Oral Administration of Trichosanthes Kirilowii Fruit Extract Ameliorates Airway Inflammation and Suppresses Th2 Cell Activities in Ovalbumin-Sensitized Mice

Abstract

Trichosanthes kirilowii is a plant used in traditional Chinese medicine. Its fruits, seeds, and roots can all be used medicinally. The fruit of T. kirilowii has a moistening effect on the lung for arresting cough. However, there is no scientific research to prove that the fruit of T. kirilowii can improve asthma. Herein, we evaluated whether T. kirilowii fruit extract (TK) could reduce airway inflammation and airway hyperresponsiveness (AHR) in a murine model of asthma. Female BALB/c mice were sensitized with ovalbumin to induce asthma and treated with varying oral doses of TK from days 14 to 27 of the experiment. Additionally, IL-4/TNF-α-stimulated BEAS-2B cells were treated with different doses of TK to investigate inflammatory cytokine and chemokine secretion. TK treatment significantly mitigated AHR, eosinophil infiltration, and airway inflammation in the lungs of asthmatic mice. It also alleviated goblet cell hyperplasia and lung ROS levels while modulating Th2-associated cytokine levels in the bronchoalveolar lavage fluid. Moreover, TK effectively alleviated proinflammatory cytokine, chemokine, and eotaxin in IL-4 and TNF-α-stimulated BEAS-2B cells. Our results indicate that TK effectively improved asthma symptoms in mice through suppressed Th2-cell activation, which mitigated airway inflammation, oxidative stress, and AHR.

Keywords: Trichosanthes kirilowii fruit extract; airway hyperresponsiveness; airway inflammation; asthma; eosinophil.

FIGURE 1

HPLC profiles of Trichosanthes kirilowii (TK) extracts. (A) HPLC fingerprint of TK extract; the major peaks on the TK profile were identified using 3,29‐dibenzoyl karounitriol (DK) as a standard. (B) HPLC fingerprint of the DK standard.

FIGURE 2

Effects of T. kirilowii fruit extract (TK) on airway hyperresponsiveness (AHR) and cell counts in bronchoalveolar lavage fluid (BALF) of asthmatic mice. (A) Mice inhaled increasing doses of methacholine, and AHR was assessed (shown as Penh values). (B) The inflammatory cells in BALF were counted, including total cells, eosinophils, neutrophils, lymphocytes, and macrophages. (C) Percentage of eosinophils in BALF. Three independent experiments were analyzed. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the OVA control group.

FIGURE 3

T. kirilowii fruit extract (TK) effects on asthmatic lung tissue. TK reduced (A) eosinophil infiltration (HE stain; 200× magnification) and (B) the inflammatory score. (C) PAS‐stained lung sections displaying goblet cell hyperplasia (200× magnification). (D) The number of PAS‐positive cells per 100 μm of basement membrane. Lung sections were stained with (E) Masson trichrome stain to detect collagen expression (200× magnification) for (F) quantitative analysis of collagen. Values are the mean ± SEM. *p < 0.05, **p < 0.01 versus the OVA group (scale bar = 100 μm).

FIGURE 4

T. kirilowii fruit extract (TK) effects on the phosphorylation of NF‐κB and oxidative stress factors. (A) Immunohistochemistry stain detected NF‐κB expression in lung tissue(scale bar = 100 μm). (B) The phosphorylation of p65 (p‐p65, NF‐κB subunit) expression in lung tissue, and (C) fold‐change relative to p‐p65/p65 expression. (D) Catalase (CAT), (E) glutathione (GSH), (F) superoxide dismutase (SOD), and (G) malondialdehyde (MDA) activities in mouse lung tissues. Values are the mean ± SEM. *p < 0.05, **p < 0.01 versus the OVA group.

FIGURE 5

Effects of T. kirilowii fruit extract (TK) on the levels of cytokines and chemokines in bronchoalveolar lavage fluid (BALF). The concentrations of (A) IFN‐γ, (B) IL‐4, (C) IL‐5, (D) IL‐13, (E) IL‐6, (F) TNF‐α, (G) CCL11, and (H) CCL24 were measured in BALF. Three independent experiments were analyzed. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the OVA control group.

FIGURE 6

Effects of T. kirilowii fruit extract (TK) on liver enzymes and OVA‐specific antibodies in serum. Serum levels of (A) GOT, (B) GPT, (C) OVA‐IgE, (D) OVA‐IgG1, and (E) OVA‐IgG2a from mice without or with TK treatment. Three independent experiments were analyzed. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the OVA control group.

FIGURE 7

Effects of T. kirilowii fruit extract (TK) on cytokine production in OVA‐activated spleen cells. (A) IFN‐γ, (B) IL‐4, (C) IL‐5, and (D) IL‐13 levels were measured by ELISA. Three independent experiments were analyzed. All data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the OVA control group.

FIGURE 8

Effects of T. kirilowii fruit extract (TK) on cytokine and chemokine production by BEAS‐2B cells. (A) Cell viability of TK‐treated, TNF‐α/IL‐4‐stimulated BEAS‐2B cells. (B–F) ELISA results show the levels of (B) IL‐6, (C) IL‐8, (D) MCP‐1, (E) CCL5, (F) CCL11, (G) CCL24, and (H) ICAM‐1 in BEAS‐2B cells treated with TNF‐α/IL‐4. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, compared with BEAS‐2B cells stimulated with TNF‐α/IL‐4 without TK pretreatment.

FIGURE 9

T. kirilowii fruit extract (TK) effects on NF‐κB and ROS production in activated BEAS‐2B cells. (A) The phosphorylation of p65 (p‐p65, NF‐κB subunit) expression, and (B) fold‐change relative to p‐p65/p65 expression. (C) Fluorescence microscopy images show ROS labeled with DCF‐DA. (D) Fluorescence‐intensity quantification of ROS production. Values are the mean ± SEM. **p < 0.01 versus TNF‐α /IL‐4 activated BEAS‐2B cells.