Antioxidative and antiphotoaging activities of neferine upon UV-A irradiation in human dermal fibroblasts

Abstract

Our daily exposure to ultraviolet radiation (UVR) results in the production of reactive oxygen species (ROS), lipids, proteins and DNA damage and alteration in fibroblast structure, thus contributing to skin photoaging. For this reason, the use of natural bioactive compounds with antioxidant activity could be a strategic tool to overcome ultraviolet A (UV-A) induced deleterious effect. Neferine is an alkaloid extract from the seed embryos of lotus (Nelumbo nucifera Gaertn). In the present study, we report the protective effect of neferine against UV-A induced oxidative stress and photoaging in human dermal fibroblasts (HDFs). HDFs subjected to UV-A irradiation showed increased production of ROS and malondialdehyde (MDA). Furthermore, it depleted the cellular enzymatic antioxidant superoxide dismutase (SOD) and non-enzymatic antioxidant glutathione peroxidase (GPx). On the other hand, HDFs treated with neferine followed by UV-A irradiation reversed the process, reduced the ROS and lipid peroxidation and restored the antioxidants pool. Moreover, neferine treatment significantly inhibited UV-A induced matrix metalloproteinase-1 (MMP-1) expression in HDFs. Remarkable morphological and ultrastructural alterations observed in HDFs upon UV-A irradiation, were also reduced with neferine treatment. Taken together, our results suggest that neferine has strong antioxidative and photoprotective properties and thus may be a potential agent for the prevention and treatment of UV-A mediated skin photoaging.

Keywords: Antioxidant; Fibroblasts; Neferine; Oxidative stress; Photoaging; Ultraviolet A.

Figure 1. Structure of neferine

The empirical formula of neferine is C₃₈H₄₄N_2O6, with a relative molecular weight of 624.

Figure 2. Chromatogram of neferine extract from lotus seeds at 280 nm

Figure 3. Cytoprotective effect of neferine (Nef) in human dermal fibroblasts

(A) Effect of neferine on the morphology of fibroblasts. Original magnification 100x; scale bar 100 µm. (B) protective effect of neferine was analyzed using the MTT assay. Data represents the ± SD mean (n=4). The significance of difference versus the control group is *P<0.05.

Figure 4. Protective role of neferine (Nef) in human dermal fibroblasts following UV-A irradiation

Magnification 100x; scale bar 100 µm, 200x; scale bar 100 µm and 400x; scale bar 50 µm.

Figure 5. TEM showing the protective effect of neferine (Nef) in HDFs upon UV-A irradiation

Magnification 4000x; scale bar 5 µm and 20000x; scale bar 1 µm.

Figure 6. TEM showing the protective role of neferine (Nef) on mitochondria, ER, and GA in human dermal fibroblasts following UV-A irradiation

Magnification 40000x; scale bar 500 µm and 80000x; scale bar 200 µm.

Figure 7. Effect of neferine (Nef) on ROS generation in human dermal fibroblasts upon UV-A irradiation

(A) DCF-DA fluorescence is an indication of the relative intensity of ROS production in mitochondria. (B) The 499 and 525 nm were the excitation and emission wavelengths respectively. Data shown are the representation in ±SD (n=4). *P<0.05 versus control group; ^#P<0.05 versus UV-A alone group.

Figure 8. Effect of neferine (Nef) on MDA levels in human dermal fibroblast upon UV-A irradiation

Data are the representation in ±SD (n=4). *P<0.05 versus control group and ^#P<0.05 versus UV-A alone group.

Figure 9. Effect of neferine (Nef) on (A) SOD and (B) GPx activities in human dermal fibroblasts following UV-A irradiation

Data are expressed as the mean ± SD of n=4.*P<0.05 versus control group and ^#P<0.05 versus UV-A alone group.

Figure 10. Inhibitory effect of neferine (Nef) on MMP-1 expression in human dermal fibroblasts upon UV-A irradiation

(A) Western blotting analysis of MMP-1 expression in HDFs. (B) The data are expressed as the mean ± SD of n=3. *P<0.05 versus control group and ^#P<0.05 versus UV-A alone group.