Anti-inflammatory effects of Capparis ecuadorica extract in phthalic-anhydride-induced atopic dermatitis of IL-4/Luc/CNS-1 transgenic mice

Abstract

Context: The natural products derived from Capparis ecuadorica H.H. Iltis (Capparaceae) could have great potential for anti-inflammation since they inhibited the inflammatory response in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells.

Object: This study investigated the anti-inflammatory effects and related mechanism of methanol extract of C. ecuadorica leaves (MCE) during atopic dermatitis (AD) responses.

Materials and methods: Alterations in the phenotypical markers for AD, luciferase signal, iNOS-mediated COX-2 induction pathway, and inflammasome activation were analysed in non-Tg (n = 5) and 15% phthalic anhydride (PA) treated IL-4/Luc/CNS-1 transgenic (Tg) HR1 mice (n = 5 per group), subsequent to treatment with acetone-olive oil (AOO), vehicle (DMSO) and two dose MCE (20 and 40 mg/kg) three times a week for 4 weeks.

Results: MCE treatment reduced the intracellular ROS level (48.2%), NO concentration (7.1 mmol/L) and inflammatory cytokine expressions (39.1%) in the LPS-stimulated RAW264.7 cells. A significant decrease was detected for ear thickness (16.9%), weight of lymph node (0.7 mg), IgE concentration (1.9 µg/mL), and epidermal thickness (31.8%) of the PA + MCE treated Tg mice. MCE treatment induced the decrease of luciferase signal derived from the IL-4 promoter and the recovery of the IL-4 downstream regulator cytokines. PA + MCE treated Tg mice showed decreasing infiltration of mast cells (42.5%), iNOS-mediated COX-2 induction pathway, MAPK signalling pathway and inflammasome activation in the ear tissue.

Conclusions: These findings provide the first evidence that MCE may have great potential to suppress chemical-induced skin inflammation through the suppression of IL-4 cytokine and the iNOS-mediated COX-2 induction pathway, and activation of inflammasome.

Keywords: COX-2; IgE; Inflammation; iNOS; luciferase signal.

Figure 1.

Detection of ROS, NO and cytokines in MCE + LPS treated RAW264.7 cells. (A) Phytochemical composition and component conformation in MCE. Total flavonoids, phenols and condensed tannin content were analysed in mixtures containing different concentrations of MCE. (B) After DCF‐DA treatment, the intensity of green fluorescence in RAW264.7 cells of subset groups was detected at 200× magnification using a fluorescence microscope (Eclipse TX100, Nikon, Tokyo, Japan). (C) Total number of DCF-DA stained cells were counted per specific area; relative level of stained cells in MCE treated groups are represented based on the stained cell number in No treated group. (D) RAW264.7 cells (5 × 10⁵ cells/mL) were treated with vehicle, MCELo or MCEHi in the absence or presence of LPS (1 µg/mL) for 24 h. After collecting the culture supernatants, NO concentration was measured using Griess reagent. (E) LPS-stimulated RAW264.7 cells were pre-treated with vehicle, MCELo or MCEHi for 2 h, and the expression levels of TNF-α, IL-1β and IL-6 mRNA were determined by RT-PCR. Intensity of each band was determined using an imaging densitometer, and the relative levels of the three mRNA genes were calculated based on the band intensity of β-actin as the endogenous control. Two to three wells were used for preparing DCF-DA stained cells, collecting culture supernatant, and RNA extraction; total cell number, NO concentration and mRNA expressions were measured in duplicate for each sample. Data represents the mean ± SD from duplicates. *p < 0.05 compared to the No treated group, #p < 0.05 compared to the LPS + vehicle treated group.

Figure 2.

Measurement of ear morphology, ear thickness and lymph node weight after MCE treatment. (A) Ear vein and colour were analysed in photographs of IL-4/Luc/CNS-1 Tg mice treated with PA + vehicle or PA + MCE. (B) Ear thickness of mice in all five groups was measured for 4 weeks using a thickness gauge, as described in Materials and methods. (C) After final treatment, the submandibular lymph node (SL), spleen and thymus were collected from all animals in the subset groups, and their weights were measured using an electronic balance. Three to four mice per group were used in the preparation of ear image and the collection of organs, and the ear thickness and weight of organs were measured in duplicate for each sample. Data presented are the means ± SD from duplicates. *p < 0.05 compared to the AOO treated group. #p < 0.05 compared to the PA + vehicle treated group.

Figure 3.

Measurement of histological structure and IgE concentration after MCE treatment. (A and B) After collecting ear tissues, histological changes were identified by staining with H&E, followed by observation at 400× magnification. (C) The concentration of IgE was quantified in serum using an enzyme linked immunosorbent assay with detectable concentration range of 10–5,000 ng/mL. Three to four mice per group were used for the collection of serum and H&E staining, and thickness measurement and ELISA was assayed in duplicate for each sample. Data shown are the means ± SD from duplicates. *p < 0.05 compared to the AOO treated group. #p < 0.05 compared to the PA + vehicle treated group.

Figure 4.

Detection of luciferase signal after MCE treatment. (A) After treatment with PA + vehicle and PA + MCE for 4 weeks, the whole body and eight organs were imaged at 24 h after the final treatment, using the Living Image software. The colour overlay in the image represents the photons per second emitted from the organs in accordance with the pseudocolor scale shown next to the image. In this image, red indicates the highest number of photons per second, while blue indicates the lowest. Abbreviations: L, lung; K, kidney; S, spleen; H, heart; SL, submandibular lymph node; ML, mesenteric lymph node; T, thymus; P, pancreas. (B) Luciferase gene of the mRNA expression was measured in the SL of PA + vehicle and PA + MCE Tg mice using RT-qPCR analyses. Three to four mice per group were used for sample preparations, and luciferase signal was assayed in duplicate. (C) The levels of TNF-α, IL-6 and IL-1α transcripts were detected in the total mRNA of SL by quantitative real time-PCR (RT-qPCR) analyses using specific primers. Three to four mice per group were used for the preparation of total RNAs, and RT-qPCR analyses were assayed in duplicate for each sample. Data shown are the means ± SD from duplicates. *p < 0.05 compared to the AOO treated group. #p < 0.05 compared to the PA + vehicle treated group.

Figure 5.

Detection of mast cell infiltration after MCE treatment. Infiltration of mast cells in the slide sections of ear tissue was identified by staining with toluidine blue, followed by observation at 400× magnification. Arrows indicate the infiltrated mast cells in the dermis of the ear tissue. Three to four mice per group were used in the preparation of toluidine blue stained sample, and the number of stained cells was counted in duplicate for each sample. Data shown are the means ± SD from duplicates. *p < 0.05 compared to the AOO treated group. #p < 0.05 compared to the PA + vehicle treated group.

Figure 6.

Expression of iNOS, COX-2 and MAPK members after MCE treatment. Western blot was used to detect iNOS, COX-2 and phosphorylation of ERK, JNK and p38 in the homogenates of ear tissue using specific antibodies. After determining the intensity of each band using an imaging densitometer, the relative levels of iNOS, COX-2, ERK, p-ERK, JNK, p-JNK, p38 and p-p38 proteins were calculated based on the band intensity of β-actin protein as the endogenous control. The relative phosphorylation levels of ERK, JNK and‐p38 in MCE treated cells were calculated based on the ratio of phosphorylated and non-phosphorylated proteins. Three to four mice per group were used in the preparation of tissue homogenate, and Western blot analyses were assayed in duplicate for each sample. Data represents the mean ± SD of duplicates. *p < 0.05 compared to the AOO treated group. #p < 0.05 compared to the PA + vehicle treated group.

Figure 7.

Expression of ASC, Casp-1 and NLRP3 protein after MCE treatment. Western blot was performed to detect ASC, Casp-1 and NLRP3 proteins in the homogenates of ear tissue, using specific antibodies. After determining the intensity of each band using an imaging densitometer, the relative levels of ASC, Casp-1 and NLRP3 proteins were calculated based on the band intensity of β-actin protein as the endogenous control. Three to four mice per group were used in the preparation of tissue homogenate, and Western blot analyses were assayed in duplicate for each sample. Data represents the mean ± SD of duplicates. *p < 0.05 compared to the AOO treated group. #p < 0.05 compared to the PA + vehicle treated group.