Figure 6.

BKFE inhibits STZ-induced production of ROS by downregulation of p-Erk/Nox4 pathway in MIN6 cells and mice. (A) The cells were treated as described in Figure 5, and the cells were stained with H₂DCFDA and analyzed by flow cytometry. Values on the representative flow cytometry data indicate the DCF fluorescence intensity of whole cells. The bar graph shows quantitative data for the percentage of intracellular ROS generation. (B) Nox4 mRNA values were analyzed by quantitative RT-PCR from three independent experiments. MIN6 cells were pretreated with 10 μg/mL of BKFE for 1 h and then further incubated with 10 mM STZ for 1 h (C) or 30 min (D) to detect Nox4, Erk, and p-Erk levels. Con, Vehicle + citrate buffer; STZ, Vehicle + STZ; BKFE, BKFE + citrate buffer; BKFE + STZ, BKFE + STZ. (E) Immunohistochemical detection of Nox4 in pancreatic sections from experimental mice treated with STZ and/or BKFE and non-diabetic controls. Representative images showing the expression of Nox4 (red) and insulin (green) by immunofluorescence staining. The nuclei were counterstained with DAPI (blue). (Scale bars, 50 μm). Con, Vehicle + citrate buffer; STZ, Vehicle + STZ; BKFE, BKFE + STZ. (F–G) MIN6 cells were pretreated with 10 μg/mL of BKFE for 1 h in the absence or presence of PD98059 (10 μM for 2 h), an Erk inhibitor, followed by 10 mM STZ treatment for 30 min to detect Erk and p-Erk (F) or 1 h to detect Nox4 levels (G). MIN6 cells were pretreated with 10 μg/mL BKFE for 1 h in the absence or presence of Erk siRNA (20 nM for 24 h), followed by treatment with 10 mM STZ for 30 min to detect Erk and p-Erk (H) or 1 h to detect Nox4 levels (I). The relative expression levels of Nox4 (C, G and I) and p-Erk (D and F) were normalized to that of β-actin or total-Erk and quantified using the ImageJ software. (F-I) +, Present; −, Absent. Data are presented as mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01 vs. Con or siCon only; # p < 0.05 vs. STZ or siCon + STZ.