A quantitative proteomic approach to evaluate the efficacy of carnosine in a murine model of chronic obstructive pulmonary disease (COPD)

Abstract

The aim of the work was to study a dose-dependent effect of inhaled carnosine (10, 50 or 100 mg/kg/day) in mice exposed to cigarette smoke as a model of chronic obstructive pulmonary disease (COPD). A dose-dependent loading of the dipeptide in lung tissue and bronchoalveolar lavage (BAL) was firstly demonstrated by LC-ESI-MS analysis. Cigarette smoke exposure induced a significant lung inflammation and oxidative stress in mice which was dose-dependently reduced by carnosine. Inflammation was firstly evaluated by measuring the cytokines content in the BAL. All the measured cytokines were found significantly higher in the smoke group in respect to control, although the data are affected by a significant variability. Carnosine was found effective only at the highest dose tested and significantly only for keratinocyte-derived cytokine (KC). Due to the high variability of cytokines, a quantitative proteomic approach to better understand the functional effect of carnosine and its molecular mechanisms was used. Proteomic data clearly indicate that smoke exposure had a great impact on lung tissue with 692 proteins differentially expressed above a threshold of 1.5-fold. Protein network analysis identified the activation of some pathways characteristic of COPD, including inflammatory response, fibrosis, induction of immune system by infiltration and migration of leukocyte pathways, altered pathway of calcium metabolism and oxidative stress. Carnosine at the tested dose of 100 mg/kg was found effective in reverting all the pathways evoked by smoke. Only a partial reverse of the dysregulated proteins was evident at low- and mid-tested doses, although, for some specific proteins, indicating an overall dose-dependent effect. Regarding the molecular mechanisms involved, we found that carnosine upregulated some key enzymes related to Nrf2 activation and in particular glutathione peroxidase, reductase, transferase, SOD, thioredoxins, and carbonyl reductase. Such mechanism would explain the antioxidant and anti-inflammatory effects of the dipeptide.

Keywords: Carnosine; Chronic obstructive pulmonary disease (COPD); Cigarette smoke; Inflammation; Nrf2; Oxidative stress; Quantitative proteomic.

Fig. 1

Carnosine content in plasma (A), BAL (B) and lung tissue homogenate (C) in control mice and after airway administration of carnosine at three doses: 10, 50 and 100 mg/kg. Statistical differences among the groups were tested by one-way ANOVA with Tukey’s multiple comparisons test.

Fig. 2

Fluorescent proteins in plasma (A) and lung tissue homogenate (B). Statistical differences among the groups were tested by one-way ANOVA with Tukey’s multiple comparisons test.

Fig. 3

Effects of carnosine airway administration on BAL cytokines in smoke exposed mice. Statistical differences among the groups were tested by one-way ANOVA with Tukey’s multiple comparisons test.

Fig. 4

Volcano plots of quantified proteins in 10 mg/kg carnosine smoker mice related to smoker mice (A), in 50 mg/kg carnosine smoker mice related to smoker mice (B), in 100 mg/kg carnosine smoker mice related to smoker mice (C) in smoker lung versus control mice (D).

Fig. 5

Pathways regulated in smoker lung versus control mice (S/C), in 50 mg/kg carnosine smoker mice related to smoker mice and in 100 mg/kg carnosine smoker mice related to smoker mice. In y axis z-scores are reported.

Fig. 6

Pathways involved in “inflammatory response” significantly altered in smoker lung versus control mice (A), in 100 mg/kg carnosine smoker mice related to smoker mice (B). In red the increased genes, in green those decreased. The color intensity is positive related to the up- or down-gene’s regulation; orange line leads to activation, yellow lines for findings inconsistent with state of downstream molecule; grey line for effect not predicted. The central functional module is orange if the function is activated and blue if it is deactivated. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Pathways involved in “fibrosis” significantly altered in smoker lung versus control mice (A), in 100 mg/kg carnosine smoker mice related to smoker mice (B).

Fig. 8

Pathways involved in “synthesis of reactive oxygen species” significantly altered in smoker lung versus control mice (A), in 100 mg/kg carnosine smoker mice related to smoker mice (B).

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