Hyaluronic acid is able to start specific signaling events due to CD44 and RHAMM (receptor of HA-mediated motility) [2]. Reduced expression of CD44 in the skin is one of the discussed mechanisms of skin fragility in aging and dermatoporosis [3].

Some studies have shown that hormone replacement therapy could increase skin thickness in postmenopausal women [4-7]. Oral hydrolyzed collagen (HC) might increase skin hydration and elasticity, collagen content and density of the dermis [8-13]. Some topical treatments, such as glycolic and retinoic acid, are useful to improve skin atrophy and fragility [14]. Topical HC formulations are yet to be clinically studied.

The aim of this study was to evaluate whether the use of oral and/or topical hydrolyzed collagen could alter gene expression of type 1 collagen, elastin, and CD44 in postmenopausal women with signs of skin aging on the forearms.

METHODS

Study Population: Postmenopausal women, aged over 60 years, with mild signs of aging of the forearms were eligible. Hormone replacement therapy, chemotherapy, immunosuppression, active infectious or inflammatory lesions in the forearms, the presence of photodermatosis and recent (3 months) oral or topical treatments or procedures for skin aging of the forearms were exclusion criteria. The study was conducted in the outpatient clinic of a public hospital in São Paulo, Brazil, from June 2016 to April 2018. All patients signed an informed consent form.

Randomization and Blinding: double-blind, randomized, placebo-controlled study. The allocation was concealed from the investigator and participants through numbered packs with the treatments inside.

Intervention: Patients were oriented to avoid sun exposure and use physical photo protection during the study and were randomized according to oral and topical treatments, through a computer software. Oral treatment was either oral placebo (Po, maltodextrin) or hydrolyzed collagen (HCo, collagen peptides, 5g) once a day for 24 weeks. Topical treatment was either topical placebo (Pt, vehicle serum) or hydrolyzed collagen (HCt, 2.5% collagen peptides serum), three pumps at night on each forearm for 24 weeks. The topical HC formulation was developed by UNIFESP’s Institute of Environmental, Chemical, and Pharmaceutical Sciences and used the same collagen peptides as the oral version in a serum containing polyacrylamide & C13-14 isoparaffin & laureth-7 (3.5%), vegetable glycerin (5%), isononyl isonanoate (3%), phenoxyethanol, and parabens (0.5%).  The oral HC was a combination of different and specific collagen peptides of type I collagen, with a molecular weight of 2kDa, from bovine and porcine origin.

For RNA extraction and qPCR analysis for col1A, CD44, and elastin, a 2-mm skin biopsy of the dorsal surface of each forearm was performed (7 cm from the antecubital fold on the midline).

For the total RNA extraction of the skin tissues, a RN easy mini kit reagent (QIAGEN®, Hilden, Germany) was used. A spectrophotometer (NanoDrop® Lite, Thermo Fisher Scientific®, Waltham, MA, USA) was used to quantify the RNA in each sample by determining the absorbance ratio at 260 and 280 nm. A 0.5-μg piece from the total extracted RNA of each sample was used for the synthesis of cDNA using the Superscript III reverse transcriptase kit with random hexamer primers and thermocycler Veriti (Thermo Fisher Scientific®, Waltham, MA, USA), following the manufacturer’s instructions. The cDNA was frozen at −20°C until tested. The qPCR assay reaction was performed using the Evagreen (Solis Biodyne) on the thermocycler apparatus Quantstudio 5 (ABI Applied Biosystems®, Foster City, CA, USA), following the manufacturer’s instructions. The Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, NM_002046 - Efficiency curve 95.848%) was used as an endogenous control for each sample. The following genes were analyzed: CD44 (NM_000610 - Efficiency curve 97.98 %), COL1A1 (NM_000088 - Efficiency curve 90.027%), and ELN (NM_001278939 - Efficiency curve 91.693%). Reactions were made in triplicate for each gene, including the endogenous control. The ∆CT values were obtained by the difference between the CT values of the target genes and the GAPDH gene. The 2^−∆LN method [15] was used to calculate the gene expression for each target gene.

The change in the expression of Col1A, CD44, and elastin from pretreatment to after six months (pre/post) was expressed as the fold change (CI 95%) through bootstrap estimation (5,000 resamplings).

RESULTS

Fourteen women with mild signs of skin aging aged 60-80 years (mean 66.4 ± 5.5years) were included. Among these, qPCR extraction was successful in 17 of the 28 forearms. Eight patients used Po, and six used CHo. Regarding topical interventions in each forearm, nine patients used Pt, and eight used CHt.

The results of gene expression profile are described in Table 1.

DISCUSSION

Oral or topical collagen did not increase gene expression of relevant dermal components after six months of intervention.

This study corroborates the findings of a randomized, double-blind, factorial study to evaluate efficacy and safety of topical and/or oral hydrolyzed collagen in women with stage 1 dermatoporosis in which no significant difference was observed for each intervention, nor their comparison, for all efficacy parameters: clinical and quality of life scores, dermal elasticity, thickness and echogenicity, and histologic and immunohistochemical markers [16].

This was a small study in a single center in Brazil.

CONCLUSION

Oral and/or topical collagen peptides have no benefits regarding gene expression of type 1 collagen, CD44, and elastin on forearm skin with mild signs of aging after six months of intervention in postmenopausal women.

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  Table 1