Browsing by Author "Zhang, Jinmin"

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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.
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, Hongli
Purpose: 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.
Developing a stable drug delivery system for the mesoionic compound MIH 2.4Bl using reconstituted high-density lipoproteins (rHDL) nanoparticles
(2021) Petty, Roland; Sabnis, Nirupama; Debnath, Dipti; Mathis, James; Zhang, Jinmin; Lacko, Andras G.; Fudala, Rafal
Purpose: In recent years, mesoionic compounds have displayed promising results as an anticancer agent due to their unique structure and properties. Specifically, the 1,3-thiazolium-5-thiolate derivative of a mesoionic compound, MIH2.4Bl, has been shown to inhibit oxidative phosphorylation in mitochondria, induce expression of autophagy-related proteins (Beclin-1 and ATG5), and promote cell cycle arrest at the G2/M phase in breast cancer cells. However, due to the hydrophobic nature of the drug, the cellular uptake could be affected. The rHDL drug delivery technology has been successful in solubilizing several drugs. In the present study, we produced a stable rHDL-MIH2.4BI formulation for efficient drug delivery to breast cancer cells. Methods: The rHDL-MIH2.4Bl nanoparticles were prepared using the cholate dialysis protocol enhanced by thermocycling. The size, polydispersity index, and zeta potential of the formulation were determined using a light scattering analysis instrument (Zetasizer, Malvern Panalytical Ltd). Anisotropy was determined by spectrofluorometry, and the entrapment efficiency was determined using the absorbance of MIH2.4Bl at 480nm. Results: rHDL-MIH2.4Bl formulations were successfully prepared with an entrapment efficiency of 20.3%, an average size of 38.7 ± 12.14nm, and a zeta potential of -14.13 ± 0.41mV. After 26 days of storage at 4℃, this formulation retained 97.6% of the drug with an increased anisotropy measurement from 0.189 on day1 to 0.323 on day 26. Conclusions: While additional studies need to be performed to optimize the current formulation, the rHDL-MIH2.4Bl nanoparticle appears suitable to enhance the solubility and bio-availability of MIH2.4Bl.
Fluorescence Characterization and Cellular Localization of the Mesoionic Compound MIH 2.4Bl
(2021) Mathis, James; Debnath, Dipti; Lacko, Andras G.; Souza, Helivaldo; Filho, Petrônio; Fudala, Rafal; Zhang, Jinmin
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in women worldwide, making this disease a critical public health problem. Mesoionic compounds, which possess a 5-membered heterocyclic aromatic ring associated with a sextet of electrons, have shown remarkable promise as anti-cancer agents due to their unique structure and reaction properties. We previously reported the synthesis of a new 1,3-thiazolium-5-thiolate derivative of the mesoionic class (MIH 2.4Bl) and characterization of its selective cytotoxicity in a panel of breast cancer cell lines. Our studies suggest that treatment with MIH 2.4Bl mediates apoptotic death in breast cancer cells through mitochondrial dysfunction. To advance potential translational studies toward therapeutic applications, we have begun studies of the fluorescence properties of MIH 2.4Bl, using steady-state and time-resolved fluorescence techniques. Our preliminary steady-state measurements showed that the absorption spectrum of the drug is similar in different tested solvents. All samples, dissolved in various solvents, exhibited maximum absorbance between 440 and 480 nm; excitation at 480 nm elicited the highest emission at approximately 615 nm. These results may allow for future detection and localization of MIH 2.4Bl in vitro and in vivo. Follow-up studies utilizing fluorescence confocal microscopy are anticipated to reveal the site(s) of drug accumulation in situ and how cytotoxicity is induced in cancer cells. In addition, fluorescence lifetime measurements will be conducted to provide assessments of changes in the macromolecular conformational and experimental dynamic range of the drug.
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-Jun
Glutaredoxins (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.
Novel functions of the Glutaredoxin (Grx) System in the Lens
(2023) Zhang, Jinmin; Dang, Terry; Yu, Yu; Wu, Hongli
Purpose: 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.
Regulation of Grxs in Cell Functions and Senescence in the Lens
(2024-03-21) Zhang, Jinmin; Yu, Yu; Wu, Hongli
Purpose: 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.
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, Hongli
Retinal 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.