Chlorogenic Acid Alleviates Allergic Inflammatory Responses Through Regulating Th1/Th2 Balance in Ovalbumin-Induced Allergic Rhinitis Mice

Abstract

BACKGROUND Allergic rhinitis (AR) is a prevalent atopic disorder caused by immune imbalance. Chlorogenic acid (CGA) has antibacterial, antiviral, antioxidative and immunoregulatory effects, but its role in anaphylactic disease remains unclear. The current study aimed to investigate the function of CGA in AR. MATERIAL AND METHODS AR mice models were induced with ovalbumin (OVA) by orally administrating the mice with 50 mg/kg (L-CGA), 100 mg/kg (M-CGA), or 200 mg/kg (H-CGA) of CGA. The number of nasal rubbings and sneezes was recorded. Afterward, the mice were sacrificed for the collection of blood, nasal lavage fluid (NALF), and nasal tissues. The cells in NALF were counted by hemocytometer and stained by Diff-Quick. Nasal mucosa was observed by H&E staining. ELISA testing was conducted for detecting the levels of anti-OVA antibodies and Th1/Th2-related cytokine. Quantitative real-time polymerase chain reaction experiments were conducted to determine mRNA expressions of Th1/Th2-related cytokines. RESULTS In the OVA-induced AR mice, CGA treatment reduced nasal rubbing and sneezing, and also suppressed the number of total cells, eosinophils, neutrophils, lymphocytes, macrophages, and epithelial cells in NALF. OVA-induced up-regulation of nasal mucosa thickness was inhibited by CGA, and the effects of OVA on IgE, IgG1, and IgG2a were reversed by CGA. The regulatory effects of OVA on mRNA expressions and levels of Th1/Th2-related cytokines were abolished by CGA treatment in AR mice. CONCLUSIONS CGA can alleviate allergic inflammatory responses through regulating Th1/Th2 balance in OVA-induced allergic rhinitis mice.

Figure 1

Chlorogenic acid (CGA) relieved the symptom of allergic rhinitis (AR) induced by ovalbumin (OVA) in mice. (A) The chemical formula of CGA is shown in the figure. (B) The mice were sensitized on days 0, 7, and 14 by OVA and Al(OH)₃, and then orally gavaged from day 15 to 25 (11 days) with CAG (50, 100, and 200 mg/kg), Cromolyn disodium salt hydrate (DSGC, 100 mg/kg), or isopycnic distilled water once a day. Next, the mice were challenged with saline containing 10% OVA via nose drops 30 minutes (min) after gavage. Before sacrifice on day 26, (C, D), nasal-rubbing and -sneezing frequencies of the mice in different groups were counted on day 25. n=10 per group. ^### P<0.001 vs. control, *** P<0.001 vs. OVA.

Figure 2

Chlorogenic acid (CGA) reduced the infiltration of differential inflammatory cells in nasal lavage fluid (NALF) and prevented epithelial cells disruption. NALF was collected immediately after sacrifice, and the cells were isolated by cytospin. (A) Cells from different groups were stained by Diff-Quick. Red arrows indicate eosinophils. Black arrows indicate epithelial cells lost from inflammatory tissues. (B) The number of total cells in NALF. (C) The number of eosinophils in NALF. (D) The number of neutrophils in NALF. (E) The number of lymphocytes in NALF. (F) The number of macrophages in NALF. Scale bar: 25μm, ×400 magnification. n=10 per group. ^### P<0.001 vs. control, *** P<0.001 vs. OVA.

Figure 3

Chlorogenic acid (CGA) reduced mucosa thickness and mucus secretions in the nasal mucosa of mice. (A) Histological features of the nasal mucosa were identified by hematoxylin-eosin (HE) staining, and the mucosa thickness of nasal septum was measured. Enzyme-linked immunosorbent assays (ELISA) were performed to detect the levels of anti-OVA specific IgE (B), anti-OVA IgG1 (C) and anti-OVA IgG2a (D) in the serum of the mice. Scale bar: 10μm, ×1000 magnification. n=10 each group. ^### P<0.001 vs. control, * P<0.05, ** P<0.01, *** P<0.001 vs. OVA.

Figure 4

Chlorogenic acid (CGA) regulated the balance of Th1/Th2-associtaed cytokines in NALF and nasal tissues of the mice. Th1-related cytokine levels of IFN-γ and IL-12, and Th2-related cytokine levels IL-4, IL-5, and IL-13 were measured by enzyme-linked immunosorbent assays (ELISA) in NALF (A–E) and nasal mucosa (F–J). n=10 each group. ^# P<0.05, ^### P<0.001 vs,. control, * P<0.05, ** P<0.01, *** P<0.001 vs. OVA.

Figure 5

Chlorogenic acid (CGA) regulated mRNA expressions of IFN-γ, IL-12, IL-4, IL-5, and IL-13 in NALF and nasal mucosa. Quantitative real-time polymerase chain reaction (qRT-PCR) experiments were performed to assess the mRNA expressions of IFN-γ, IL-12, IL-4, IL-5, and IL-13 in NALF (A) and nasal mucosa (B). n=10 each group. ^### P<0.001 vs, control, * P<0.05, ** P<0.01, *** P<0.001 vs. OVA.