Therapeutic effects of chlorogenic acid on allergic rhinitis through TLR4/MAPK/NF-κB pathway modulation

Abstract

Chlorogenic acid (CGA) exhibits promising anti-inflammatory properties, making it a potential therapeutic agent for inflammatory conditions and allergic rhinitis (AR). This study aimed to evaluate the therapeutic effects of CGA on inflammation in RAW264.7 macrophage cells and on AR in mice. RAW264.7 cells were treated with lipopolysaccharide (LPS) to induce inflammation and cultured with varying concentrations of CGA, a Tlr4-silenced gene (shTlr4) transfection, and the MAPK/NF-κB pathway activator diprovocim. Cell viability was assessed using the CCK8 assay, while levels of nitric oxide (NO), TNF-α, and IL-6 were measured by Griess colorimetry, immunofluorescence, and ELISA. Expression and phosphorylation levels of the MAPK/NF-κB pathway were evaluated using qPCR and western blotting. Additionally, ovalbumin (OVA)-induced AR mice received different doses of CGA, and Toll-like receptor-4 (Tlr4) overexpression was induced. In vitro, CGA treatment significantly reduced LPS-induced cell activity, NO, TNF-α, and IL-6 secretion, and downregulated Tlr4, p-p38, p-p65, and p-IκB expression. Tlr4 inhibition suppressed cell activity and inflammation by blocking MAPK/NF-κB pathway activation. Conversely, Tlr4 overexpression counteracted the effects of CGA, increasing cell activity and inflammatory factor concentration. In OVA-induced AR mice, CGA effectively alleviated allergic symptoms, reduced inflammatory factor secretion, and inhibited TLR4/MAPK/NF-κB pathway activity. These findings suggest CGA's potential as an anti-inflammatory agent in RAW264.7 cells and AR models through modulation of the TLR4/MAPK/NF-κB pathway.

Figure 1.

Effect of culture with CGA on LPS-induced RAW264.7 cell function. (A) Cytotoxicity analysis of effects of CGA culture on cells; (B) Effects of CGA and LPS on cell proliferation; (C) The secretion of NO in cells was detected by the Griess method; (D) The secretion of TNF-α and IL-6 was detected by ELISA; (E) Effect of CGA on the protein expression of TLR4, p38, p-p38, p65, p-p65, IκB, and p-IκB in cells; (F) Effect of CGA on the mRNA expression of TLR4, p38, p65, and IκB in cells. N ═ 3, *P < 0.05, compared with control group; ^#P < 0.05, compared with LPS group. LPS: Lipopolysaccharide; CGA: Chlorogenic acid; TLR4: Toll-like receptor-4.

Figure 2.

Effect of downregulation of TLR4 expression on LPS-induced RAW264.7 cell function. (A) Transfection efficiency of shTlr4 was detected; (B) Effect of shTlr4 transfection on cell proliferation was determined by CCK8 assay; (C) The protein expression of TLR4, p38, p-p38, p65, p-p65, IκB, and p-IκB were detected by western blotting; (D) The mRNA expression of TLR4, p38, p65, and IκB in cells was detected by qPCR; (E) The NO secretion in the cells was detected by immunofluorescence staining; (F) The secretion of TNF-α and IL-6 in cells was detected by ELISA. N ═ 3, *P < 0.05, compared with control group; ^#P < 0.05, compared with LPS group. LPS: Lipopolysaccharide; TLR4: Toll-like receptor-4; shTLR4: Short hairpin RNA targeting TLR4; NO: Nitric oxide.

Figure 3.

Effect of TLR4/MAPK/NF-κB pathway activity on LPS-induced cell function. (A) Over-TLR4 expression efficiency detection; (B) The effects of CGA and over-TLR4 transfection on the TNF-α and IL-6 levels in cells were detected by ELISA; (C) The changes of cell proliferative activity were detected by CCK8; (D) The NO secretion from the cells was detected by immunofluorescence staining; (E) The levels of TNF-α and IL-6 were determined by ELISA; (F) The protein expression of TLR4, p38, p-p38, p65, p-p65, IκB, and p-IκB was detected by western blotting; (G) The mRNA expression of TLR4, p38, p65, and IκB in cells was detected by qPCR. N ═ 3, ^&P < 0.05, compared with control group; *P < 0.05, compared with the LPS group; ^#P < 0.05, compared with the LPS+CGA group. LPS: Lipopolysaccharide; TLR4: Toll-like receptor-4; CGA: Chlorogenic acid; NO: Nitric oxide.

Figure 4.

Effects of different doses of CGA on mice with allergic rhinitis. (A) Rhinitis behavioral score in mice; (B and C) The numbers of mice scratching their nose and sneezing after treatment; (D) Pathological staining analysis of nasal mucosa of mice (Red arrow points to eosinophils; blue arrow points to lamina propria of mucosa); (E) Analysis of blood eosinophil count in mice. N ═ 5, *P < 0.05, ***P < 0.001, compared with control group; ^#P < 0.05, ^###P < 0.001, compared with model group. CGA: Chlorogenic acid.

Figure 5.

(A) Effect of CGA on inflammatory response and TLR4/MAPK/NF-κB pathway activity in mice with allergic rhinitis. (B) Determination of serum histamine and IgE levels in mice. Detection of serum IL-4, IL-5, IFN-γ, and TNF-α in mice. The protein (C) and mRNA (D) expression of TLR4, p38, p-p38, p65, p-p65, IκB, and p-IκB was detected by western blotting and qPCR. N ═ 5, *P < 0.05, ***P < 0.001, compared with control group; ^#P < 0.05, ^###P < 0.001, compared with LPS group; ^&P < 0.05, compared with CGA+over-NC group. CGA: Chlorogenic acid; TLR4: Toll-like receptor-4.