Liposome-Encapsulated Baicalein Suppressed Lipogenesis and Extracellular Matrix Formation in Hs68 Human Dermal Fibroblasts

Abstract

The dermis of human skin contains large numbers of fibroblasts that are responsible for the production of the extracellular matrix (ECM) that supporting skin integrity, elasticity and wound healing. Previously, an in vivo study demonstrated that dermal fibroblasts siting in the lower dermis are capable to convert into skin adipose layer and hence fibroblast lipogenesis may vary the structure and elasticity of dermis. In the present study, Hs68 human dermal fibroblasts were utilized as an in vitro model to study the lipogenesis via using adipogenic differentiation medium (ADM). Baicalein, isolated from Scutellaria baicalensis, is one of the flavonoids to inhibit adipocyte differentiation due to high antioxidant activity in vitro. In order to develop a suitable formulation for baicalein (a poorly water-soluble drug), soybean phosphatidylcholine (SPC) was used to prepare baicalein-loaded liposomes to enhance drug bioavailability. Our results demonstrated that liposome-encapsulated baicalein protected cell viability and increased cellular uptake efficiency of Hs68 fibroblasts. Lipid accumulation, triglyceride synthesis and gene expressions of lipogenesis enzymes (FABP4 and LPL) were significantly increased in ADM-stimulated Hs68 fibroblasts but subsequently suppressed by liposome-encapsulated baicalein. In addition, ADM-induced TNF-α expression and related inflammatory factors was down-regulated by liposome-encapsulated baicalein. Through ADM-induced lipogenesis, the protein expression of elastin, type I and type III collagens increased remarkably, whereas liposome-encapsulated baicalein can down-regulate ADM-induced ECM protein synthesis. Taken together, we found that liposome-encapsulated baicalein can inhibit ADM-induced lipid accumulation and ECM formation in Hs68 fibroblasts through the suppression of lipogenesis enzymes and inflammatory responses. Liposome-encapsulated baicalein may have the potential to improve wound healing and restore skin structure after skin injury.

Keywords: Hs68 human dermal fibroblast; adipogenic differentiation medium; baicalein; lipogenesis; liposomes.

FIGURE 1

Stability of liposomal formulations. Empty and baicalein (BC)-loaded liposomes were stored at 4°C over different time intervals.

FIGURE 2

The effect of baicalein, empty and baicalein-loaded liposomes on cell viability of Hs68 fibroblasts. Cells were treated with different concentrations of baicalein and baicalein-loaded liposomes for 24 h and then measured by MTT assay. Cell viability of control was expressed as 100%. The data are presented as means ± standard deviation (n = 4–8). ^∗p < 0.05 relative to 20 μg/ml Baicalein-treated cells.

FIGURE 3

Cellular uptake of DiI-loaded liposomes in Hs68 fibroblasts. (A) Hs68 fibroblasts were incubated with DiI and DiI-loaded liposomes at different time intervals (1–24 h). The fluorescent image was photographed under fluorescent microscopy paired with CCD system. (B) The fluorescent intensity of DiI-loaded liposomes inside the cells was quantified using Image J. The data are expressed in relative index compared with control. The results are presented as the means ± standard deviation (×100 magnification, scale bar = 200 nm, n = 4).

FIGURE 4

The effect of empty and baicalein-loaded liposomes on cell viability and nitrite production of RAW264.7 macrophages. (A) Cells were incubated with baicalein, empty and baicalein-loaded liposomes for 24 h, and then cell viability was measured using MTT assay. ^∗p < 0.05 relative to 20 μg/ml Baicalein-treated cells (B) RAW264.7 macrophages were induced to inflammation by 500 ng/ml LPS. The inhibitory effect of baicalein and baicalein-loaded liposomes on nitrite production of LPS-induced RAW264.7 macrophages was determined after 24 h of incubation. The total nitrite production in LPS-stimulated cells is expressed as 100%. The data are presented as the means ± standard deviation. ^#P < 0.05 relative to control and ^∗p < 0.05 relative to LPS-stimulated cells (n = 3).

FIGURE 5

Lipid accumulation in human dermal fibroblasts, Hs68 (A) after the differentiation, lipid accumulation in the cells was stained with Oil Red O dye and visualized under a microscope at 100x of magnification. (Scale bar = 200 μm). (B) The quantification of lipid accumulation in Hs68 cells was measured using an ELISA reader at 500 nm. The data are presented as relative index to control, and the results were expressed as means ± standard deviation (n = 3–5). (C) Intracellular triglyceride content were determined with TG adipogenesis kit at 570 nm in Hs68 cells after 14 and 21 days of incubation with 20% ADM. The results were expressed by the mean intensity of triglyceride compared to control ± standard deviation (n = 3–8). ^#P < 0.05 relative to control, ^∗p < 0.05 relative to cells treated with ADM and ^$p < 0.05 relative to cells co-treated with ADM and empty liposomes.

FIGURE 6

The effect of baicalein, empty and baicalein-loaded liposomes on adipogenic marker genes, LPL (A) and FABP4 (B) in the presence of ADM. The mRNA expressions of LPL and FABP4 were measured by real-time PCR analysis after 3 days incubation with ADM. Levels of LPL and FABP4 mRNA expression are presented relative to control gene expression. The data were presented as the mean ± standard deviation (n = 3). ^#P < 0.05 relative to control, ^∗p < 0.05 relative to cells treated with ADM only and ^$p < 0.05 relative to cells co-treated with ADM and empty liposomes.

FIGURE 7

The effect of ADM, Baicalein and baicalein-loaded liposomes on gene expression of TNF-α. The level of TNF-α mRNA expression was measured using RT-PCR. The level of TNF-α mRNA expression is presented relative to control gene expression. The data were presented as the mean ± standard deviation (n = 3). ^#P < 0.05 relative to control, ^∗p < 0.05 relative to cells treated with ADM only and ^$p < 0.05 relative to cells co-treated with ADM and empty liposomes.

FIGURE 8

The effect of baicalein, empty and baicalein-loaded liposomes on TNF-α-induced inflammatory responses in Hs68 fibroblasts. Hs68 fibroblasts were treated with test samples in the presence or absence of TNF-α for an hour. Afterward, the gene expressions of COX-2 (A), IL-6 (B) and IL-8 (C) were determined by real-time PCR. (D) The mRNA expressions of MMP-1 and MMP-3 were also measured after 4 h incubation with test samples in the presence or absence of TNF-α. Levels of COX-2, IL-6, IL-8, MMP-1 and MMP-3 mRNA expression are presented relative to control gene expression. The results were expressed as the mean ± standard deviation (n = 3). ^#P < 0.05 relative to control and ^∗p < 0.05 relative to cells treated with TNF-α only.

FIGURE 9

The effect of ADM, baicalein and baicalein-loaded liposomes on elastin, type I and type III collagen protein expressions in Hs68 fibroblasts. Immunofluorescence staining was carried out using specific antibody. (A) The immunofluorescent image of elastin, type I and type III collagen were photographed under microscopy paired with CCD system (×100 magnification, Scale bar = 200 μm). Quantification of fluorescent intensity for protein expression levels of (B) elastin, (C) type I collagen and (D) type III collagen was determined by Image J. The levels of protein expression are presented relative to control. The results were expressed as the mean ± standard deviation (n = 3). ^#P < 0.05 relative to control, ^∗p < 0.05 relative to cells treated with ADM and ^$p < 0.05 relative to cells co-treated with ADM and empty liposomes.

FIGURE 10

Protein expressions of elastin (A) and collagen (B) were measured by ELISA. The protein level was presented in relative proportion compared with control. The data were expressed as the mean ± standard deviation (n = 4). ^#P < 0.05 relative to control, ^∗p < 0.05 relative to cells treated with ADM only and ^$p < 0.05 relative to cells co-treated with ADM and empty liposomes.

FIGURE 11

The hypothetic model of how adipogenesis, inflammation and ECM remodeling are regulated in Hs68 human dermal fibroblasts. Physiological stimuli such as ADM may signal through LPL or FABP4 to activate adipogenic and inflammatory pathways, which may be responsible for the upregulation of ECM remodeling.