Topical nerve growth factor attenuates streptozotocin-induced diabetic cataracts via polyol pathway inhibition and Na+/K+-ATPase upregulation.

The purpose of this study was to investigate whether and how topical nerve growth factor (NGF) attenuates streptozotocin (STZ)-induced diabetic cataracts in vivo. Rats were randomly divided into three groups, including the normal control rat group, STZ-induced diabetic cataract rat group (DM group), and STZ-induced diabetic cataract rat group treated with 200 µg/mL recombinant rat ß-NGF (DM + NGF group). Cataract formation was evaluated by portable slit lamp biomicroscopy following pupil dilation at 8 weeks. The expression levels of NGF, aldose reductase (AR), and Na+/K+-ATPase in the lens epithelial cells (LECs) of the three groups were measured in the presence or absence of topical NGF. TUNEL-positive LECs were quantified to determine if hyperglycemia caused LEC apoptosis. At 8 weeks, the mean cataract score in the control group was significantly lower than that in DM and DM + NGF groups, and the score in the DM + NGF group was significantly lower than that in the DM group. At the equatorial zone and anterior central zone of lens, NGF and Na+/K+-ATPase expression levels were significantly decreased in the DM group; however, they were partially restored in the DM + NGF group. At the equatorial zone and anterior central zone of lens, AR expression and TUNEL-positive apoptotic LECs were significantly increased in the DM group compared with the control group, however, they were significantly decreased in the DM + NGF group. In conclusion, topical NGF could delay the progression of diabetic cataracts by attenuating polyol pathway activation and increasing Na+/K+-ATPase protein levels.

Selection of iPSCs without mtDNA deletion for autologous cell therapy in a patient with Pearson syndrome.

Screening for genetic defects in the cells should be examined for clinical application. The Pearson syndrome (PS) patient harbored nuclear mutations in the POLG and SSBP1 genes, which could induce systemic large-scale mitochondrial genome (mtDNA) deletion. We investigated iPSCs with mtDNA deletions in PS patient and whether deletion levels could be maintained during differentiation. The iPSC clones derived from skin fibroblasts (9% deletion) and blood mononuclear cells (24% deletion) were measured for mtDNA deletion levels. Of the 13 skin-derived iPSC clones, only 3 were found to be free of mtDNA deletions, whereas all blood-derived iPSC clones were found to be free of deletions. The iPSC clones with (27%) and without mtDNA deletion (0%) were selected and performed in vitro and in vivo differentiation, such as embryonic body (EB) and teratoma formation. After differentiation, the level of deletion was retained or increased in EBs (24%) or teratoma (45%) from deletion iPSC clone, while, the absence of deletions showed in all EBs and teratomas from deletion-free iPSC clones. These results demonstrated that non-deletion in iPSCs was maintained during in vitro and in vivo differentiation, even in the presence of nuclear mutations, suggesting that deletion-free iPSC clones could be candidates for autologous cell therapy in patients. [BMB Reports 2023; 56(8): 463-468].

Anti-inflammatory and anti-apoptotic effects of N-acetylcysteine in diabetic rat corneal epithelium.

AIM: To characterize the anti-inflammatory and anti-apoptotic effects of N-acetylcysteine (NAC) in streptozotocin (STZ)-induced diabetic rat corneal epithelium and human corneal epithelial cells (HCECs) exposed to a high-glucose environment. METHODS: HCECs were incubated in 0, 5, 50 mmol/L glucose medium, or 50 mmol/L glucose medium with NAC for 24h. Diabetes was induced in rats by intraperitoneal injection of 65 mg/kg STZ and some of these rats were topically administered NAC to corneas with 3 mice per group. We characterized receptor for advanced glycation end-products (RAGE) expression using immunofluorescence, and interleukin (IL)-1ß and cleaved caspase-3 (CCAP-3) expression using immunohistochemistry. Circulating tumor necrosis factor (TNF)-α concentration was measured by ELISA and cleaved poly-ADP ribose polymerase (PARP) concentration was quantified by Western blotting. Apoptotic cells were detected using TUNEL assay and annexin V and propidium iodide staining. RESULTS: Diabetic rats had higher expression of RAGE (2.46±0.13 fold), IL-1ß, and CCAP-3 in apoptotic cells of their corneas than control rats. The expression of RAGE (1.83±0.11 fold), IL-1ß, and CCAP-3, and the number of apoptotic cells, were reduced by topical NAC treatment. HCECs incubated in 50 mmol/L glucose medium showed high concentrations of TNF-α (310±2.00 pg/mL) and cleaved PARP (7.43±0.56 fold), and more extensive apoptosis than cells in 50 mmol/L glucose medium. However, the addition of NAC reduced the concentrations of TNF-α (153.67±2.31 pg/mL) and cleaved PARP (5.55±0.31 fold) and the number of apoptotic cells. CONCLUSION: NAC inhibits inflammation and apoptosis in the corneas of diabetic rats and HCECs maintained in a high-glucose environment.

Protective Effects of Cyclosporine A Emulsion Versus Cyclosporine A Cationic Emulsion Against Desiccation Stress in Human Corneal Epithelial Cells.

PURPOSE: To compare the protective effects of cyclosporine A emulsion (Restasis: 0.05% cyclosporine A) (CsAE) and cyclosporine A cationic emulsion (Ikervis: 0.1% cyclosporine A) (CsACE) on cellular inflammation, apoptosis, proliferation, and survival in an in vitro dry eye model. METHODS: The concentration of CsA in CsAE and CsACE was verified using a liquid chromatography tandem mass spectrometry system. Human corneal epithelial cells were subjected to desiccation stress. Human corneal epithelial cells were incubated with or without 3 groups of cyclosporine A medium (CsAE 1:50, CsACE 1:50, and CsACE 1:100). p-NF-κB p65, p-IκBα, Bax, Bcl-xL, p-Erk1/2, and p-Akt levels were determined using Western blots, and TNF-α levels were quantified using an enzyme-linked immunosorbent assay. RESULTS: The CsA concentration of CsACE 1:100 was nearly the same as that of CsAE 1:50. Compared with CsAE 1:50 (0.78 ± 0.19 fold), the p-NF-κB p65 level was further reduced in CsACE 1:50 (0.38 ± 1.20 fold) and 1:100 (0.29 ± 0.11 fold) as well as p-IκBα. Levels of TNF-α were also lower in CsACE 1:50 and 1:100 than in CsAE 1:50. Induction of the apoptotic protein Bax was significantly decreased in CsACE 1:50 and 1:100 compared with CsAE 1:50, whereas that of the antiapoptotic protein Bcl-xL was increased in CsACE 1:50 and 1:100. p-ERK1/2 and p-Akt levels were higher in CsACE 1:50 and 1:100 than in CsAE 1:50. CONCLUSIONS: CsACE had more potent anti-inflammatory and antiapoptotic effects than CsAE in a transwell desiccation stress model. CsACE also enhanced proliferation and survival factors under desiccation stress compared with CsAE in this in vitro dry eye model.