112 SKIN PROTECTION
ROS production on 45 J/cm2 a
Control
Blue light control
10 25 50
* * *
0 100 200 300 400 500 600 700 800 900 1000 Relative fluorescence units
* LED-BL exposure
ROS production on 5 J/cm2 b
Control
Blue light control
10 25 50
100 0 200 * * * * 400 600 800 Relative fluorescence units
Figure 4: LED-BL significantly increased ROS in keratinocytes (a) and fibroblasts (b) as measured by 2’,7’dichlorodihydrofluorescein diacetate (H2- DCFDA). Pre-treatment with olive fruit extract significantly reduced LED-BL- associated ROS generation. Error bars represent ± SEM. *p< 0.05 vs untreated control.
LED-BL- 45 J/cm2 a
Control
Blue light control
10 25 50
0 0.2 0.4 * * * 0.6 A.D.U. 0.8 1 1.2 (1.5 hours) 8-OHdG PCNA Control
Blue light control
*
10 25 50
100 0 0.2 * * 0.4 0.6 A.D.U.
Figure 5: Relative protein expression (mean ± SEM) of 8-OHdG and PCNA in keratinocytes (a) and fibroblasts (b) after LED-BL exposure and treatment with olive fruit extract. Data were calculated as arbitrary densitometric units (ADU) collected from three independent experiments and normalised vs. α-tubulin. *p< 0.05 vs. untreated control.
by measuring the oxidation of H2-DCFDA (2’,7’-dichlorodihydrofluorescin diacetate). H2- DCFDA diffuses through the cell membrane and is enzymatically hydrolysed by intracellular esterase to the non-fluorescent H2-DCF. Intracellular ROS are able to oxidise H2-DCF to the fluorescent 2′-7′-dichlorofluorescein (DCF), whose intensity of fluorescence is directly proportional to the levels of intracellular ROS. Fluorescence was measured with a
spectrofluorometer at excitation/emission wavelengths of 480 nm-535 nm and the signals were compared to the untreated control.
Results
LED-BL exposed keratinocytes and fibroblasts exhibited increased fluorescence compared with non-exposed control cells, with a ROS production more than four and three times higher, respectively, than control (Fig 4a, b).
LED-BL exposed keratinocytes, pre-
treated with 10, 25 and 50 μg/ml of olive fruit extract, showed a minor level of ROS with a reduction from 25 to 50% (comparison with the blue light control) (Fig 4a). Decrease of ROS expression in
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fibroblasts, pre-treated with 10, 25, 50 and 100 μg/ml of olive fruit extract, ranged from 58,5 to 66,36% (Fig 4b). These results clearly showed the
remarkable radical scavenging property of olive fruit extract, and its capability to protect skin cells from blue light-induced oxidative stress.
Effect of olive fruit extract on blue light-induced DNA damage
8-OHdG and PCNA evaluation by Western Blot
Design
8-OHdG and PCNA were quantified through specific antibodies, which were detected using a chemiluminescent system. The signal intensity of the bands was calculated on the basis of the density of the α-tubulin and the values were expressed as arbitrary densitometric unit (ADU).
Results
Cellular DNA damage, induced by LED-BL, was highlighted by Western Blot with an increased expression of PCNA and ex novo
synthesis of 8-OHdG in both cell lines (Fig 5a, b). Western Blot analysis showed that, in keratinocytes, pre-treated with olive fruit extract, the expression of PCNA decreases in a concentration-dependent manner, whereas 8-OHdG disappears (Fig 5a). A comparable behaviour was highlighted in fibroblasts, where the level of PCNA decreases and the expression of 8- OHdG was completely suppressed (Fig 5b).
8-OHdG and PCNA evaluation by immunocytochemistry Design
The markers of DNA damage were also evaluated with a qualitative technique. In order to visualise the expression of 8-OHdG and PCNA, keratinocytes and fibroblasts were stained with antibodies against PCNA and 8-OHdG, green and red respectively (Figs 6a, b; Fig 7a, b). The nuclei of cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI), a blue colorant. Digital images were acquired using Leica fluorescence microscope connected to a digital camera. Magnification ×40.
April 2018 0.8 1 1.2 * *
LED-BL- 5 J/cm2 b
(15 mins) 8-OHdG PCNA 1000 1200 * LED-BL exposure
*
Olea-HT 10
(µg/mL)
Olea-HT 10
(µg/mL)
Olea-HT 10
(µg/mL)
Olea-HT 10
(µg/mL)
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