Short-Term l-arginine Treatment Mitigates Early Damage of Dermal Collagen Induced by Diabetes

Abstract

Changes in the structural properties of the skin due to collagen alterations are an important factor in diabetic skin complications. Using a combination of photonic methods as an optic diagnostic tool, we investigated the structural alteration in rat dermal collagen I in diabetes, and after short-term l-arginine treatment. The multiplex approach shows that in the early phase of diabetes, collagen fibers are partially damaged, resulting in the heterogeneity of fibers, e.g., "patchy patterns" of highly ordered/disordered fibers, while l-arginine treatment counteracts to some extent the conformational changes in collagen-induced by diabetes and mitigates the damage. Raman spectroscopy shows intense collagen conformational changes via amides I and II in diabetes, suggesting that diabetes-induced structural changes in collagen originate predominantly from individual collagen molecules rather than supramolecular structures. There is a clear increase in the amounts of newly synthesized proline and hydroxyproline after treatment with l-arginine, reflecting the changed collagen content. This suggests that it might be useful for treating and stopping collagen damage early on in diabetic skin. Our results demonstrate that l-arginine attenuates the early collagen I alteration caused by diabetes and that it could be used to treat and prevent collagen damage in diabetic skin at a very early stage.

Keywords: AFM; Raman spectromicroscopy; dermal collagen I; diabetes; l-arginine; polarization-resolved SHG imaging.

Figure 1

pSHG images of dermal collagen (a) and calculated β values (b) of control (non-diabetic), l-arginine (l-arginine-treated non-diabetic), diabetic, and diabetic l-arginine-treated rats. Different colors in panel (a) indicate the different values of the β-coefficient (as indicated by the color bar in the leftmost panel). Thus, greener images indicate a higher value of the β-coefficient and, therefore, more arranged collagen. The opposite is true for red images. The values represent the mean ± SEM, for each point n = 27, * p < 0.05. Scale bars: 50 µm.

Figure 2

Mean Raman spectra of dermal collagen I structure of control (non-diabetic), l-arginine (l-arginine-treated non-diabetic), diabetic, and diabetic l-arginine-treated rats. The light gray highlighted areas are magnified and present the most intensive changes described in Table S1.

Figure 3

Raman spectra of dermal collagen I of control (non-diabetic), l-arginine (l-arginine−treated non-diabetic), diabetic, and diabetic l-arginine-treated rats.

Figure 4

Young’s modulus measured by AFM in control (non-diabetic), l-arginine (l-arginine-treated non-diabetic), diabetic, and diabetic l-arginine-treated rats. The values represent the mean ± SEM, * p < 0.05.

Figure 5

Micrographs of the rat skin of control (non-diabetic), l-arginine (l-arginine-treated non-diabetic), diabetic, and diabetic l-arginine-treated rats stained with picrosirius red and analyzed without polarizers (left images) and with linear polarizers (right images). Magnification: 10×; original scale bars: 250 µm.