis abilities of human retinal microvascular endothelial cells were measured by transwell, EdU and tube formation assays., Results: FGF2 was significantly upregulated in retinal tissues of the oxygen-induced retinopathy mouse model and it markedly enhanced tube formation, migration, and proliferation abilities of human retinal microvascular endothelial cells in vitro. The proteomic analysis identified 287 differentially expressed proteins in endothelial cells in response to FGF2 stimulation, characterized by a notable upregulation of the glycolysis pathway, among which we confirmed that the enolase 2 (ENO2) levels were elevated after FGF2 stimulation, and its knockdown resulted in diminished glycolytic activity and impaired angiogenic processes. Furthermore, the use of the ENO2 inhibitor AP-Ⅲ-a4 alleviated angiogenesis in vivo and in vitro., Conclusions: Our findings underscore the pivotal role of ENO2-mediated glycolysis in FGF2-induced angiogenesis, suggesting that ENO2 may serve as a promising therapeutic target for managing pathological neovascularization.

Purpose: To investigate the effect of Ca2+/calmodulin-dependent protein kinase II (CAMKII) δ subtypes (CAMK2D) on sodium iodate (NaIO3)-induced retinal degeneration in mice., Methods: Bioinformatics analysis and Western blot experiments were used to screen the significantly differentially expressed genes in age-related macular degeneration (AMD) disease. CAMK2D knockdown and overexpression models were constructed by lentivirus (LV) infection of adult retinal pigment epithelial cell line-19 (ARPE-19) cells in vitro. Flow cytometry was used to detect ARPE-19 cell apoptosis induced by NaIO3. In vivo, CAMK2D knockdown and overexpression mouse models were generated by infecting mouse retinal pigment epithelium (RPE) with adeno-associated virus (AAV). Retinography, optical coherence tomography (OCT), and histological analysis (hematoxylin and eosin staining) were used to detect NaIO3-induced retinal structural changes in mice. Electroretinography (ERG) was used to detect NaIO3-induced retinal function changes in mice. TdT-mediated dUTP nick-end labeling (TUNEL) staining was used to detect the apoptosis of retinal cells induced by NaIO3. RNA sequencing (RNA-Seq) and bioinformatics analysis were used to screen for target genes affected by CAMK2D in CAMK2D-overexpressing ARPE-19 cells. And flow cytometry, OCT, and ERG were used to evaluate the regulatory effect of CAMK2D on target genes., Results: Bioinformatics analysis found the expression of genes related to Ca2+ signal was significantly reduced in AMD patients. Western blot showed that in a mouse model of dry AMD induced by NaIO3, CAMK2D expression in RPE-Choroid tissue significantly lower than normal mice. In vitro, our results showed that overexpression of CAMK2D in ARPE-19 cells decreased apoptosis induced by NaIO3 and knockdown increased apoptosis. In vivo, CAMK2D overexpression in RPE cells can attenuate the retina degeneration induced by NaIO3 and CAMK2D knockdown aggravated degeneration. The bioinformatics analysis indicated that CAMK2D might affect AMD pathology through complement factor I (CFI). In vitro, knockdown of CFI in ARPE-19 cells increased apoptosis induced by NaIO3. In knockdown CFI ARPE-19 cells, overexpression of CAMK2D reduced the above apoptosis. In mice retina, CFI knockdown can aggravate the retina degeneration induced by NaIO3. In knockdown CFI mice, overexpression of CAMK2D in RPE can attenuate the above retina degeneration. Western blot confirmed that CAMK2D regulated the expression of CFI in mice., Conclusions: CAMK2D can attenuate the retinal degeneration induced by NaIO3, which was achieved by regulating the CFI.

Purpose: This study aimed to investigate the role of SIRT4 in retinal protection, specifically its ability to mitigate excitotoxic damage to Müller glial cells through the regulation of mitochondrial dynamics and glutamate transporters (GLASTs)., Methods: A model of retinal excitatory neurotoxicity was established in mice. Proteins related to mitochondrial dynamics, GLAST, and SIRT4 were analyzed on days 0, 1, 3, and 5 following toxic injury. The influence of SIRT4 on mitochondrial dynamics-related proteins and GLAST was examined by inducing SIRT4 overexpression through intraperitoneal injection of resveratrol or by using SIRT4 knockout (KO) mice. Additionally, the effects of upregulating and downregulating SIRT4 expression in rat Müller glial cell lines (rMC-1) were explored via lentiviral vector transfection to assess changes in mitochondrial morphology and GLAST expression., Results: After excitotoxic injury to the mouse retina, the retinal thickness and structure were disrupted, the number of retinal ganglion cells (RGCs) decreased, and Müller glial cells were activated by day 1. The levels of OPA1, GLAST, and SIRT4 proteins peaked on the first day after injury and then gradually decreased, indicating a synchronized dynamic trend. The upregulation of SIRT4 expression promoted OPA1 and GLAST protein expression, thereby alleviating retinal excitotoxic injury. Furthermore, the upregulation of SIRT4 expression promoted mitochondrial fusion and increased GLAST expression in rMC-1 cells, reducing cellular excitotoxic damage. Conversely, downregulation of SIRT4 had the opposite effect., Conclusions: SIRT4 plays a significant role in mitigating excitotoxic damage in the retina, modulating Müller glial cell injury by regulating mitochondrial dynamics and glutamate transporter expression, ultimately influencing retinal health.

Purpose: Fungal keratitis (FK) remains a treatment challenge, necessitating new therapeutic targets. Piezo1, a mechanosensitive ion channel, regulates calcium signaling and immune cell function. This study investigates its role in macrophage-mediated antifungal responses in FK., Methods: Piezo1 and Pyrin expression in corneas and bone marrow-derived macrophages (BMDMs) were assessed by RNAseq, quantitative real-time PCR (qRT-PCR), Western blot, and immunofluorescence. Intracellular calcium ion concentration was detected by Fluo-4 AM fluorescent probe staining. Heterozygous Piezo1 deficiency (Piezo1+/-) mice and Yoda1 were performed to regulate the expression of Piezo1., Results: Our investigation demonstrates elevated expression of Piezo1 in the corneas of patients with FK and infected mice. This upregulation of Piezo1 corresponded with the swift recruitment of macrophages via the limbus. Additionally, Piezo1+/- mice exacerbate the progression of FK in the infection model. Furthermore, Piezo1 knockdown in macrophages exhibit a notable reduction phagocytic capacity, accompanied by an increase in viable colony-forming units in an in vitro model of fungal infection. Moreover, using a pharmacologic activator of Piezo1 (Yoda1), a calcium ion (Ca2+) chelator of BAPTA or Piezo1+/- mice, we demonstrate that Piezo1 activation triggers the Pyrin inflammasome via augmented calcium ion influx, which is required for protection against FK in murine hosts., Conclusions: Piezo1 is crucial for innate immunity in FK, enhancing macrophage recruitment, activation, and Pyrin inflammasome-mediated antifungal activity via calcium signaling. Using Piezo1+/- mice and Yoda1, we confirm Piezo1's role in fungal clearance. Targeting Piezo1 offers a novel strategy to improve FK outcomes by boosting macrophage function and immune response.

Purpose: R-spondin3 (RSPO3), a mammalian-specific amplifier of WNT signaling pathway, maintains the homeostasis of various adult stem cells. However, its expression at the limbus and the effect on limbal epithelial stem cells (LESCs) remains unclear. We investigated the impact of RSPO3 on the proliferation and self-renewal of LESCs and explored its molecular mechanisms., Methods: The expression of four RSPO subtypes at the limbus were detected. Co-cultured with RSPO3 in vitro, the cell outgrowth area and cell density of human LESCs (hLESCs) were measured, along with EdU assay and evaluation of biomarkers of cell proliferation (Ki67) and stemness (△Np63 and ABCG2). The expression of key molecules in WNT/β-catenin signaling pathway were investigated in RSPO3-co-incubated hLESCs and controls. The effect of RSPO3 on corneal epithelium wound recovery in vivo was investigated in a mouse model of corneal epithelium injury., Results: Among four subtypes of RSPO protein, only the RSPO3 isoform was stably expressed at the human limbus. RSPO3 promoted the proliferation and stemness maintenance of hLESCs in vitro in a dose-dependent manner when its concentration ≤ 100 ng/mL, and this effect was not impaired when the activation of β-catenin was inhibited by XAV939, indicating that the effect of RSPO3 on hLESCs was not dependent on canonical WNT/β-catenin signaling pathway. Exogenous RSPO3 accelerated epithelial wound healing by enhancing the proliferation and self-renewal of residual LESCs., Conclusions: RSPO3 promotes the proliferation and self-renewal of LESCs through a WNT/β-catenin-independent signaling pathway which might have translational significance in the treatment of corneal epithelium injury and limbal stem cell deficiency.

Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs., Competing Interests: Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: Abcam, Aviva Systems Biology, Bio-Techne, Cell Signalling Technology, Developmental Studies Hybridoma Bank, GeneTex, Horizon Discovery, Proteintech, Synaptic Systems, Thermo Fisher Scientific., (Copyright: © 2025 Fanti R et al.)

Purpose: To investigate the therapeutic efficacy of BEZ235, a dual PI3K/mTOR inhibitor, in suppressing pathological neovascularization in an oxygen-induced retinopathy (OIR) mouse model and explore the role of cyclin D1 in endothelial cell cycle regulation., Methods: Single-cell RNA sequencing was performed to analyze gene expression and cell-cycle alterations in retinal endothelial cells under normoxic and OIR conditions. The effects of BEZ235 on human umbilical vein endothelial cells (HUVECs) and human retinal microvascular endothelial cells (HRMECs) were evaluated by assessing cell viability, cell-cycle progression, proliferation, migration, and tube formation. In the OIR mouse model, retinal neovascularization was evaluated by retinal flatmount immunofluorescence staining, hematoxylin and eosin (H&E) staining, quantitative reverse-transcription polymerase chain reaction (RT-qPCR), and western blot analyses. The in vivo toxicity of BEZ235 was evaluated by electroretinography (ERG) and histological examination of the heart, liver, spleen, lungs, and kidneys., Results: In vitro, BEZ235 significantly inhibited cell cycle progression by downregulating cyclin D1 at both mRNA and protein levels, inducing G0/G1 phase arrest. This led to significant reductions in cell viability, proliferation, migration, and tube formation. In the OIR model, BEZ235 substantially decreased neovascularization and improved vascular organization. BEZ235 mediates its effects by inhibiting the PI3K/Akt/mTOR pathway, reducing Akt and 4E-binding protein 1 (4EBP1) phosphorylation levels, thus downregulating cyclin D1 expression. ERG and histological examination suggested that BEZ235 did not induce evident retinal or systemic toxicity at the dosage used to inhibit retinal neovascularization., Conclusions: BEZ235 effectively inhibits retinal neovascularization by downregulating cyclin D1 via 4EBP1 phosphorylation inhibition, highlighting its potential as a promising therapeutic agent for retinal neovascularization diseases.

Purpose: Although mechanical injury to the cornea (e.g. chronic eye rubbing) is a known risk factor for keratoconus progression, how it contributes to loss of corneal integrity is not known. Here, we set out to determine how eye rubbing can influence keratoconus progression by exploring the expression of known disease markers in mechanically stressed corneal epithelial cells., Methods: To explore the effects of mechanical stress on the expression of genes implicated in keratoconus (e.g. WNT10A, COL12A1, and TGFB1), we measured their expression using an in vitro model that simulates eye rubbing by cyclic stretching of an immortalized human corneal epithelial cell line (hTCEpi) for 16 hours. We further examined the influence of WNT10A expression in hTCEpi cells using loss-of-function approaches., Results: Mechanical strain led to a marked reduction in WNT10A mRNA and protein expression, as well as decreased collagen XII mRNA and protein expression, in hTCEpi cells. Reduced expression of WNT10A protein in WNT10A knockdown cells resulted in reduced protein expression of collagens I and XII, and reduced mRNA expression of MMP9 and TGFB1. Conversely, primary keratocytes treated with recombinant WNT10A protein increased TGFB1 mRNA expression., Conclusions: We provide a molecular explanation for how mechanical strain results in reduced expression of WNT10A in the corneal epithelium, which, in turn, leads to depletion of collagen type I and XII, and TGFβ1 expression. These results provide a molecular link among mechanical strain, WNT10A expression, and the biomechanical failure of the keratoconus cornea.

Purpose: The purpose of this study was to investigate the activated core kinases involved in the DNA damage responses (DDR) during ferroptosis of retinal pigment epithelial (RPE) cells in vitro and their regulatory effects on ferroptosis., Methods: Ferroptosis was induced by erastin in induced RPE (iRPE) cells derived from human umbilical cord mesenchymal stem cells (hUCMSCs), hUCMSCs, and induced pluripotent stem cell-derived RPE (iPSC-RPE) cells. CCK8 was employed to measure the cell viability. Calcein/PI staining was used to detect the ferroptotic cells. The γ-H2AX, 8-oxoG, and phosphorylated DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were determined through immunostaining. The phosphorylation of DNA-PKcs and ERK1/2 was determined by Western blotting. Lipid peroxides were detected by BODIPY581/591-C11 staining., Results: The iRPE cells exhibited a stronger ability to resist ferroptosis compared to hUCMSCs. Ferroptosis induced DNA damage in cells, and DNA-PKcs was rapidly phosphorylated in iRPE cells on the treatment of erastin. In addition, inhibition of DNA-PKcs phosphorylation promoted ferroptosis in iRPE cells, suggesting that DNA-PKcs prevents ferroptosis. Meanwhile, DNA-PKcs inhibited ERK1/2 phosphorylation only at the early stage of ferroptosis induction, whereas ERK1/2 phosphorylation played a protective role in iRPE cells. Furthermore, erastin inducing DNA-PKcs phosphorylation and inhibition of its phosphorylation promoting ferroptosis were also verified in iPSC-RPE cells., Conclusions: The present study elucidates that the key DDR kinase DNA-PKcs is activated and plays protective role during ferroptosis in RPE cells in vitro, which will provide new research targets and strategies for inhibiting ferroptosis in RPE cells.

Purpose: Sulfur mustard gas (SM) exposure to eyes causes multiple corneal injuries including stromal cell loss in vivo. However, mechanisms mediating stromal cell loss/death remains elusive. This study sought to test the novel hypothesis that SM-induced toxicity to human corneal stromal fibroblasts involves ferroptosis mechanism via p38 MAPK signaling., Methods: New Zealand white rabbit corneas, naïve and SM exposed (200 mg-min/m3 for eight minutes and collected after three days) were used to examine the levels of cell death and reactive oxygen species (ROS) for in vivo studies. Donor human corneas were used to generate primary human corneal stromal fibroblasts (hCSF) for in vitro studies. The hCSFs were exposed to nitrogen mustard (NM; SM analogue) at various timepoints (30 minutes, eight hours, and 24 hours). A p38 MAPK specific inhibitor, SB202190, was also used. Quantitative reverse transcription polymerase chain reaction, Western blotting, reactive oxygen species (ROS), lipid peroxidation, live/dead assay, and RNASeq were used in various investigations., Results: SM caused a significant increase in cell death and ROS production three days after SM exposure in rabbit corneas. NM exposure to hCSF demonstrated a significant increase in ROS, lipid peroxidation, and ferroptosis biomarkers ACSL4 (inducer) and significant decrease in reducer (SLC7A11 and GPX4) compared to controls in a time-dependent manner. The inhibition of p38 MAPK promoted cell survival and reduced ROS production following mustard gas exposure., Conclusions: The results of in vivo and in vitro investigations uncovered a novel mechanism that mustard gas toxicity to the cornea involves ferroptosis pathway and p38 MAPK activation.

Proliferative vitreoretinopathy (PVR) is a multifactorial ocular condition characterized by the development of fibrotic membranes inside the vitreous cavity and on the detached retina, which can result in severe blindness. Semaphorin7A (Sema7a) is involved in axon growth, inflammatory responses, and immune regulation; however, its role in PVR and regulatory mechanisms in retinal pigment epithelium (RPE) cells remains unclear. This study aimed to examine Sema7a in PVR and the underlying mechanisms. Transcriptome sequencing was used to investigate the changes in mRNA expression profiles. Western blotting, immunofluorescence, and real-time polymerase chain reaction (RT-PCR) were utilized to investigate the potential mechanism of Sema7a on epithelial-mesenchymal transition (EMT) in RPE cells. Stimulating RPE cells with transforming growth factor beta-1 (TGF-β1) decreased the levels of epithelial markers but increased those of mesenchymal markers. Based on transcriptome sequencing, many molecules associated with PVR progression were regulated. PVR vitreous fluid proteomics data analysis showed that Sema7a significantly changed at different levels. Silencing Sema7a in RPE cells attenuated TGF-β1-induced EMT and their ability to induce experimental PVR; in contrast, recombinant Sema7a (rSema7a) directly triggered EMT in RPE cells. TGF-β1 induction mechanically activated the PI3k-AKT and MAPK pathways, while Sema7a knockdown by short interfering RNA lowered the phosphorylation of the PI3k-AKT/MAPK signaling pathway. Therefore, Sema7a may be a viable therapeutic target for PVR due to its crucial role in the TGF-β1-induced EMT of RPE cells., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Objectives: As dental pulp therapy evolves towards regenerative approaches, biomolecules such as icariin, derived from Epimedium flowers, are being evaluated for their therapeutic potential. This study investigates icariin's effectiveness in promoting odontogenic differentiation in human dental pulp stem cells (hDPSCs) in vitro and as a pulp-capping agent in vivo., Design: The study explored the effects of icariin on hDPSCs at concentrations of 10, 20, and 40 µM. Cell viability and migration assays were conducted to evaluate cytotoxicity and chemotaxis. Odontogenic differentiation was assessed using alkaline phosphatase staining and alizarin red S (ARS) staining, complemented by real-time PCR and Western blot analyses of key markers such as RUNX family transcription factor 2 (RUNX2), collagen type I alpha 1 chain (COL1A1), alkaline phosphatase (ALPL), and dentin sialophosphoprotein (DSPP). Additionally, the in vivo effects of icariin were tested in a rat maxillary molar model, where icariin-treated collagen sponges were used for direct pulp capping to evaluate its potential to induce reparative dentin formation., Results: Icariin showed no cytotoxic effects on hDPSCs at any tested concentration, enhanced migratory activity in a dose-dependent manner, and significantly increased alkaline phosphatase activity and calcium deposition. Gene and protein expression analyses revealed a dose-dependent increase in odontogenic differentiation markers in icariin-treated hDPSCs. In vivo, icariin effectively promoted reparative dentin formation in exposed rat pulp., Conclusions: Icariin enhances odontogenic differentiation of hDPSCs and has promising potential as a pulp-capping agent for vital pulp therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)