Stimulation of the Fibrillar Collagen and Heat Shock Proteins by Nicotinamide or Its Derivatives in Non-irradiated or Uva Radiated Fibroblasts, and Direct Anti-oxidant Activity of Nicotinamide Derivatives

In skin aging, from intrinsic factors or exposure to ultraviolet (UV) radiation, there is loss of structural fibrillar collagen and regulatory heat shock proteins. Phenolic compounds, with hydroxyl groups attached to an aromatic ring, have antioxidative and anti-inflammatory properties. Nicotinamide is an amide derivative of niacin or vitamin B3, with an amide linked to an aromatic ring, with UV absorptive, antioxidant, anti-inflammatory and anti-cell death/apoptosis properties. The goal of this research was to investigate the anti-skin aging mechanism of nicotinamide and its derivatives, 2,6-dihydroxynicotinamide, 2,4,5,6-tetrahydroxynicotinamide, and 3-hydroxypicolinamide (collectively niacin derivatives), through the stimulation of fibrillar collagens (type I, III and V, at protein and/or promoter levels) and the expression of heat shock proteins (HSP)-27, 47, 70, and 90 in non-irradiated or UVA radiated dermal fibroblasts; and from its direct antioxidant activity. UVA radiation inhibited the expression of types I and III collagen, and HSP-47 in dermal fibroblasts. The niacin derivatives significantly and similarly stimulated the expression of types I (transcriptionally), III and V collagens in non-irradiated, and UVA radiated fibroblasts indicating predominant effects. The 2,6-dihydroxynicotinamide had greater stimulatory effect on types I and III collagen in the non-irradiated, and UVA 147 radiated fibroblasts, as well as greater direct antioxidant activity than the other niacin derivatives. The niacin derivatives, with a few exceptions, stimulated the expression of HSP-27, 47, 70 and 90 in non-irradiated, and UVA radiated fibroblasts. However, they had varied effects on the expression of the different HSPs in non-irradiated, and UVA radiated fibroblasts indicating non-predominant, albeit stimulatory, effect. Overall, nicotinamide and its derivatives have anti skin aging potential through the stimulation of fibrillar collagen and HSPs.

The chaperone proteins that facilitate the formation of the ECM and the prevention of molecular oxidative damage are the heat shock proteins (HSP).The HSPs facilitate proper protein folding and prevent their degradation.HSP-27 increases cellular antioxidant score by increasing cellular reduced glutathione, and reducing oxidized proteins [10].HSP-70, which is reduced with cellular aging, provides anti-inflammatory and skin protective properties [11,12].HSP-47, a collagen specific chaperone protein, is co-stimulated with fibrillar collagen by ECM strengthening agents, such as copper, in dermal fibroblasts [8].HSP-90 facilitates the migration of dermal fibroblasts, and the maturation of the ECM, imperative to wound healing [13].
The goal of this research was to determine the beneficial regulation of the fibrillar collagen (types I, III, V) and HSPs (27,47,70,90) by nicotinamide, and three of its derivatives, 2,6-dihydroxynicotinamide, 2,4,5,6-tetrahydroxynicotinamide, and 3-hydroxypicolinamide (collectively niacin derivatives) in non-irradiated or UVA-radiated dermal fibroblasts; as well as the direct antioxidant activity of the nicotinamide derivatives.We have reported that nicotinamide exhibits direct antioxidant activity in millimolar concentrations, up to 50% of control [6].The hypothesis of this research was that these niacin derivatives would stimulate fibrillar collagen and HSPs, and exhibit direct antioxidant property, with the hydroxyl derivatives providing greater effectiveness.The niacin derivatives significantly stimulated the expression of type I collagen at 0.1% and 1% (except 3-hydroxypicolinamide at 0.1%), type I collagen promoter activity at 0.01%, 0.1% and 1%, and types III and V collagen at 1% in non-irradiated fibroblasts, in comparison with control (p < 0.05) (Figure 1).In addition, 2,6-dihydroxynicotinamide significantly stimulated expression of type III collagen at 0.1% and type V collagen at 0.01%, and 0.1% (p < 0.05) (Figure 1c,d).The niacin derivatives had similar activities, except for the significantly greater stimulation of types I and III collagen by 2,6-dihydroxynicotinamide.
UV radiation inhibited the expression of HSP-47 in dermal fibroblasts.The niacin derivatives had varied effects on the expression of the different HSPs in non-irradiated and UVA radiated fibroblasts.The HSP-27 was stimulated by 2,6-dihydroxynicotinamide and 3-hydroxypicolinamide at 0.01%, 0.1% and 1% in non-irradiated cells, but only at 1% in the UVA-irradiated cells.While nicotinamide did not alter HSP-27 in non-irradiated fibroblasts, it stimulated HSP-27 at 0.01%, 0.1% and 1% in UVA-irradiated fibroblasts.The HSP-47 was stimulated by 1% 2,4,5,6-tetra hydroxynicotinamide and 3-hydroxypicolinamide in non-irradiated fibroblasts, and by 1% of all the niacin derivatives in UVA radiated fibroblasts.The HSP-70 was stimulated by all the niacin derivatives at 1% in non-irradiated fibroblasts, but also at 0.01% in UVA radiated fibroblasts.Nicotinamide and 2,6-dihydroxynicotinamide were stimulatory to the expression of HSP-90 in non-irradiated and UVA radiated fibroblasts, however the effect of nicotinamide was less stimulatory in the UV radiated cells.It is inferred that the effects of the niacin derivatives on the expression of HSPs do not predominant in the UVA exposed fibroblasts.

Type I Collagen Promoter Activity
Fibroblasts were co-transfected with COL1α1 promoter-firefly luciferase plasmid (pGL4 vector) (gift from Dr. Joel Rosenbloom, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA) and thymidine kinase (TK) promoter-hRenilla luciferase plasmid (Promega) (for normalization of transfection efficiency) using Escort (Sigma) for 24 h, prior to dosing with or without UVA-radiation +/− niacin derivatives for 24 h.The cells were measured for luminescence from firefly, and renilla luciferase activities with specific substrates and quantitated using recombinant luciferase as standard (Promega).

Antioxidant Activity
The direct antioxidant activity of nicotinamide derivatives was determined by incubating them with ABTS ® (2,2'-azino-di-(3-ethylbenzthiazoline sulphonate)) and metmyoglobin, and determining the inhibition of the oxidation of ABTS ® to ABTS ® radical by metmyoglobin by the nicotinamide derivatives, spectrophotometrically at 405 nm (Cayman Chemical Antioxidant Assay kit, Ann Arbor, MI, USA).

Data Analysis
The significant effects of UVA or niacin derivatives were analyzed relative to respective controls (without niacin derivatives) by ANOVA and student t-tests at 95% confidence interval.The effects of UVA radiation on dermal fibroblasts were statistically analyzed relative to non-irradiated control cells.The significant effects of the niacin derivatives on non-irradiated cells were analyzed relative to non-irradiated control cells (0% niacin derivative).The significant effects of each of the niacin derivatives on UVA-radiated fibroblasts were analyzed relative to UVA radiated control cells (0% niacin derivative).

Conclusion
Ultraviolet (UV) radiation, more so UVA, damages the dermal collagen fibers and regulatory heat shock proteins.The effects of UVA radiation are primarily through the induction of oxidative stress and inflammation.Phenolic compounds exhibit antioxidant and anti-inflammatory properties, which are dependent on the number and location of hydroxyl groups.This research examined the potential of niacin derivatives, nicotinamide, 2,6-dihydroxynicotinamide, 2,4,5,6-tetrahydroxynicotinamide, and 3-hydroxypicolinamide, to beneficially regulate types I, III and V collagen, and heat shock proteins 27, 47, 70 and 90 in non-irradiated or UVA-radiated dermal fibroblasts; as well as exhibit direct antioxidant activity.The niacin derivatives, more so 2,6-dihydroxynicotinamide, stimulated collagen fibers and differentially up-regulated heat shock proteins in non-irradiated, and UVA radiated fibroblasts, and exhibited direct antioxidant activity.It is inferred that the niacin derivatives have photoprotective and anti-skin aging potential in cosmetics.