Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/30810
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Browsing Eye / Vision by Author "Dang, Terry"
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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 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.