Browsing by Author "Wu, Hongli"
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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 Mechanism study toward understanding the protective effects of glutaredoxin 2 (Grx2) on light-induced retinal damage(2018-03-14) Liu, Xiaobin; Xavier, Christy; Ananti, Princess; Liu, Yang; Wu, Hongli; Wang, Duen-ShianPurpose: Glutaredoxin 2 (Grx2), located mainly in the mitochondria, is a glutathione-dependent oxidoreductase which is known to reduce S-glutathionylated proteins. In previous study, we have found that Grx2 could protect the retina from light-induced retinal degeneration. However, the molecular mechanisms that coordinate mitochondrial energy production with thiol-repair processes 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. Methods: Wild type (WT) and Grx2 KO mice were exposed to white light at 12,000 lux for 1 hour after dark adaptation. 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 by performed the whole transcriptome shotgun sequencing (RNA-seq). The gene network was analyzed using DESeq2 pathway analysis software and the selected genes of interest were further confirmed by real-time PCR and Western Blot. 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 statistical 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 and inflammation reaction were significantly upregulated. These genes include complement C3, C4a, C4B (C4B), Bcl-3, NF-kappa B, Jak3, and STAT3. Conclusions: Collectively, our results suggest that Grx2 could protect the retina from light-induced retinal degeneration. It plays an important role in regulating light-induced retinal inflammation which may be associated with its ability to reduce S-glutathionylated substrates.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 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 Emerging functional crosstalk between the Grx system and Nrf2 pathway: evidence from UV radiation-induced cataract formation(2022) Dang, Terry; Wu, HongliGlaucoma, cataracts, age-related macular degeneration (AMD), are linked to oxidative stress by the external and internal environment. Ocular tissues are more susceptible to oxidative stress due to daily exposure of UV light and high oxygen consumption. The human body has several antioxidant enzymes such as catalase, superoxide dismutase (SOD), and thioredoxin. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an antioxidant enzyme transcription factor that regulates the downstream antioxidant genes. It also has glutathione (GSH) that searches for the free radicals in our body and oxidizes to form glutathione mixed disulfide (GSSG). As the GSSG levels increase, it naturally adds to other proteins causing protein glutathionylation (PSSG). PSSG is an important post-translational modification linked to oxidative stress. Research has shown that the glutaredoxin (Grx) system is capable of reversing PSSG formation which can be assumed to cause less oxidative stress. To take a closer look at the function of the Grx system in protecting the lens against ultraviolet (UV)- induced cataract formation, glutaredoxin (Grx1) and glutaredoxin 2 (Grx2) is studied in a Grx1/Grx2 double knockout (DKO) mice model. By intercrossing Grx1 knockout (KO) and Grx2 KO mice, Grx1/Grx2 DKO mice resulted. The study population was half male and half female, one month old Grx1/Grx2 DKO and age-matched wild type (WT) mice. They were exposed to 20.6 kJ/m2 UV radiation for 15 mins 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. To further define the crosstalk between the Grx system and nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway, Nrf2 and its downstream target proteins were examined by using Western blot analysis. The results showed 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, a marker for protein thiol oxidation, 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). The Nrf2 dependent antioxidant response can no longer function with combined Grx1 and Grx2 deletion. This will cause more oxidative stress and 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 Retinal Pigment Epithelial Cells from Oxidative Damage by Regulating Autophagy(2016-03-23) Liu, Xiaobin; Nguyen, Yen; Tran, Tyler; Wu, Hongli; Xavier, ChristyPurpose: Glutaredoxin 2 (Grx2) is an oxidoreductase present in the mitochondria where it protects the organelle from oxidative damage and maintains its redox homeostasis.The purpose of this study is to evaluate the cytoprotective effects of Grx2 in human retinal pigment epithelial (RPE) cells and characterize its potential function in regulating autophagy.Methods: Primary RPE cells were isolated from Grx2 knockout (KO) mice and treated with or without 400 µM H2O2 for 4 h. Human retinal pigment epithelial (ARPE-19) cells were transfected with either a human Grx2 cDNA-containing plasmid (pCR3.1-hGrx2) or an empty vector pCR3.1. Cells were treated with or without 200 µM H2O2 for 16 h. Grx2 protein expression was detected by western blot analysis. Cell viability was measured by a colorimetric assay with WST8. The morphology of nuclear chromatin was assessed by staining with Hoechst 33342. Apoptosis was quantitatively analyzed by flow cytometry. The level of protein glutathionylation (PSSG) and autophagy pathway proteins were measured by immunoblotting.Results: Primary RPE cells that lack Grx2 were more sensitive to oxidative damage. On the other hand, Grx2 overexpression protected RPE cells from H2O2-induced cell viability loss. Assessment of apoptosis indicated that cells transfected with Grx2 were more resistant to H2O2 with fewer cells undergoing apoptosis as compared to vector control cells. PSSG accumulation was also attenuated by Grx2 overexpression with acute H2O2 challenge. Furthermore, protein levels of LC3II and Beclin-1, which are key molecules to initiate autophagy, were inhibited in Grx2 overexpressed cells with H2O2 treatment. Conversely, primary Grx2 KO RPE cells showed higher levels of LC3II and Beclin-1 under oxidative stress.Conclusion: Grx2 rescues RPE cells from lethal oxidative damage, possibly through alleviation of ROS-triggered autophagy and prevention of PSSG accumulation.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 INCREASED EXPRESSION OF GLUTAREDOXIN 1 (Grx1) PROTECTS HUMAN RETINAL PIGMENT EPITHELIAL CELLS FROM OXIDATIVE DAMAGE(2014-03) Liu, Xiaobin; Jann, Jamieson; Wu, HongliOxidative stress is believed to contribute to the pathogenesis of many diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial (RPE) cells are considered as major targets. It is widely accepted that the RPE cells have enormous number of thiol-containing proteins, which can undergo modifications to change retinal protein functions. In contrast, the mechanism of thiol redox regulation in the retina and its association with AMD are still very poorly understood. In particular, the function of glutaredoxin 1 (Grx1), a thiol repair enzyme in cytosol, is virtually unknown. This project seeks to address this paucity in a comprehensive and physiological relevant fashion, and therefore is both novel and innovative. Furthermore, the ability to identify novel therapeutic targets for further research is the first critical step in finding new treatments for AMD. The overall success of this project will raise new hope that Grx1 or its mimic may be a potential therapeutic agent for AMD, and perhaps for other ocular diseases induced by oxidative stress. Purpose (a): The retina is constantly exposed to oxidative stress, which is countered by well-designed antioxidant systems present in retinal pigment epithelial (RPE) cells. Disruption of these systems may lead to the development of age-related macular degeneration (AMD). In this study, we explored the strategy of overexpressing glutaredoxin 1 (Grx1), a component of the endogenous antioxidant defense system, to combat oxidative damage in RPE cells. Methods (b): Human retinal pigment epithelial (ARPE-19) cells were transfected with either a Grx1-containing plasmid or an empty vector. Normal ARPE-19 cells and transfected cells were treated with or without 200 µM H2O2 for 24 h. Grx1 protein expression was detected by western blots and enzyme activity was measured by spectrophotometry. Cell viability was measured by a colorimetric assay with WST8. The morphology of nuclear chromatin was assessed by staining with Hoechst 33342. Apoptosis was quantitatively analyzed by flow cytometry. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Results (c): Grx1 protein level and enzyme activity in Grx1 transfected cells were significantly increased as compared to non-transfected and vector transfected cells. Grx1 overexpression protected ARPE-19 cells from H2O2-induced cell viability loss. Assessment of apoptosis indicated that cells transfected with Grx1 were relatively more resistant to H2O2 with fewer cells undergoing apoptosis as compared to vector control or non-transfected cells. Furthermore, PSSG accumulation was also dramatically attenuated by Grx1 overexpression. Conclusions (d): Grx1 can protect human retinal pigment epithelial cells against H2O2-induced cell death. The mechanism of this protection is likely associated with its ability to prevent lethal accumulation of PSSG.Item Inhibition of the Glutaredoxin System Increases Doxorubicin Sensitivity in Hepatocellular Carcinoma by Impairing the Nrf2-dependent Antioxidant Response(2015-03) Xavier, Christy; Wu, Hongli; Liu, Xiaobin; Nguyen, BenjaminPurpose: Hepatocellular carcinoma (HepG2) is the most common type of liver cancer, causing approximately 1.25 million deaths annually. Even with premier anti-cancer drugs like doxorubicin, the lethality of hepatocellular carcinoma has increased and is mainly attributed to growing drug resistance. Specifically, overexpression of key antioxidant enzymes such as the glutaredoxin system (Grxs) may enable drug resistance. Glutaredoxin is a powerful protective thiol repair enzyme that increases cancer cell survival. In this study, we explored a new anti-cancer strategy, the inhibition of Grxs, as a way to both increase doxorubicin sensitivity and reverse resistance in HepG2 by impairing the Nrf2-dependent antioxidant response. Methods: HepG2 cells (Sigma) were transfected with Grxs or scramble shRNA vector. HepG2 was treated with doxorubicin in a dose and time-dependent manner. Cell viability was measured by the WST-8 colorimetric assay. Western blot was performed to test expression levels of pro- and anti-apoptotic proteins like Bax, Bcl2, and cleaved caspase-3. The level of protein glutathionylation (PSSG) was measured by immunoblotting using anti-PSSG antibody. Western blot was used to also examine the expression levels of Nrf2 and its downstream genes in Grxs-inhibited cells before and after doxorubicin treatment. Nrf2 translocation assay and co-immunoprecipitation with Grxs and PSSG was also performed. Antioxidant gene screening for 91 Nrf2-pathway related genes for scramble and Grxs shRNA after doxorubicin treatment was analyzed. Results: shRNA transfection gave a 50-70% Grxs knockdown. Grxs inhibition caused increased doxorubicin sensitivity with lower cell viabilities, higher pro-apoptotic protein expression levels, and increased glutathionylation than control. Grxs inhibition also decreased the expression of antioxidant enzyme transcription factor regulator, Nrf2, and its downstream antioxidant genes like HO-1, catalase, thioredoxin, and NQO1 especially after doxorubicin treatment. Nrf2’s presence in the nucleus and cytoplasm decreased with glutaredoxin knockdown. Glutaredoxin inhibition also significantly increased Nrf2 glutathionylation. Gene screening also showed significant decrease in mRNA levels of Nrf2-pathway related genes with Grxs inhibition after doxorubicin treatment. Conclusions: Grxs inhibition causes increased doxorubicin sensitivity and apoptosis of hepatocellular carcinoma by attenuating Nrf2 and its downstream antioxidant genes activation.Item Inhibition of the Glutaredoxin System Increases Doxorubicin Sensitivity in Hepatocellular Carcinoma By Impairing the Nrf2-Dependent Antioxidant Response(2015-05-01) Xavier, Christy; Wu, Hongli; Di Pasqua, Anthony J.; Yan, Liang-JunPurpose: Hepatocellular carcinoma (HepG2) is the most common type of liver cancer, causing approximately 1.25 million deaths annually. Even with premier anti-cancer drugs like doxorubicin, the lethality of hepatocellular carcinoma has increased and is mainly attributed to growing drug resistance. Specifically, overexpression of key antioxidant enzymes such as the glutaredoxin system (Grxs) may enable drug resistance. Glutaredoxin is a powerful protective thiol repair enzyme that increases cancer cell survival. In this study, we explored a new anti-cancer strategy, the inhibition of Grxs, as a way to both increase apoptosis and doxorubicin sensitivity in HepG2 by impairing the Nrf2-dependent antioxidant response. Methods: Hepatocellular carcinoma tissue and neighboring healthy liver tissue was obtained from five patients from Sun-Yat-Sen Hospital and tested for Grx1 and Grx2 expression levels using western blot. HepG2 cells were transfected with Grxs or scramble shRNA vector. Scramble sh-RNA, Grx1 shRNA, and Grx2shRNA HepG2 cells were treated with 1 uM and 10 uM doxorubicin, and cell viability was measured by the WST-8 colorimetric assay. Western blot was performed to test expression levels of pro- and anti-apoptotic proteins like Bax, Bcl2, and cleaved caspase-3. The level of protein glutathionylation (PSSG) in whole cell lysates and mitochondrial fractions were measured by immunoblotting using anti-PSSG antibody. Western blot was used to also examine the expression levels of Nrf2 and its downstream proteins in Grxs-inhibited cells before and after doxorubicin treatment. Nrf2 translocation assay and co-immunoprecipitation with Grxs and PSSG was also performed. Immunostaining was done to confirm Grx2’s presence in the nucleus and Grx2-Nrf2 binding. NucBlue live cell staining was performed to analyze Grx2’s possible function in the nucleus. Antioxidant gene screening for 48 Nrf2-pathway related genes for scramble and Grxs shRNA after doxorubicin treatment was analyzed. Results: All five patients in our clinical study showed much higher Grx1 and Grx2 levels in cancerous tissue than in normal liver tissue. shRNA transfection gave a 50-70% Grxs knockdown. Grxs inhibition caused increased doxorubicin sensitivity with approximately a 20% lower cell viability at 1 and 10 uM doxorubicin treatment, higher pro-apoptotic protein expression levels, and increased glutathionylation than control. Moreover, actin glutathionylation increased in Grxs shRNA cells. Grxs inhibition also decreased the expression of antioxidant enzyme transcription factor regulator, Nrf2, and its downstream antioxidant proteins like HO-1, catalase, thioredoxin, and NQO1 especially after doxorubicin treatment. Nrf2’s presence in the nucleus and cytoplasm decreased with glutaredoxin knockdown. Doxorubicin treatment enhanced Grx1 and Grx2 binding with Nrf2 in scramble shRNA HepG2 cells. Glutaredoxin inhibition also significantly increased Nrf2 glutathionylation, indicating lower Nrf2 activation. Immunostaining showed abundant amount of Grx2 in the nucleus and that Grx2 specifically interacts and repairs Nrf2 in the nucleus. Grx2 may also be involved in DNA repair and synthesis in the nucleus. Gene screening also showed significant decrease in mRNA levels of Bcl2, GSTs, and Prdx6 with Grxs inhibition after doxorubicin treatment. Conclusions: Grx1 is vital to repair Nrf2 in the cytoplasm, whereas Grx2 repairs Nrf2 in the nucleus, and if inhibited, can cause an increase in Nrf2 glutathionylation and inactivation. Grxs inhibition causes increased doxorubicin sensitivity and apoptosis of hepatocellular carcinoma by attenuating Nrf2 and its downstream antioxidant genes’ activation, making it an ideal pharmacological target for future anti-liver cancer treatment.Item Involvement of Nrf2 in Ocular Diseases(Hindawi, 2017-03-27) Batliwala, Shehzad; Xavier, Christy; Liu, Yang; Wu, Hongli; Pang, Iok-HouThe human body harbors within it an intricate and delicate balance between oxidants and antioxidants. Any disruption in this checks-and-balances system can lead to harmful consequences in various organs and tissues, such as the eye. This review focuses on the effects of oxidative stress and the role of a particular antioxidant system-the Keap1-Nrf2-ARE pathway-on ocular diseases, specifically age-related macular degeneration, cataracts, diabetic retinopathy, and glaucoma. Together, they are the major causes of blindness in the world.Item New Insights into the Roles of Glutaredoxins in the Lens(2024-05) Zhang, Jinmin; Wu, Hongli; Ellis, Dorette Z.; Green, Kayla; Prokai-Tatrai, Katalin; Yan, Liang-JunGlutaredoxins (Grxs) play a crucial role in reversing protein glutathionylation. Glutaredoxin 1 (Grx1) and Glutaredoxin 2 (Grx2) are two main members of Grxs. Our prior studies have demonstrated that the Grx1 and Grx2 double knockout (DKO) mice develop cataracts prematurely at three months of age, and they are more susceptible to UV radiation. Therefore, these findings have highlighted the importance of Grx1 and Grx2 in preserving the transparency of the lens. However, the precise mechanisms underlying the faster development of cataracts in response to simultaneous deletion of Grx1 and Grx2 remain unknown. Lens epithelial cells (LECs) are pivotal for preserving lens transparency and overall lens functionality. Consequently, a comprehensive understanding of the antioxidant defenses and cell repair mechanisms in LECs is vital for cataract prevention and treatment strategies. We hypothesized that the absence of Grx1 and Grx2 could alter LECs function, triggering cataractogenesis. To test the hypothesis, we isolated primary LECs from WT and DKO mice and conducted a range of in vitro experiments to assess the effects of Grxs deletion on the epithelial phenotype, cellular proliferation, apoptosis, and mitochondrial function in LECs. We also conducted histology analysis of lens tissues using hematoxylin and eosin (H&E) staining. Our results revealed that Grx1 and Grx2 deficiency altered epithelial phenotype, reduced proliferation rate, and aberrant cell cycle distribution of DKO LECs compared to WT LECs. The deficiency also induced cellular senescence in cultured DKO LECs, which is consistent with our H&E staining data showing that LECs in the lens tissue from DKO mouse had accelerated senescence. Additionally, DKO LECs displayed compromised mitochondrial function and a compensatory metabolic shift towards glycolysis, indicating an adaptive response to Grx deficiency. Importantly, we also found that the OHPy2N2 activated Grxs and prevented the lens from H2O2-induced lens opacification. In conclusion, the findings in this study indicate that Grxs are important in regulating the aging process in the lens. Compounds that can activate Grxs may be promising candidates for preventing cataracts.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 Novel Strategy for RPE Protection: Glutaredoxin-Targeting Natural Products(2015-03) Liu, Xiaobin; Xavier, Christy; Jann, Jamieson; Giordano, Dante; Wu, HongliPurpose: Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is known as an important factor in the pathogenesis of retinopathies, such as age-related macular degeneration (AMD). In our previous study, we identified glutaredoxin 1 (Grx1), a thiol-disulfide oxidoreductase, as a new cytoprotective enzyme in RPE cells. In this study, we searched for small molecule Grx1 inducers from natural products to protect RPE cells from oxidative damage. Methods: Five natural antioxidant phenolics, including Salvianolic acid A (Sal A), Salvianolic acid B (Sal B), total salvianolic acid, curcumin, and epigallocatechin gallate (EGCG) were screened for their Grx1-inducing activities and cytoprotective effects in primary human RPE cells. Grx1 expression was examined by Western blot. Cell viability was evaluated with the WST8 assay. The level of protein glutathionylation (PSSG) was measured by using anti-PSSG antibody. Results: Among all the tested compounds, Sal B was found to be the most potent Grx1 inducer, which upregulated Grx1 by ~3 fold at 50 µM after 24 h. In both a time and dose-dependent manner, Sal B protected cells from H2O2-induced cell viability loss. Sal B also reduced annexin V positive cells, decreased Bax/Bcl-2 ratio, prevented caspase-3 cleavage, and inhibited ROS production. Additionally, H2O2-induced PSSG accumulation was markedly decreased by Sal B treatment. Moreover, knockdown of Grx1 by siRNA significantly reduced the cytoprotective effects of Sal B. Conclusions: Sal B protects primary human RPE cells from oxidative stress-induced damage by upregulating Grx1. Naturally occurring Grx1 inducers may be used as new therapeutic strategies to treat oxidative stress-related retinopathies like AMD.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.Item RETINA-TARGETED ESTROGEN PRODRUG: A NEW CONCEPT FOR RETINAL PROTECTION(2022-05) Lal, Kevin; Wu, Hongli; Prokai-Tatrai, Katalin; Liu, YangRetinal 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, a bioprecursor prodrug was developed, 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 cells from light-induced damage, male C57BL/6J mice were injected with or without 100 μg/kg DHED (n=15), 200 μg/kg DHED (n=15), 400 μg/kg DHED (n=15) and 200 μg/kg E2 (n=15) 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) in groups that received no treatment. However, DHED treatment significantly prevented light-induced retinal structural changes and light-induced a- and b-wave reduction. Additionally, photoreceptor loss was decreased as indicated by increased outer nucellar layer thickness and SD-OCT data. The photoreceptor protective effects upon DHED-derived E2 treatment are stronger than that of the direct E2 treatment, consistent with our earlier observation that targeted E2 delivery via DHED prodrug produces more robust neuroprotection than direct administration of E2. 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.