Fig 1.
Hyperglycemia induces ROS production and NOX2 gene expression.
Raw cells were treated for 24h with: 5 mM D-glucose (LG), or 25 mM D-glucose (HG). (A) ROS levels were measured using the Cellular Reactive Oxygen Species Detection Assay Kit. The data is represented as Mean ± SD, n = 5 per treatment group. (B) RT-qPCR analysis of NOX2 gene expression. Results show increased NOX2 gene expression under high glucose conditions.
Fig 2.
Hyperglycemia reduces Lethe gene expression.
RNA analyses by RT-qPCR (mean ± SD, n = 5 per group) showed significantly decreased Lethe gene expression in RAW cells under high glucose conditions (25 mM D-glucose). However, no difference in Lethe gene expression was observed between the L-glucose treated groups compared to the low D-glucose (LG) treated groups.
Fig 3.
Overexpression of Lethe in RAW macrophage cells decreases ROS production.
(A) Overexpression of Lethe gene expression was achieved by plasmid transfection and confirmed by RNA analyses by RT-qPCR (mean ± SD, n = 3 per group), Lethe gene expression was significantly induced in the RAW macrophages transfected with pcDNA-Lethe compared to the RAW macrophages transfected with pcDNA3.1. (B) Under HG condition, ROS production was prevented following transfection of RAW macrophages with pcDNA-Lethe (mean ± SD, n = 5 per group). ROS levels were measured by the Cellular Reactive Oxygen Species Detection Kit.
Fig 4.
Overexpression of Lethe decreases ROS production in BMM.
(A)BMM was confirmed as macrophage by Flow cytometry analysis. Non-diabetic and diabetic BMM showed more than 97% F4/80 and CD11b double positive. (B) Lethe expression was measured by RT-qPCR (n = 3), and Lethe was significantly higher in non-diabetic BMM (p < 0.05) at baseline. (C) NOX2 gene expression was significantly higher in diabetic BMM at baseline (n = 3, p < 0.05), inversely associated with Lethe expression. (D) Lethe overexpression was achieved by pcDNA-Lethe transfection in BMM compared to pcDNA3.1 transfection as control (n = 3, p<0.01). (E) Lethe overexpression significantly reduced NOX2 expression in BMM (n = 3, p<0.05). (F) Diabetic BMM had significantly higher ROS production in the group transfected with the control plasmid pcDNA3.1, compared to the group treated with pcDNA-Lethe. In pcDNA-Lethe transfected groups, both non-Db and Db BMM had significantly lower ROS production compared to the pcDNA3.1 transfected controls (the data are mean ± SD, n = 5 per group).
Fig 5.
Lethe overexpression reduces NOX2 gene expression.
RNA analyses by RT-qPCR (mean ± SD, n = 5 per group) showed no significant change in SOD2 (A), SOD3 (B), and Catalase (C) gene expression, while NOX2 (D) gene expression was significantly decreased following overexpression of Lethe in the RAW macrophages transfected with pcDNA-Lethe.
Fig 6.
Lethe overexpression prevents translocation of p65 to nucleus.
(A) Western blot analysis showed that hyperglycemia induced nuclear p65, while overexpression of Lethe significantly reduced nuclear p65. For protein in nuclear fraction, we used TATA binding protein (TBP) as loading control; for protein in cytoplasmic fraction, we used GADPH as loading control. (B) Protein levels were normalized to cytoplasmic GAPDH and nuclear TBP by the software ImageJ. Results indicated that there was no change in total p65 protein level, while Lethe overexpression significantly reduced nuclear p65 (mean ± SD, n = 3 per group), while no significantly difference in total p65 protein levels between groups.
Fig 7.
Lethe is down-regulated while NOX2 is up-regulated in diabetic wounds.
mRNA analyses by RT-qPCR (mean ± SD, n = 5 per group) showed that Lethe is significantly downregulated in diabetic wounds 1, and 3 days after wounding (A), while NOX2 gene expression is significantly upregulated at the same time point (B), (mean ± SD, n = 5 per group).