The role of synthetic and naturally derived isothiocyanates in experimental models of skin photo-aging and psoriasis

Lolou, Vasiliki (2021) The role of synthetic and naturally derived isothiocyanates in experimental models of skin photo-aging and psoriasis. Doctoral thesis, Northumbria University.

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Abstract

Isothiocyanates, phytochemicals found in cruciferous vegetables, are known for their health benefits and their antioxidant and anti-inflammatory effect. They are known to act indirectly as antioxidant, through the induction of Nrf2 and phase II enzymes, while they are shown to reduce cytokine production, leading to reduced inflammation. Isothiocyanates are studied on their effects on skin disease, such as dermatitis and photo-aging, and are shown to reduce inflammation and reduce UV damage, respectively. This project aims to provide further insight of the effect of isothiocyanates on photo-aging and psoriasis through models of photo aged HaCaT cells and a novel therapeutic approach using UVB, respectively.

Initially and for the determination of the total isothiocyanates in watercress, a determination method was developed using the cyclocondensation assay. Measurements were taken using the spectrophotometer, while validation of the method was conducted using a hexane extraction method from the literature. The standard curves of five ITCs studied in this work (SFN, IBN, AITC, BITC and PEITC) gave a response factor from 0.028 to 0.035, indicating limited variation in the reaction of ITCs, while the limit of detection (LOD) was 1-60 μg / mL or 1-400 μM or 1-60 mg / g dry matter. Compared to the literature (Y. Zhang et al., 1992), the maximum LOD presented in the current work is significantly higher with an actual confidence level of 98.4%.

During the determination of total ITC content in watercress matrix component interference was examined, revealing a macromolecule interference that was successfully resolved by the addition of a clarification step that includes addition of methanol and centrifugation, improving the reliability of the method by reducing the coefficient of variation from 23.6% to 14% (n = 6). No fibre or metal interference is shown, while plastic is shown to interfere with the quantification and glassware increased recovery of ITCs by 35% giving a 7-fold decrease in the corresponding coefficient of variation from 14% to 2%. The developed methodology, with an 88.95% recovery, detected 1.54 ± 0.038 μmol / g of total ITCs content in freeze-dried watercress powder, while significantly higher quantities of 20.6 ± 0.31 μmol / g were detected in fresh watercress.

Photo-aging is characterised from physiological changes of the skin and overall increased wrinkles that gradually appear due to sustainably prolonged UVR exposure of the skin. sulforaphane, one of the most studied isothiocyanates, is known to increase viability in photo-aged cells and reduce the effect of UV oxidation in human immortal keratinocyte cells. In this project both phytochemicals had a photo-protective effect on the UVB-induced photo-aging models of 25 and 50 mJ / cm2 with significantly increased cell viability at 72 hours by at least 10% compared to controls. For the role of ITCs and UVB on psoriasis HaCaT cells were treated with low doses of UVB and post-treated with either of the five isothiocyanates; sulforaphane, Iberin, allyl isothiocyanate, benzyl isothiocyanate and phenethyl isothiocyanate. All but allyl isothiocyanate were shown to further reduce viability and growth, when combined with 5 and 10 mJ / cm2 UVB, while all phytochemicals mentioned above were shown to induce apoptosis, increase mitochondrial membrane permeability and affect the cell cycle. ROS induction from the combinational treatments was also studied and it was shown that the viability of the cells increased, reversing the effect of the treatment and indicating the main mechanism of the combination therapy. All ITCs but AITC (20 μM) studied showed a significant reduction of viability when combined with 5 mJ / cm2 UVB at 48h or 10 mJ / cm2 UVB at 24h, while BITC was shown to be the least potent. HaCaT cell growth was also shown to reduce significantly after their treatment with all ITC combinations but AITC. All ITCs studied showed an increase of apoptotic cells through the evaluation of caspase 3/7 activity, while AITC only showed an increase of apoptotic cells after the treatment with 10 mJ / cm2 UVB and not with 5 mJ / cm2 UVB. Moreover, treatments with combinations of UVB and PEITC, AITC, IBN and SFN showed necrotic cells significantly high indicating damage to membranes, while PEITC was shown to be the most potent. AITC reduced necrotic cells significantly compared to 10 mJ / cm2 UVB but not with the AITC alone, while BITC combination did not show any increase to necrotic cells compared to the controls. ΔΨm was shown to decrease in all the combinational treatments indicating intracellular ROS induction and mitochondrial dysfunction. SFN and PEITC were the most potent on reducing ΔΨm. ROS induction as the main mechanism of action of the combinational treatment was shown to reverse after the addition of GSH strengthening the hypothesis of reduced viability being mediated by the induction of intracellular ROS. SFN, IBN and BITC were shown to arrest G2/M phase at 24h / 10 mJ / cm2 UVB, when compared to the individual controls of UVB or ITC alone, while PEITC significantly arrested G2/M phase only when compared to UVB control. 48h / 5 mJ / cm2 UVB only showed a significant G2/M phase arrest after treatments with IBN and AITC when compared to their corresponding ITC controls. SubG1 phase was arrested by SFN, IBN and BITC combinational treatments at 24h, while similar effect was shown by SFN, IBN, AITC at 48h. No effect on S phase was shown from either ITC, while SFN and PEITC arrested G1 phase at 24h, when compared to SFN alone control and UVB control, respectively. The treatment of 10 mJ / cm2 UVB in combination with 20 μM of SFN, IBN, BITC or PEITC were the most effective of the two treatments overall, with the most significant effect on cell viability, apoptosis, ΔΨm, cell cycle and ROS induction.

When watercress was used for the treatment of HaCaT cells post-UVB exposure in the optimised combinational treatment it was shown to have a similar effect on cell growth (SRB123 assay), apoptosis (caspase 3/7), mitochondrial membrane permeability (JC-1), cell cycle (PI) and ROS induction (DHR assay and addition of antioxidant glutathione). 20 μM watercress extract expressed in PEITC significantly (p<0.0001) reduced cell growth, while its combination with 10 mJ / cm2 UVB further (p<0.001) enhanced the effect. The comparison of the combinational treatments using watercress extract or synthetic PEITC, reveals a similar effect of the two, on caspase 3/7 activation, increasing apoptotic cells significantly compared to the controls of UVB alone and ITC alone. Significant reduction of the ratio of JC-1 aggregates over JC-1 monomers (ΔΨm < 6) on HaCaT cells from watercress extract and synthetic PEITC is shown, while a further enhanced effect (ΔΨm < 2) is shown when watercress or PEITC are combined with UVB.

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