Browsing by Author "Yu, Yu"
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Item A macrocyclic molecule with multiple antioxidative activities protects the lens from oxidative damage(Frontiers Media S.A., 2022-11-18) Zhang, Jinmin; Yu, Yu; Mekhail, Magy A.; Wu, Hongli; Green, Kayla N.Growing evidence links oxidative stress to the development of a cataract and other diseases of the eye. Treatments for lens-derived diseases are still elusive outside of the standard surgical interventions, which still carry risks today. Therefore, a potential drug molecule (OH)Py(2)N(2) was explored for the ability to target multiple components of oxidative stress in the lens to prevent cataract formation. Several pathways were identified. Here we show that the (OH)Py(2)N(2) molecule activates innate catalytic mechanisms in primary lens epithelial cells to prevent damage induced by oxidative stress. This protection was linked to the upregulation of Nuclear factor erythroid-2-related factor 2 and downstream antioxidant enzyme for glutathione-dependent glutaredoxins, based on Western Blot methods. The anti-ferroptotic potential was established by showing that (OH)Py(2)N(2) increases levels of glutathione peroxidase, decreases lipid peroxidation, and readily binds iron (II) and (III). The bioenergetics pathway, which has been shown to be negatively impacted in many diseases involving oxidative stress, was also enhanced as evidence by increased levels of Adenosine triphosphate product when the lens epithelial cells were co-incubated with (OH)Py(2)N(2). Lastly, (OH)Py(2)N(2) was also found to prevent oxidative stress-induced lens opacity in an ex vivo organ culture model. Overall, these results show that there are multiple pathways that the (OH)Py(2)N(2) has the ability to impact to promote natural mechanisms within cells to protect against chronic oxidative stress in the eye.Item A Systematic Pharmacology Analysis of the Age-Related Eye Disease Study 2 (AREDS2) formula and its role in preventing Age-Related Macular Degeneration (AMD)(2021) Au, My-Lien; Yu, Yu; Lou, Alexander; Garcia, Luis; Tran, Myhoa; Wu, HongliPurpose: According to the major clinical trial sponsored by the National Eye Institute (NEI), oral supplementation with the Age-Related Eye Disease Study 2 (AREDS2) formulation (vitamins C and E, zinc, copper, lutein, and zeaxanthin) has been shown to delay the progression of advanced age- related macular degeneration (AMD). However, the detailed pharmacological mechanisms of AREDS2 are not fully understood at the molecular level. In this study, we intend to develop a systematic approach to predict AREDS2-associated targets and to map the drug-disease-target network. Methods: Genes of interest were identified via the NCBI database for compounds in the AREDS2 formula. Cytoscape software was used to visually create a network of source and target nodes to analyze the similarities between them. The formula's relation to AMD was analyzed via the Gene2Function and GeneCard databases. Results: A total of 158 genes were identified as the targets of the AREDS2 formula. 27 of these genes were a result of multiple components of the AREDS2 formula. The main pathways that these genes affect were identified and mapped out to include lipid metabolism, DNA damage responses, and oxidative stress. The top 5 genes regulated by the most components of the AREDS2 formula are GSTP1, Nrf2, VEGFA, HIF1A, and CXCL8. Conclusions: A systematic pharmacology-based approach provides beneficial information for elucidating the potential mechanisms of action of the AREDS2 formula in treating AMD. Furthermore, it provides future direction for AMD treatment which may focus on anti-adipogenic, anti-inflammatory, and anti-oxidant pathways.Item A Systemic Pharmacology Analysis of the Age-Related Eye Disease Study 2 (AREDS2) formula and its role in preventing Age-Related Macular Degeneration (AMD)(2020) Wu, Hongli; Yu, Yu; Lou, Alexander; Garcia, Luis; Tran, Myhoa; Duong, Anh; Wang, Miki; Brown, EricaPurpose: According to the major clinical trial sponsored by the National Eye Institute (NEI), oral supplementation with the Age-Related Eye Disease Study 2 (AREDS2) formulation (vitamins C and E, zinc, copper, lutein, and zeaxanthin) has been shown to delay the progression of advanced age-related macular degeneration (AMD). However, the detailed pharmacological mechanisms of AREDS2 have not been fully understood at the molecular level, other than its well-known antioxidative effects. In this study, we intend to develop a systemic approach to predict the AREDS2-associated targets and to build the drug-disease-target network. Methods: Genes of interest were identified via the NCBI database for all compounds in the AREDS2 formula. Cytoscape software was used to visually create a network of source and target nodes to analyze similarities between nodes. Cytoscape was again used to identify major pathways the AREDS2 formula targeted. Results: A total of 158 genes were identified to be targets of the AREDS2 formula. 27 out of 158 genes were a result of multiple components of the AREDS2 formula. The main pathways these genes affect were identified and mapped out to include adipogenesis, angiogenesis, apoptosis cascade, DNA damage response, fluid shear stress and atherosclerosis, glutathione metabolism, HIF1 signaling pathway, innate immune pathway, lipoprotein metabolism, Nrf2 pathway and oxidative stress pathway. The top 5 target genes were GSTP1, Nrf2, VEGFA, HIF1A, and CXCL8. Conclusion: The systemic pharmacology-based approach provides beneficial information for elucidating the potential mechanisms of action of the AREDS2 formula in treating AMD.Item Biological Characteristics of Lens Epithelial Cells from Grx1 and Grx2 Double Knockout Mice(2022) Zhang, Jinmin; Yu, Yu; Lal, Kevin; Dang, Terry; Ezugwu, Chimdindu; Tran, Myhoa; Wu, HongliPurpose: Glutaredoxins are glutathione (GSH) dependent enzymes that play an important role in repairing oxidized proteins, preventing subsequent protein misfolding and disrupting protein aggregation. The Grx system has two major isozymes: glutaredoxin 1 (Grx1) and the recently discovered glutaredoxin 2 (Grx2). To achieve a comprehensive understanding of the Grx system in the lens, our lab recently created a Grx1 and Grx2 double knockout (DKO) mouse model to observe how the double deletion of the enzymes may affect the lens epithelial cell (LEC) survival and lens transparency. Methods: Primary LECs were cultured from wild-type (WT) and DKO mice. Cell proliferation was tested via various assay kits, and cell cycle distribution was evaluated using flow cytometry analysis. Cell apoptotic markers including Bcl-2, Bax, and caspase 3 were detected using Western Blot. The mitochondrial function was evaluated via ATP concentration. Cytoskeletal arrangement and its intercellular connection were also examined by using fluorescent microscopy. Results: Compared to WT cells, DKO cells displayed a much slower growth. The number of DKO cells arrested in the M phase was twofold higher than that of WT cells. The population of DKO cells arrested in the S phase was 50% less than that of WT cells. For the apoptotic pathway, we found DKO cells have higher levels of Bax and cytochrome c with lower ATP production. Furthermore, we also found that DKO cells had higher levels of vimentin expression, which may lead to cytoskeleton reorganization and polarity. Conclusions: In conclusion, our data suggest that Grx function loss may inhibit cell proliferation, disrupt the normal cell cycle, trigger apoptosis pathway, and damage mitochondrial functions.Item Decoding the anti-cataractogenic mechanism of grapes via a systemic pharmacology approach(2019-03-05) Liu, Xiao; Wang, Duen; Garcia, Luis; Ssentamu, Frank; Lou, Alexander; Wu, Hong; Yu, YuIntroduction: Our previous study has indicated that grapes may be able to protect against in vivo ultraviolet B (UV-B) radiation-induced cataract. To better understand the mechanism of action of grapes in cataract prevention, this follow-up study was designed to identify the molecular targets of grapes in the lens by using a systemic pharmacology approach. Methods: As recommended by the California Table Grape Commission (CTGC), we selected four compounds including resveratrol, catechin, quercetin, and anthocyanins as the major phytoconstituents of grapes for target prediction. All genes that can be regulated by grapes were obtained from NCBI (www.pubmed.gov) and TCMSP (http://lsp.nwu.edu.cn/tcmsp.php). Genes that are associated with cataracts were collected from GeneCards (www.GeneCards.org). The comparison between grape-related targets and cataract-associated genes was conducted using Cytoscape 3.2.1 with ClueGo plugin. Gene Ontology (GO) enrichment analysis of grape-regulated genes was conducted using Database for Annotation, Visualization, and Integrated Discovery (www.david.ncifcrf.gov). Results: A total of 332 targets that are grape regulated were identified and visualized by protein network. Subsequently, 147 GO functional pathways were clustered, including anti-apoptotic, anti-inflammation, PI3K-Akt signaling, ATP binding, and FOXO pathways. Among these protein targets, X-linked inhibitor of apoptosis (XIAP), heat shock protein (HSP) 90, and prostaglandin-endoperoxide synthase (PTGS) were correlated with all of the active ingredients of grapes. Comparison between grape targets and cataract disease genes showed that thirteen grape targets overlapped with cataract associated genes, including PTGS2, HSP90AA1, HSP90AA2P, mitogen-activated protein kinase 1 (MAPK1), MAPK14, MAPK3, amyloid precursor protein (APP), glycogen synthase kinase 3B (GSK3B), protein kinase α (PRKCA), protein kinase C delta (PRKCD), B-cell lymphoma 2 (BCL2), BCL2L1, and K-ras (KRAS). Conclusions: The anticataractogenesis effects of grapes may involve not only directly scavenging free radicals but also activating the antiapoptotic pathway.Item Emerging functional crosstalk between the glutaredoxin system and Nrf2 antioxidant pathway: evidence from ultraviolet radiation-induced cataract formation(2020) Yu, Yu; Lou, Alexander; Garcia, Luis; Tran, Myhoa; Duong, Anh; Wu, Hongli; Liu, XiaobinPurpose: To determine the function of glutaredoxin (Grx) system, both glutaredoxin 1 (Grx1) and glutaredoxin 2 (Grx2), in protecting the lens against ultraviolet (UV)-induced cataract formation by using Grx1/Grx2 double knockout (DKO) mice as a model. Methods: One-month old Grx1/Grx2 DKO and age-matched wild-type (WT) mice were exposed to UV radiation to induce cataracts. Mice were euthanized at 4 days post-exposure. The degree of the cataract and lens morphology were evaluated under a dissecting microscope. Glutathione (GSH), free protein thiol(PSH), and protein glutathionylation (PSSG) levels were measured as general markers of oxidative damage. Nrf2 and its downstream target proteins were examined using Western blot. Results: We found that UV radiation caused more severe anterior subcapsular cataract in Grx1/Grx2 DKO than that of WT mice. The opacity of the lenses in DKO mice appeared to extend deeper into the cortical and even nuclear regions. Lenses of Grx1/Grx2 DKO mice contained significant lower levels of GSH and PSH. On the other hand, the accumulation of PSSG was much higher in Grx1/Grx2 DKO group. Deletion of Grx1 and Grx2 also decreased the expression of antioxidant enzyme transcription factor regulator, Nrf2, and its downstream antioxidant genes, including catalase, superoxide dismutase (SOD), and another redox regulator of thioredoxin (Trx). These changes were especially extensive in the lens after UV exposure. Conclusions: With combined Grx1 and Grx2 deletion, the Nrf2-dependent antioxidant response is severely impaired, causing elevation of oxidative stress that may increase the lens susceptibility to UV-induced damage.Item Functions of glutaredoxin 2 (Grx2) in the retina: Mechanisms and Protection(2021) Tran, Myhoa; Yu, Yu; Wu, HongliPurpose: Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase that reduces S-glutathionylated proteins. Previously, we found that Grx2 could protect the retina from light-induced retinal degeneration (LIRD). However, the mechanisms that coordinate thiol-repair processes in the damaged retina remain unknown. To better understand the protective effects of Grx2, our study was extended to analyze the transcriptome changes of retinal tissue in light-exposed Grx2 knockout (KO) mice. Methods: Wild type (WT) and Grx2 KO mice were exposed to white light at 23,000 lux for 1 hour after dark adaptation for 10 hours. Retinal damage confirmed by electroretinogram (ERG) and spectral domain optical coherence tomography (SD-OCT). The transcriptome of the retinal tissue in WT and Grx2 KO mice were compared using transcriptome shotgun sequencing (RNA-seq). DESeq2 software utilized to analyze gene network. Real-time PCR and Western Blot further confirmed the genes of interest. Results: Light-exposed Grx2 KO mice showed compromised visual function indicated by loss of a- and b-wave amplitudes and thinning of the outer nuclear layer (ONL). Thousands of genes identified with statistically significant expression changes and were then classified into cellular processes and molecular pathways. Among these pathways, many genes that contribute to complement activation, inflammation, and cell survival system were significantly upregulated. Conclusions: Our results suggest that Grx2 could protect the retina from LIRD. Grx2 plays an important role in regulating light-induced retinal inflammation which may be associated with its ability to repair S-glutathionylated substrates.Item Glutaredoxin 2 (Grx2) protects the retina from light-induced photoreceptor damage via regulating the endothelin receptor B (Ednrb) pathway(2019-03-05) Liu, Xiaobin; Ssentamu, Frank; Aguilera Garcia, Luis; Yu, Yu; Li, Yousong; Wu, Hongli; Wang, DuenShian1.Purpose : Glutaredoxin 2 (Grx2) is a glutathione-dependent oxidoreductase which is known to reduce S-glutathionylated proteins. In a previous study, we have found that Grx2 could protect the retina from light-induced retinal degeneration. However, the molecular mechanisms that coordinate thiol-repair processes and cell survival systems in the damaged retina remain largely unknown. To better understand the protective effects of Grx2 in the retina, our study was thus extended to analyze the full transcriptome changes of the retinal tissue in light-exposed Grx2 knockout (KO) mice. 2.Methods : Wild type (WT) and Grx2 KO mice were exposed to white light at 23,000 lux for 1 hour after dark adaptation for 10 hours. The retinal damage was confirmed by the electroretinogram (ERG) recording and spectral domain optical coherence tomography (SD-OCT) measurement. Protein glutathionylation level was evaluated by Western Blot. We then compared the full transcriptome of the retinal tissue in WT and Grx2 KO mice using transcriptome shotgun sequencing (RNA-seq). The gene network was analyzed using DESeq2 pathway analysis software, and real-time PCR and Western Blot further confirmed the selected genes of interest. 3.Results : Light-exposed Grx2 KO mice showed compromised visual function as indicated by severe loss of both a- and b-wave amplitudes and the thinning of the outer nuclear layer (ONL). Protein glutathionylation level was elevated in light-exposed Grx2 KO mice. We identified thousands of genes with statistically significant expression changes in light-exposed Grx2 KO mice and classified them into cellular processes and molecular pathways. Among these pathways, many genes that are related to complement activation, inflammation, and cell survival system were significantly upregulated. These genes include Bcl-3 and Fgf2 in NF-KappaB family pathway, C4b in classical activation pathway, Jak3 and STAT3 in JAK-STAT signaling pathway, Cdkn1a in DNA damage response pathway, Edn2 and Ednrb in endothelin 2 signaling pathway. 4.Conclusions : Collectively, our results suggest that Grx2 could protect the retina from light-induced retinal degeneration. Grx2 plays an important role in regulating light-induced retinal inflammation which may be associated with its ability to repair S-glutathionylated substrates.Item Novel functions of the Glutaredoxin (Grx) System in the Lens(2023) Zhang, Jinmin; Dang, Terry; Yu, Yu; Wu, HongliPurpose: The purpose of this study is to evaluate the function and therapeutic potential of the glutaredoxin (Grx) system, both glutaredoxin 1 (Grx1) and glutaredoxin 2 (Grx2), using Grx1/Grx2 double knockout (DKO) mice as a model. Methods: We isolated primary LECs from wild-type (WT) and DKO mice for in vitro studies including cell proliferation assays, cell cycle distribution analysis via flow cytometry, cell apoptosis via western blot and ELISA kit, mitochondrial function evaluation via ATP bioluminescence assay, expression levels of mitochondrial complexes I-V, and seahorse mito stress test, cell cytoskeleton visualization using a fluorescence microscope. Results: We found that DKO cells displayed a much slower proliferation rate compared to WT cells. The population of DKO cells in the G2/M phase was two-fold higher than that of WT cells. On the other hand, the population of DKO cells in the S phase was 50% less than that of WT cells. Additionally, DKO cells are pro-apoptotic under non-stressed condition as indicated by higher levels of Bax and cytochrome C. For the mitochondrial function, lower ATP production, less expression of mitochondrial complex III subunit UQCRC2 and complex IV subunit MTCO1 (CIV-MTCO1), lower coupling efficiency, and higher proton leak were presented in DKO cells as compared to WT cells, indicating multi-dimensional mitochondrial dysfunction in DKO cells. As for the cell cytoskeletal organization, we found that DKO cells had microtubule polarization because of the higher levels of vimentin expression which is an indicator of nuclei degeneration inhibition during the lens cell differentiation. Conclusion: Overall, we found slow cell proliferation, cell cycle arrest, and mitochondrial dysfunction in the LECs from DKO mice. Our data indicate Grx system plays an important role in maintaining the normal function of mLECs, and Grx system activation might serve as a new therapeutic strategy for cataract prevention.Item Regulation of Grxs in Cell Functions and Senescence in the Lens(2024-03-21) Zhang, Jinmin; Yu, Yu; Wu, HongliPurpose: Glutaredoxins (Grxs), a family of thiol transferases, can reverse protein glutathionylation using glutathione (GSH) as an electron donor. Therefore, it can regulate protein redox state and enzymatic activity. Specifically, Glutaredoxin 1 (Grx1) and Glutaredoxin 2 (Grx2) are predominantly localized in the cytoplasm and mitochondria, respectively. The Grx1/Grx2 double knockout (DKO) mice showed early onset of cataracts and were more sensitive to UV radiation, highlighting the importance of Grx1 and Grx2 in maintaining lens transparency. Lens epithelial cells (LECs) are crucial to lens transparency and functionality. Our current study is to explore novel roles of the Grxs in the lens using the Grx1-/-/Grx2-/- mouse model. Methods: We isolated LECs from the lenses of WT and DKO mice and conducted a range of in vitro experiments to study the effects of Grx depletion on the epithelial morphology, cell proliferation, cell death, and mitochondrial function of LECs. We also did lens tissue sectioning and hematoxylin and eosin (H&E) staining to visualize lens tissue. Results: Loss of Grx1/Grx2 led to stress fiber formation, cytoskeleton reorganization, and higher protein expression of mesenchymal markers (N-cadherin and vimentin) in LECs. The DKO LECs exhibited a lower proliferation rate and cell cycle arrest in comparison with WT LECs. Resistance to apoptosis and elevated levels of β-galactosidase activity of DKO LECs indicated that DKO LECs underwent cell senescence. DKO LECs also displayed compromised mitochondrial function, characterized by decreased ATP production, reduced expression levels of mitochondrial complexes III and IV, and increased proton leak. A compensatory metabolic shift towards glycolysis was observed in DKO LECs, indicating an adaptive response to Grx1 and Grx2 deficiencies. The HE staining data showed that the DKO mouse had aging lens epithelium characterized by less LEC density, remained flat in shape, and aligned less regularly. Conclusions: Our study demonstrates that the Grx1 and Grx2 DKO in LECs results in cytoskeletal reorganization, lower cell proliferation rate, cell cycle arrest, resistance to apoptosis, compromised mitochondrial function, and accelerated senescence. These findings underscore the importance of Grx1 and Grx2 in preventing LECs from undergoing premature aging.Item Retina-Targeted Estrogen Prodrug: A New Concept for Retinal Protection(2022) Lal, Kevin; Yu, Yu; Zhang, Jinmin; Tran, Myhoa; Ezugwu, Chimdindu; Prokai-Tatrai, Katalin; Liu, Yang; Wu, HongliRetinal injury due to excessive light exposure during military duties often results in serious vision damage to soldiers including irreversible loss of visual function. However, therapeutic interventions that can promote retinal protection or reverse retinal damage are very limited. This unmet clinical need also persists in the public when strong lasers, light, or fire cause trauma in ocular tissues. It is well known that estrogen has been shown to exhibit various beneficial actions in the central nervous system, including positively affecting mood and protecting the neuronal cells against neurodegenerative diseases. Despite estrogen's potential, its detrimental side effects prevent its clinical uses for neurotherapy. To overcome this challenge, we developed a bioprecursor prodrug, called 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED), that is selectively converted to E2 only in the neuronal cells, including retinal cells. To determine if treatment with DHED can sufficiently protect the photoreceptor from light-induced damage, male C57BL/6J mice were injected with or without 200 µg/kg DHED (n=9) and 200 µg/kg E2 (n=9) for 10 days before the light injury. Seven days after the light exposure, the visual function and retinal structure were examined by the spectral-domain optical coherence tomography (SD-OCT) and electroretinogram (ERG). After light exposure, we found massive photoreceptor loss as indicated by thinning of the outer nuclear layer (ONL) and retinal detachment. Additionally, DHED significantly prevented light-induced retinal structural changes and light-induced a- and b-wave reduction. The photoreceptor protective effects upon DHED treatment are stronger than that of E2, consistent with our earlier observation that targeted E2 delivery via DHED prodrug produces more robust neuroprotection than direct administration of E2. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based bioassay indicated that DHED delivers the biologically active estrogen to the neuronal cells including the retinal cells without affecting other tissues - unlike the systemic exposure that is seen with estrogen. In conclusion, our study supported our hypothesis that DHED is an efficacious and safe site-specific delivery agent to produce robust estrogen-mediated retinal neuroprotection.