Figure 1.
The protection of bFGF on HG-stimulated inflammation and apoptosis.
Cell proliferation under HG conditions with 10% (A) or 0.35% (B) FBS (fetal bovine serum) was measured by CCK-8 assay after a 72-hour incubation (**P<0.01, ***P<0.001, t test). (C) Immunoblotting analysis for detection of p53, c-Caspase-3, PAI-1 and TNF-α in HSFs treated with the indicated concentrations of glucose for 72 hours. (D) HSFs were pretreated with 30 mM glucose (HG) for 72 hours and then incubated with 100 ng/mL bFGF (b) or bFGF together with FGFR1 inhibitor PD173074 (PD, 50 nM) for another 1 hour. The expression of levels of TNF-α, PAI-1, c-Caspase-3and p53 were analyzed by immunoblotting. LG means 5.5 mM glucose in culture medium and GAPDH was used as an internal control. Densitometry for p53 (E) or c-Caspase-3 (F) or PAI-1 (G) or TNF-α (H) shown in (C) was normalized to the amount of GAPDH. Densitometry for p53 (I) or c-Caspase-3 (J) or PAI-1 (K) or TNF-α (L) shown in (D) was normalized to the amount of GAPDH. The results are presented as fold change as compared with fibroblasts grown in the 5.5 mM(LG)glucose containing medium. Data represent mean values ±SE of three independent experiments (*P<0.05, t test).
Figure 2.
Effects of inhibition of FGFR1 on fibroblast migration and JNK phosphorylation.
(A) Wound healing assay was performed to analyzed effects of 50 nM of FGFR1 inhibitor PD173074 (PD) on human fibroblast cell migration and LG means 5.5 mM glucose in the culture medium. (B) Cell migration distance was measured according to the data shown in (A). Data represent mean values ±SE of 10 replicates, as compared to the HG group (*P<0.001, t test). (C) Phosphorylation levels of AKT and JNK were analyzed after 30 min of bFGF (b) and PD173074 (PD) stimulation. All experiments were performed after 5 µg/mL mitomycin-C (cell proliferation inhibitor) application for one day. Densitometry for p-AKT (D) and p-JNK (E) was normalized to the amount of total AKT and JNK. The results are presented as fold change as compared to fibroblasts grown in the 5.5 mM glucose (LG) containing medium. Data represent mean values ±SE of three independent experiments (*P<0.05, t test).
Figure 3.
Effects of high glucose, bFGF and FGFR1 inhibitor on fibroblast cell migration and JNK phosphorylation.
(A) Wound healing assay was performed to analyze effects of HG, 100 ng/mL of bFGF (b) and 50 nM of FGFR1 inhibitor PD173074 (PD) in human fibroblasts and mannitol was used as an osmotic control (5.5 mM glucose plus 24.5 mM mannitol) and LG means 5.5 mM glucose in the culture medium. (B) Cell migration distance was measured according to the data shown in (A). Data represent mean values ±SE of 10 replicates, as compared to the HG group (*P<0.001, t test). (C) Phosphorylation levels of AKT and JNK were analyzed after 30 min of HG, HG+bFGF (b) and HG+bFGF+PD173074 (PD) stimulation. All experiments were performed after 5 µg/mL mitomycin-C (cell proliferation inhibitor) application for one day. Densitometry for p-AKT (D) and p-JNK (E) was normalized to the amount of total AKT and JNK. The results are presented as fold change as compared to fibroblasts grown in the 5.5 mM glucose (LG) containing medium. Data represent mean values ±SE of three independent experiments (*P<0.05, t test).
Figure 4.
Diabetes and bFGF effects on rat skin wound repair.
(A) The representative images of skin wounds from normal and diabetic rats with or without bFGF treatment. (B) Wound areas of each rat presented in (A) are counted. Wound areas are measured using TINA2.0 software (*P<0.01 t test). (C) Phosphorylation levels of JNK was analyzed after 4 days of bFGF treatment. Densitometry for p-JNK (D) was normalized to the amount of total GAPDH. The results are presented as fold change as compared to control group (N). bFGF was supplied every day. Data represent mean values ±SE of three independent experiments (**P<0.01, t test). N, control; N+b, control add bFGF; DM, diabetes; DM+b, diabetes add bFGF.
Figure 5.
(A) Activities of Rac1 were analyzed after HG, HG+bFGF (b, 100 ng/mL) and HG+b+PD173074 (PD, 50 nM) stimulation by using a pull-down system in the presence of 5 µg/mL mitomycin-C in the culture medium. (B) Densitometry for Rac1-GTP was normalized to the amount of total Rac1. The results are presented as fold change as compared to fibroblasts in the absence of bFGF. Data represent mean values ±SE of three independent experiments (*P<0.05, t test).
Figure 6.
Effects of bFGF on decrease of HG-induced accumulation of intracellular ROS levels.
(A) Cells were pretreated with HG (30 mM glucose) for 72 hours and then incubated with bFGF (b, 100 ng/mL) or bFGF together with FGFR1 inhibitor PD173074 (PD, 50 nM) for an additional 1 hour. LG means 5.5 mM glucose in the culture medium. Intracellular ROS levels were measured by using DCFH-DA dye. Bar = 100 µm. (B) Fluorescence levels were measured from 10 different cells in each samples using Image Pro-plus software (n = 10, *P<0.01 t test).
Figure 7.
Modulation of protein nitration levels in HG and bFGF-fed fibroblast cells.
Protein nitration was analyzed by immunoblotting and 3-NT antibody in HG-treated cells. bFGF (b, 100 ng/mL, 60 min) supplies repressed HG-induced increase of protein nitration levels. Numbers 1–6 on the right indicate the different nitration proteins listed in Table 1. HG and LG indicate 30 mM and 5.5 mM glucose in culture medium, respectively.
Figure 8.
MS/MS spectra for tryptic digestion of Annexin A2 nitrated peptides.
The extracted un-nitrated peptides of Annexin A2 and the corresponding nitrated peptides with +45 shift are illustrated in the top of the panel of figures for each peptide. The MS/MS spectra for the nitrated Tyr238 (A) and the un-nitrated Tyr238 (B) were shown. (C) The hexagonal indicates Annexin domain and nitration was detected at Try238 which is located at the third Annexin domain (A3) of Annexin A2 protein. Nitrated tyrosin residue is marked with a star. (D) Protein homology modeling of Annexin A2 using 3-D structure 1XJL as a template, Tyr238 located at the surface of Annexin A2.
Table 1.
List of the identified nitrated peptides from different samples in SDS-PAGE with LC/MS.
Figure 9.
Effects of inhibition of FGFR1 and JNK on Annexin A2 nitration.
(A) The Annexin A2 nitration was analyzed by immunoblotting with treatment of bFGF (b, 100 ng/mL, 30 min) or FGFR1 inhibitor PD173074 (PD, 50 nM, 30 min) under LG growth conditions (5.5 mM glucose). 3-NT and Annexin A2 antibodies were used to detect the nitrated and total Annexin A2 protein, respectively. The asterisk indicates nitrated Annexin A2. (B) The p-JNK and Annexin A2 nitration levels with or without SP600125 (SP, 25 µM, 60 min) were analyzed by immunoblotting. GAPDH protein was used as a control for normalizing of loading. (C) Analysis of the migration rate of JNK-inhibited cells was performed. Data represent mean values ±SE of 10 replications (*P<0.001, t test).
Figure 10.
Effects of SP600125 and bFGF on cellular ROS accumulation.
(A) Cells were incubated with bFGF (b, 100 ng/mL), JNK inhibitor SP600125 (SP, 25 µM) or bFGF together with SP600125 for 1 hour. All experiments were performed in low glucose (5.5 mM) containing medium. Intracellular ROS levels were measured using DCFH-DA dye. Bar = 100 µm. (B) Fluorescence levels in 10 different cells from each samples shown in (A) were measured using Image Pro-plus software (n = 10, *P<0.01 t test).
Figure 11.
bFGF and SP600125 effects on rat skin wound repair.
(A, B) Skin wounds from normal rats with or without bFGF treatment. (C, D) Skin wounds from normal rats with or without JNK inhibitor SP600125 treatment. (E, F) Skin wounds from normal rats treated with or without JNK inhibitor SP600125 together with bFGF. bFGF was supplied every day. Data represent mean values ±SE of three independent experiments (**P<0.01, t test). Wound healing was monitored until 16 days after the treatment. Wound areas are measured using TINA2.0 software. N, control; N+b, control add bFGF; DM, diabetes; DM+b, diabetes add bFGF.