Anti-Inflammatory Effect of Titrated Extract of Centella asiatica in Phthalic Anhydride-Induced Allergic Dermatitis Animal Model

Abstract

Centella asiatica has potent antioxidant and anti-inflammatory properties. However, its anti-dermatitic effect has not yet been reported. In this study, we investigated the anti-dermatitic effects of titrated extract of Centella asiatica (TECA) in a phthalic anhydride (PA)-induced atopic dermatitis (AD) animal model as well as in vitro model. An AD-like lesion was induced by the topical application of five percent PA to the dorsal skin or ear of Hos:HR-1 mouse. After AD induction, 100 μL of 0.2% and 0.4% of TECA (40 μg or 80 μg/cm²) was spread on the dorsum of the ear or back skin three times a week for four weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and NF-κB activity, which were determined by electromobility shift assay (EMSA). We also measured TNF-α, IL-1β, IL-6, and IgE concentration in the blood of AD mice by enzyme-linked immunosorbent assay (ELISA). TECA treatment attenuated the development of PA-induced atopic dermatitis. Histological analysis showed that TECA inhibited hyperkeratosis, mast cells and infiltration of inflammatory cells. TECA treatment inhibited expression of iNOS and COX-2, and NF-κB activity as well as the release of TNF-α, IL-1β, IL-6, and IgE. In addition, TECA (1, 2, 5 μg/mL) potently inhibited Lipopolysaccharide (LPS) (1 μg/mL)-induced NO production, expression of iNOS and COX-2, and NF-κB DNA binding activities in RAW264.7 macrophage cells. Our data demonstrated that TECA could be a promising agent for AD by inhibition of NF-κB signaling.

Keywords: IgE; NF-κB; atopic dermatitis; cytokine; skin inflammation; titrated extract of Centella asiatica.

Figure 1

Differences in body weight, ear thickness, ear phenotypes, and back phenotypes. Phthalic anhydride (PA) solution was repeatedly applied to the dorsum of ear and back three times a week during topical application of Titrated extract of Centella asiatica (TECA). After four weeks, body weight (A) and ear thickness (B) were observed at least three times by following the procedure described in Materials and Methods. Phenotypes (C) were randomly selected by one mouse/group. Data shown are the mean ± SD (n = 10).

Figure 2

Changes in auricular lymph node weight, expression level of iNOS and COX-2 protein in lymph node, and serum cytokine concentration. After final treatment, mice from each group were sacrificed under anesthesia. The auricular lymph nodes were then harvested from the neck regions of the mice using a microscissor, after which they were weighed (A); Alteration of the expression of the two proteins was measured by Western blotting (B); Serum used to measure the cytokine concentration was prepared from blood samples collected from the abdominal veins of mice. Serum IgE (C), TNF-α, IL-6, and IL-1β (D) concentration were quantified by enzyme-linked immunosorbent assay (ELISA). Data shown are gained from the same mice treated shown in Figure 1. Data shown are the mean ± SD (n = 10). * p < 0.05 is the significance level compared to the control group. ^# p < 0.05 is the significance level compared to the PA treated group.

Figure 3

Histopathological analysis of ear tissue and the inhibitions of NF-κB DNA binding activity by topical application of TECA in ear skin. Histopathology of ear skin in control (A-1), vehicle (A-2), PA (A-3), PA + TECA 0.2% (A-4), and PA + TECA 0.4% (A-5). PA solution was repeatedly applied to the dorsum of ears during topical application of TECA. Histopathological changes in the slide sections of ear tissue were identified by staining with hematoxylin and eosin followed by observation at 200× magnification (Scale bars, 100 μm). (A) Histological images and (B) thickness of the epidermis and dermis. Alteration of the expression of iNOS and COX-2 proteins were measured by Western blotting (C); (D) Effect of TECA on NF-κB DNA binding activity in ear skin. The activation of NF-κB was investigated using electromobility shift assay (EMSA) as described in Materials and Methods. Nuclear extracts from homogenized ear skin tissue were incubated in binding reactions of ³²P-end-labeled oligonucleotide containing the NF-κB sequence (numbers: relative expression). (E) Effect of TECA on translocation of the subunits of NF-κB (p50 and p65) into nucleus, and phosphorylation of IκBα in cytosol in ear skin. Equal amounts of nuclear proteins (20 μg/lane) or total proteins (20 μg/lane) were subjected to 10% SDS-PAGE, and expression of p50, p65, IκBα, and p-IκBα protein were detected by Western blotting using specific antibodies. Histone h1 protein and β-actin protein were used here as an internal control. Data shown are gained from the same mice treated shown in Figure 1. Data shown are the mean ± SD (n = 10). * p < 0.05 is the significance level compared to the control group. ^# p < 0.05 is the significance level compared to the PA treated group.

Figure 4

Histopathological analysis of back tissue and the inhibitions of NF-κB DNA binding activity by topical application of TECA in back skin. Histopathology of back skin in control (A-1), vehicle (A-2), PA (A-3), PA + TECA 0.2% (A-4), and PA + TECA 0.4% (A-5). PA solution was repeatedly applied to the back skin during topical application of TECA. Histopathological changes in the slide sections of back tissue were identified by staining with hematoxylin and eosin followed by observation at 200× magnification (Scale bars, 100 μm). (A) Histological images and (B) thickness of the epidermis. Alteration of the expression of iNOS and COX-2 proteins were measured by Western blotting (C); (D) Effect of TECA on NF-κB DNA binding activity in back skin. The activation of NF-κB was investigated using EMSA as described in Materials and Methods. Nuclear extracts from homogenized back skin tissue were incubated in binding reactions of ³²P-end-labeled oligonucleotide containing the NF-κB sequence (numbers: relative expression); (E) Effect of TECA on translocation of the subunits of NF-κB (p50 and p65) into nucleus, and phosphorylation of IκBα in cytosol in back skin. Equal amounts of nuclear proteins (20 μg/lane) or total proteins (20 μg/lane) were subjected to 10% SDS-PAGE, and expression of p50, p65, IκBα, and p-IκBα protein were detected by Western blotting using specific antibodies. Histone h1 protein and β-actin protein were used here as an internal control. Data shown are gained from the same mice treated shown in Figure 1. Data shown are the mean ± SD (n = 10). * p < 0.05 is the significance level compared to the control group. ^# p < 0.05 is the significance level compared to the PA treated group.

Figure 5

Effects of TECA on LPS-induced NO production, and iNOS and COX-2 expression in RAW264.7 cells. RAW 264.7 cells were pre-treated with different concentration (1, 2, and 5 μg/mL) of TECA for 2 h and then stimulated with LPS (1 μg/mL) for 24 h. Effect of TECA on LPS-induced NO production was measured by the Griess reaction as described in Materials and Methods (A); The expression of iNOS and COX-2 after stimulated 24 h was determined by Western blot (B); Effect of TECA on LPS-indcued NF-κB DNA binding activity was measured by EMSA as described in Materials and Methods (C); Effects of TECA on LPS-induced phosphorylation of IκBα in cytosol, and translocation of the subunits of NF-κB (p50 and p65) into nucleus were measured by Western blot (D). Data shown are gained from the same mice treated shown in Figure 1. Data shown are the mean ± SD (n = 10). * p < 0.05 is the significance level compared to the control group. ^# p < 0.05 is the significance level compared to the PA treated group.