Yuye Jinhua Qingre Tablets Attenuate Acute Pharyngitis by inhibiting the Complement Cascade and C5a/C5aR1 Axis

Abstract

Background: Acute pharyngitis (AP) is a common upper respiratory tract infection, primarily characterized by symptoms such as throat pain, redness, swelling, and difficulty swallowing. It is typically caused by viral infections, bacterial infections, or physical and chemical irritants. Yuye Jinhua Qingre Tablets (YYJH) are recognized for their ability to clear heat, detoxify, reduce swelling, and alleviate pain, making them a common treatment option for acute pharyngitis. However, research on their specific mechanisms of action is still inadequate.

Methods: Using UPLC-Q-Exactive-Orbitrap-MS technology combined with serum pharmacochemical analysis, the main chemical components and blood components of YYJH were identified. The anti-inflammatory activity was verified through the ammonia-induced acute pancreatitis (AP) model in SD rats and the LPS-stimulated NP69SV40T cell inflammation model. Integrating transcriptomics, proteomics, and bioinformatics analysis revealed the mechanism of YYJH in treating AP, which was further validated by molecular biology experiments.

Results: Twelve blood-entry components were identified, and their anti-inflammatory effects were validated using the SD rat acute pancreatitis (AP) model and the NP69SV40T cell inflammation model. The study results indicated that the drug significantly improved the pathological damage of the pharyngeal mucosa in rats with the AP model, reducing the levels of inflammatory cells in peripheral blood and serum inflammatory factors. The combined analysis of transcriptomics and Astral DIA proteomics revealed that the anti-inflammatory effects of YYJH are associated with the regulation of the classical complement pathway, characterized by the downregulation of complement components C1q, C3, C5, C9, and the modulation of macrophage infiltration and pro-inflammatory cytokine release through the C5a/C5aR1 axis. Gene set enrichment analysis further suggested that YYJH can alleviate AP-related metabolic disorders and immune dysregulation. Molecular biology experiments demonstrated that after YYJH intervention, the complement cascade reaction was significantly inhibited, with downregulated expression levels of C5a and C5aR1, and decreased membrane localization signals of the macrophage marker F4/80, along with reduced expression levels of inflammatory factors.

Conclusions: Research indicates that YYJH exerts anti-inflammatory effects by regulating the classical complement pathway and the C5a/C5aR1 axis, inhibiting the production of inflammatory mediators and the activation of immune cells. This provides a theoretical basis for the molecular mechanisms underlying traditional Chinese medicine in the treatment of acute pharyngitis.

Keywords: Acute pharyngitis; C5a/C5aR1 axis; Complement pathway; Yuye Jinhua Qingre Tablets.

Fig. 1

Schematic diagram of experimental protocol for YYJH treatment of AP rats

Fig. 2

Twelve blood-entry components were identified in YYJH. A Total ion chromatogram (TIC) of drug containing serum; B Classification and statistical chart of 12 constituents absorbed into blood; C The chemical structural formulas of 12 constituents absorbed into blood

Fig. 3

YYJH intervenes in multiple pathophysiological indicators in vivo. A Changes in body weight of rats across each group throughout the experiment, n = 9; B Food intake of rats in each group during the 3-day modeling period, n = 3; C Apparent index scores of rats in each group during the 3-day modeling period, n = 9; D Apparent index scores of rats in each group following 5 days of drug administration, n = 9; E Number of white blood cells and differential counts in the blood of rats in each group, n = 3; F H&E staining results of pharyngeal tissue in rats from each group (bar = 50 μm), n = 6; G Changes in serum inflammatory factor levels in rats across each group, n = 3; H Effects of YYJH-containing serum on inflammatory factor levels (YYJH represents medicated serum), n = 3. Data were presented as mean ± SD. Compared to control: ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001. Compared to model: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

Exploring the mechanism of YYJH treatment for AP based on transcriptome sequencing. A Box plot of normalized raw data; B Heatmap of sample correlation analysis; C Volcano plot of differentially expressed genes between the model group and the normal group; D MA plot of differentially expressed genes between the model group and the normal group; E Heatmap of clustering of differentially expressed genes between the model group and the normal group; F, G Enrichment results of differentially expressed genes between the model group and the normal group; H Enrichment results of up-regulated differentially expressed genes between the model group and the normal group; I Enrichment results of down-regulated differentially expressed genes between the model group and the normal group. NC normal group, N normal group, MX model group, L low dose group of YYJH, M medium dose group of YYJH, H high dose group of YYJH

Fig. 5

Results of GO and KEGG enrichment analysis of DEPs. A GO enrichment analysis results of differentially expressed proteins in acute pharyngitis; B KEGG enrichment analysis results of differentially expressed proteins in acute pharyngitis; C KEGG enrichment analysis results of up-regulated and down-regulated proteins in acute pharyngitis; D GO enrichment analysis results of differentially expressed proteins in acute pharyngitis treated with Yuye Jinhua Qingre Tablets; E KEGG enrichment analysis results of up-regulated and down-regulated proteins in acute pharyngitis treated with Yuye Jinhua Qingre Tablets; F KEGG enrichment analysis results of differentially expressed proteins in acute pharyngitis treated with Yuye Jinhua Qingre Tablets

Fig. 6

Key molecular screening and functional pathway enrichment analysis results of YYJH regulation of AP abnormal expression. A Venn diagram of YYJH-regulated AP abnormally expressed gene screening; B Bar chart of functional pathway enrichment for YYJH-regulated AP abnormally up-regulated and down-regulated genes; C Venn diagram of YYJH-regulated AP abnormally expressed protein screening; D Bar chart of functional pathway enrichment for YYJH-regulated AP abnormally up-regulated and down-regulated proteins; E Venn diagram of YYJH-regulated AP abnormally expressed key molecule screening; F PPI network diagram of key molecules; G Bubble chart of functional and pathway enrichment for key molecules. NC normal group, MX model group, H high dose group of YYJH

Fig. 7

Gene set enrichment analysis results. A Bubble chart of enriched abnormally expressed genes in AP; B Enrichment network diagram; C Enrichment GSEA plot; D Bubble chart of gene set differences between abnormally expressed genes in AP and YYJH-treated abnormally expressed genes in AP; E Heatmap of gene set differences; F Bubble chart of enrichment differences in gene sets between abnormally expressed genes in AP and YYJH-treated abnormally expressed genes in AP; G Heatmap of gene set differences. N2 normal group, MX model group, MX1 model group, H high dose group of YYJH

Fig. 8

YYJH inhibits activation of the classical complement pathway. A Western blot results of complement pathway-related proteins; B Statistical results of expression levels of complement pathway-related proteins; C RT-qPCR experimental results. Data were presented as mean ± SD, n = 3. Compared to control: ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001. Compared to model: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 9

YYJH inhibits the activation of complement C5a/C5aR1 axis to exert anti-inflammatory effects. A IF experimental results of YYJH intervention on the expression of molecules related to the complement pathway (bar = 20 μm); B Western blot experimental results of YYJH intervention on the expression levels of molecules related to the complement pathway; C ELISA detection results of YYJH intervention on the expression levels of molecules related to the complement pathway; D IHC experimental results of YYJH regulation on the levels of inflammatory factors (bar = 20 μm); E Western blot experimental results of YYJH regulation on the expression levels of inflammatory factors; F IF experimental results of YYJH regulation on the expression of macrophage markers (bar = 50 μm); G Western blot experimental results of YYJH regulation on the expression levels of molecules related to macrophage activation. Data were presented as mean ± SD, n = 3. Compared to control: ^#p < 0.05, ^##p < 0.01, ^###p < 0.001. Compared to model: *p < 0.05, **p < 0.01

Fig. 10

YYJH inhibits the activation of complement C5a/C5aR1 axis to exert anti-inflammatory effects