Figure 1.
Construction and purification of PEP-1-PON1 protein.
A representative diagram of the constructed PEP-1-PON1 protein (A). PEP-1-PON1 protein contains a His tag consisting of six histidine residues. Expressed and purified fusion proteins were analyzed by 12% SDS-PAGE (B) and subjected to Western blot analysis with an anti-rabbit polyhistidine antibody (C).
Figure 2.
Transduction of PEP-1-PON1 proteins into Raw 264.7 cells.
Cells were cultured in a 60-1-PON1 (0.1–0.3 µM) and control PON1 proteins were added to the culture media for 1 h (A), PEP-1-PON1 (0.3 µM) and control PON1 proteins were added to the culture media for 10–60 min (B), and analyzed by Western blot analysis. Transduction of PEP-1-PON1 stability was assessed after various time periods (1–60 h). Cells pretreated with 0.3 µM PEP-1-PON1 for 1 h and analyzed by Western blot analysis (C). The intracellular distribution of the transduced PEP-1-PON1 was observed by confocal microscopy (D). Scale bar = 20 µm.
Figure 3.
Inhibitory effect of PEP-1-PON1 protein on LPS-induced inflammatory response in Raw 264.7 cells.
Raw 264.7 cells were stimulated with LPS with or without pretreatment with PEP-1-PON1 protein for 1 h. Cell lysates (A: 10 ng/ml LPS for 24 h, B: 1 µg/ml LPS for 12 h) were prepared and analyzed for COX-2 and iNOS protein expression by Western blotting and band intensity by densitometer (a). After total RNA was extracted from the cells (10 ng/ml and 1 µg/ml LPS for 24 h), cytokines (IL-1β, L-6, TNF-α) and β-actin mRNA were analyzed by RT-PCR using specific primers and band intensity by densitometer (b). *P<0.01, compared with LPS treated cells.
Figure 4.
Inhibitory effect of PEP-1-PON1 on LPS-induced MAPK and NF-kB activations.
Raw 264.7 cells were stimulated with 1 µg/ml LPS for 15 min with or without pretreatment with PEP-1-PON1 protein for 1 h. Cells extract prepared and analyzed for MAPK protein activation by Western blotting and band intensity by densitometer (A). Phosphorylation and the degradation of p65 and IkBα were analyzed by Western blotting and band intensity by densitometer (B). ‘p’ indicates the phosphorylated form of the protein. *P<0.01, compared with LPS treated cells.
Figure 5.
Effect of PEP-1-PON1 on LPS- or H2O2-induced ROS production.
After the cells were treated with PEP-1-PON1 protein for 1 h, cells were stimulated with LPS 10 ng/ml for 50 min and 1 µg/ml for 30 min or H2O2 1 mM for 20 min and 2 mM for 10 min. Intracellular ROS levels were measured after staining with DCF-DA and the fluorescent intensity was measured by an ELISA plate reader. Scale bar = 25 µm. *P<0.01, compared with only LPS or H2O2 treated cells.
Figure 6.
Effect of transduction of PEP-1-PON1 proteins against H2O2-induced cell viability and DNA fragmentation.
H2O2 (1 mM and 1.5 mM, 16 h) was added to Raw 264.7 cells pretreated with PEP-1-PON1 (0.3 µM) for 1 h. Cell viabilities were estimated by with a colorimetric assay using MTT (A). **P<0.01, compared with H2O2 treated cells. Cells were treated with PEP-1-PON1 protein (0.3 µM) for 1 h, and then exposed to H2O2 (1 mM for 15 h and 5 mM for 4 h). DNA fragmentation was detected by TUNEL staining (B). Scale bar = 50 µm.
Figure 7.
Effect of transduced PEP-1-PON1 protein against H2O2-induced activation of caspase-3 and mitochondrial membrane potential.
Cells were treated with PEP-1-PON1 protein (0.1–0.3 µM) for 1 h, and then exposed to H2O2. Caspase-3 and cleaved caspase-3 was detected by Western blotting and band intensity by densitometer (A). Akt, p53, phosphorylation Akt and p53 were protein expression levels were measured by Western blotting and band intensity by densitometer (B). *P<0.01, compared with H2O2 treated cells. ‘p’ indicates the phosphorylated form of the protein. Mitochondrial membrane potential was detected using a mitochondrial membrane potential assay kit (C). Scale bar = 50 µm.
Figure 8.
Effect of transduced PEP-1-PON1 protein on TPA-induced ear edema.
Ears of mice were treated with TPA (1 µg/ear) and PEP-1-PON1 protein and controls (PON1 protein and PEP-1 peptide) was topically applied to mice ears 1 h after TPA treatment. The inhibition of TPA-induced ear edema was analyzed by hematoxylin and eosin immunostaining (A) and measuring changes in ear thickness (B) as well as weight of 5 mm ear biopsy (C). Scale bar = 50 µm for A (top panel), except for 25 µm in high magnifications in A (bottom panel). **P<0.01, compared with TPA treated mice.
Figure 9.
Inhibitory effect of PEP-1-PON1 protein against TPA-induced COX-2 and cytokine expression in edema model.
Mice were stimulated with TPA (1 µg/ear) and PEP-1-PON1 protein topically applied to mice ear. Mice ear extracts were prepared and analyzed for COX-2 protein (A) and COX-2 mRNA (B) expression by Western blotting and RT-PCR using specific primers. The band intensity was measured by densitometer. Total RNA was extracted from ear biopsies. TNF-α, IL-1β, IL-6, and GAPDH mRNA were analyzed by RT-PCR using specific primers and band intensity by densitometer (C). *P<0.01, compared with TPA treated mice.
Figure 10.
Inhibitory effect of transduced PEP-1-PON1 protein against TPA-induced MAPK and NF-kB activation in edema model.
Ears of mice were treated with TPA (1 µg/ear) and PEP-1-PON1 protein was topically applied to mice ears 1 h after TPA treatment. Ear biopsies were prepared and analyzed for MAPK protein activation by Western blotting and band intensity by densitometer (A). The degradation and phosphorylation of p65 and IkBα was analyzed by Western blotting and band intensity by densitometer (B). ‘p’ indicates the phosphorylated form of the protein. *P<0.01, compared with TPA treated mice.