Oxidative Stress Modulation by Carnosine in Scaffold Free Human Dermis Spheroids Model: A Proteomic Study

Abstract

Carnosine is an endogenous β-alanyl-L-histidine dipeptide endowed with antioxidant and carbonyl scavenger properties, which is able to significantly prevent the visible signs of aging and photoaging. To investigate the mechanism of action of carnosine on human skin proteome, a 3D scaffold-free spheroid model of primary dermal fibroblasts from a 50-year-old donor was adopted in combination with quantitative proteomics for the first time. The label free proteomics approach based on high-resolution mass spectrometry, integrated with network analyses, provided a highly sensitive and selective method to describe the human dermis spheroid model during long-term culture and upon carnosine treatment. Overall, 2171 quantified proteins allowed the in-depth characterization of the 3D dermis phenotype during growth and differentiation, at 14 versus 7 days of culture. A total of 485 proteins were differentially regulated by carnosine at 7 days, an intermediate time of culture. Of the several modulated pathways, most are involved in mitochondrial functionality, such as oxidative phosphorylation, TCA cycle, extracellular matrix reorganization and apoptosis. In long-term culture, functional modules related to oxidative stress were upregulated, inducing the aging process of dermis spheroids, while carnosine treatment prevented this by the downregulation of the same functional modules. The application of quantitative proteomics, coupled to advanced and relevant in vitro scaffold free spheroids, represents a new concrete application for personalized therapies and a novel care approach.

Keywords: carnosine; dermis spheroids; high-resolution mass spectrometry; network analyses; oxidative stress.

Figure 1

(A) Immunofluorescence for collagen type III (in red) on 3D whole mount tissues. Mag. 20×. (B) The 3D rendering of Z stack maximum projection: nuclei (stained in blue) and collagen type III signal (stained in red).

Figure 2

Scatter plots of log2 ratio on x-axis against -log10 p value on y-axis of significantly quantified proteins. (A) Quantified proteins in human dermis spheroids over time, from 7 to 14 days (C14 vs. C7). (B) Quantified proteins in carnosine pre-treated dermis versus control at 7 days (PD7 vs. C7). Green color indicates downregulation (log2 ratio ≤ −0.55), red color represents upregulation (log2 ratio ≥ 0.55). Statistical parameters (p < 0.05; q < 0.05, q = FDR adjusted + value) were set to identify the differentially expressed proteins between samples.

Figure 3

(A) Subcellular distribution of identified proteins. (B) Main pathways involving extracellular matrix organization and plasma membrane proteins.

Figure 4

Functional module of matrix reorganization obtained by Reactome (a section of the entire picture) (Supplementary Figure S2). The brown color indicates all the extracellular and plasma proteins identified in analyses of controls and carnosine treated samples.

Figure 5

Functional modules related to pathways of reactive oxygen species obtained by IPA. (A) dermis spheroids at 14 days related to those at 7 days. (B) Carnosine treated spheroids at 7 days versus controls at 7 days. The orange and blue color of the central hub indicate an up and downregulation of the module, respectively, while red and green indicate the up and downregulated proteins, respectively.