Pharmacological effects and mechanism of Kaihoujian Throat Spray (children's type) in the treatment of pediatric acute pharyngitis and tonsillitis

Abstract

Context: Kaihoujian Throat Spray children's type (KHJSC) is a Chinese medicine prescription for treating pediatric acute pharyngitis and tonsillitis (APT). However, its relevant mechanisms remain unclear.

Objective: To investigate the pharmacological effects of KHJSC on APT in vitro and in vivo, and explore the possible mechanism and target proteins.

Materials and methods: The antiviral and antibacterial effects in vitro were evaluated by IC₅₀ and MICs. Thirty-six Japanese white rabbits were averagely divided into control group, model group, amoxicillin group and 3 dose groups of KHJSC (720, 540 and 360 μL/kg/d). The model rabbits were injected with β-hemolytic Streptococcus solution into the tonsils for 2 consecutive days. KHJSC treatment started on the third day. The whole blood, serum, tonsil tissues and pharyngeal mucosa tissues were collected for routine blood tests, proteomic, ELISA and other tests on the sixth day.

Results: The IC₅₀ of KHJSC on HCoV-229E, influenza PR8 and Ad3 were 1.99, 1.99 and 4.49 mg/mL, respectively; MICs of MDR-PA, MRSA and β-hemolytic Streptococcus were 350, 350, and 175 mg/mL. KHJSC markedly decreased the number of white blood cells, lymphocytes, neutrophils, and the level of IL-1β, IL-5, IL-6, IL-18, TNF-α and MCP-1; increased the content of IL-2 and IFN-γ. Proteomic analysis and ELISA revealed that PI3K-Akt signaling pathway, NF-κB signaling pathway and Toll-like receptor signaling pathway were the potential mechanisms of KHJSC against APT.

Discussion and conclusion: These results provided the reference and scientific basis for the application of KHJSC in clinic and further mechanisms study.

Keywords: Antibacterial; Antiviral; TMT quantitative proteomic; Traditional Chinese medicine; β-hemolytic Streptococcus.

Fig. 1

Representative base peak intensity (BPI) chromatograms of KHJSC in positive (A) and negative (B) ion modes.

Fig. 2

Antiviral activity of KHJSC on different cells by CPE assay. Typical CPE characteristics, cell rounding and crimpling were observed in the Hep-2 (A) and A549 cells (B) infected by Ad3 and PR8, fibrous cells break off and curl up in MRC-5 induced by HCoV-229E (C). The cellular damage were significantly alleviated by KHJSC (TC₀) treatment group.

Fig. 3

Effect of KHJSC for APT in rabbits infected by β-hemolytic Streptococcus. (A) Pathological changes of pharyngeal tissues (HE 10 × 10); (B) pathological changes of tonsil tissues (HE 10 × 40); (C) levels of WBC, LYMPH, and NEUT in whole blood (n = 6). Data were expressed as the mean ± SD. ^##P < 0.01 vs. Control, **P < 0.01 vs. Model.

Fig. 4

The effects of KHJSC on the expression levels of IL-1β(A), IL-5 (B), IL-6 (C), IL-18 (D), TNF-α(E), MCP-1 (F), IL-2 (G) and IFN-γ(H) inflammatory cytokines in rabbits (n = 6). Data were expressed as the mean ± SD. ^##P < 0.01 vs. Control, **P < 0.01 vs. Model.

Fig. 5

Characterization of differentially expressed proteins in 3 sample groups. (A) Volcano plot of differentially expressed proteins in Model group vs. Control group. Green dots showed downregulated proteins, red dots showed upregulated proteins, and the gray dots showed proteins with no statistically significant difference between the Control and Model rabbits; (B) Volcano plot of differentially expressed proteins in Model group vs. KHJSC group. Green dots showed downregulated proteins, red dots showed upregulated proteins, and the gray dots showed proteins with no statistically significant difference between the Model and KHJSC group; (C) The common differentially expressed proteins in Model group vs. Control groups and Model group vs. KHJSC. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

(A) Bioinformatics analysis for GO annotation of differentially expressed proteins in acute pharyngitis rabbits; (B) Bioinformatics analysis for GO enrichment of differentially expressed proteins in acute pharyngitis rabbits; (C) Bioinformatics analysis for KEGG pathway enrichment of differentially expressed proteins in acute pharyngitis rabbits; (D) Protein-protein interactions among differentially expressed proteins analyzed using the String database.

Fig. 7

The Elisa analysis demonstrated the expression levels of PI3K (A), AKT (B), NF-κB (C) and TLR2 (D) in the Control, Model, KHJSC groups. Data were expressed as the mean ± SD. ^##P < 0.01 vs. Control, **P < 0.01 vs. Model.

Fig. 8

The chemical structure and formula of Bergeninum (A), Matrine (B), Sophocarpine (C) and Genistein (D).