Anti-Inflammatory and Proliferative Properties of Luteolin-7-O-Glucoside

Abstract

Flavonoids display a broad range of structures and are responsible for the major organoleptic characteristics of plant-derived foods and beverages. Recent data showed their activity, and in particular of luteolin-7-O-glucoside (LUT-7G), in reduction of oxidative stress and inflammatory mechanisms in different physiological systems. In this paper, we tried to elucidate how LUT-7G could exert both antioxidant and anti-inflammatory effects in endothelial cells cultured in vitro. Here, we showed that LUT-7G is able to inhibit the STAT3 pathway, to have an antiproliferative action, and an important antioxidant property in HUVEC cells. These properties are exerted by the flavone in endothelial through the transcriptional repression of a number of inflammatory cytokines and their receptors, and by the inhibition of ROS generation. ROS and STAT3 activation has been correlated with the production of oxysterols and other hydroxylated fatty acids, and they have been recognized important as players of atherogenesis and cardiocirculatory system diseases. The analysis of the general production pathway of these hydroxylated species, showed a strong decrease of cholesterol hydroxylated species such as 7-alpha-hydroxicholesterol, 7-beta-hydroxicholesterol by the treatment with LUT-7G. This confirms the anti-inflammatory properties of LUT-7G also in the endothelial district, showing for the first time the molecular pathway that verify previous postulated cardiovascular benefits of this flavone.

Keywords: STAT3; flavonoids; hydroxylation; inflammation; luteolin-7-O-glucoside.

Figure 1

The picture shows the derivatives of phenylbenzo-pyrone representing the basic structure of a wide class of molecules.

Figure 2

LUT-7G treatment of endothelial cells. (A) WB showing the regulation of STAT3 protein before (left lanes) and after LUT-7G treatment (right lanes, two independent experiments). (B,C) Immunofluorescence analysis showing STAT3 in the nucleus (active STAT3) in untreated cells and STAT3 nuclear depletion after 48 h of LUT-7G treatment (bars = 15 μm). (D,E) Cell Growth and Cell cycle analysis showing proliferation reduction and G1 enrichment in treated samples. (F) Scratch assay in untreated cells, (G) same assay from 4 to 20 h (bars = 60 μm).

Figure 3

LUT-7G treatment in endothelial cells. (A) immunofluorescence analysis with Ki67 staining (green) of HUVEC cells in proliferating condition. (B) After 48 h without treatment. (C) After 48 h and LUT-7G treatment. (D–F) Same time course and treatment with vWF staining (green) (bars = 60 μm). (G,H) Higher enlargement of vWF staining, showing a cytoplasmic location in untreated cells (G) and a membrane staining in treated cells. Indeed, treated cells show an important reduction of vWF synthesis. In the picture, phalloidin is stained in red, and nuclei in blue (DAPI (4′,6-diamidino-2-phenylindole); bars = 60 μm; Prolif = proliferating endothelial cells).

Figure 3

LUT-7G treatment in endothelial cells. (A) immunofluorescence analysis with Ki67 staining (green) of HUVEC cells in proliferating condition. (B) After 48 h without treatment. (C) After 48 h and LUT-7G treatment. (D–F) Same time course and treatment with vWF staining (green) (bars = 60 μm). (G,H) Higher enlargement of vWF staining, showing a cytoplasmic location in untreated cells (G) and a membrane staining in treated cells. Indeed, treated cells show an important reduction of vWF synthesis. In the picture, phalloidin is stained in red, and nuclei in blue (DAPI (4′,6-diamidino-2-phenylindole); bars = 60 μm; Prolif = proliferating endothelial cells).

Figure 4

Inflammatory gene expression regulated by LUT-7G treatment, and clusterization analysis. (A) schematic representation of modulated genes after IL-22 treatment. The array contains in total 189 genes. On the right site of the gradient diagram, are represented the number of genes of which the treatment with LUT-7g reverts the effects of IL-22. (B) Clusterization function pathway obtained using the regulated genes. FDR is False Discovery Rate; N total number of genes; B total number of genes associated with a specific GO term; n is the flexible cut-off. Enrichment is defined as (b/n)/(B/N).

Figure 5

Oxidative stress and fatty acids hydroxylation analysis. (A) ROS production analysis, the results are presented in % units considering the control as 100%. (B) LUT-7G treatment affects cholesterol levels, which increase, and levels of hydroxylated species, which decrease dramatically. Data are shown as mean and standard deviation. Welch’s two-sample t-test was used to identify biochemicals that differed significantly between experimental groups. A p < 0.05 was considered significant (see Section 4).

Figure 6

LUT-7G treatment affects linoleic acid levels, which increase, while its hydroxylated species is not detectable. Data are shown as mean and standard deviation. Welch’s two-sample t-test identified biochemical differences between experimental groups. A p < 0.05 was considered significant (see Section 4).