Tinosporae Radix attenuates acute pharyngitis by regulating glycerophospholipid metabolism and inflammatory responses through PI3K-Akt signaling pathway

Abstract

Introduction: With the onset of the COVID-19 pandemic, the incidence and prevalence of acute pharyngitis (AP) have increased significantly. Tinosporae Radix (TR) is a vital medication utilized in the treatment of pharyngeal and laryngeal ailments, especially AP. The study endeavors to explore unclear molecular mechanisms of TR in addressing AP.

Methods: Network pharmacology and metabolomics analyses of effect of TR on AP were conducted, and apossible pathway was validated both in vivo using the acute pharyngitis rat model and in vitro using the LPS-induced RAW264.7 cells model, through techniques such as histopathological examinations, immunohistochemical technology, ELISA, RT-qPCR, and Western blotting to systematically explore the possible mechanisms underlying the inhibition of AP by TR.

Results and discussion: Network pharmacology analysis identified several key targets, including PIK3CA, IL6, AKT1, TNF, and PTGS2, alongside pivotal signaling pathways such as IL-17, TNF, Hepatitis B, nuclear factor kappa B (NF-κB), Influenza A, and the PI3K-Akt pathway. Most of them are closely associated with inflammation. Then, wide-target metabolomics analysis showed that TR downregulated substances within the glycerophospholipid metabolic pathway, and modulated the PI3K-Akt pathway. The integrated findings from network pharmacology and metabolomics underscored the pivotal role of the PI3K-Akt signaling pathway and the attenuation of inflammatory responses. Finally, in vitro and in vivo experiments have shown that TR can inhibit inflammatory factors such as IL-6, TNF - α, and COX-2, downregulate targets such as PI3K and AKT on the PI3K-Akt signaling pathway, and thereby alleviate the inflammatory response of AP. Our study demonstrated that TR exerts an anti-AP effect through suppression of release of inflammatory factors and modulation of glycerophospholipid metabolism via suppressing the PI3K-Akt signaling pathway.

Keywords: PI3K-Akt signaling pathway; Tinosporae Radix; acute pharyngitis; glycerophospholipid metabolism; inflammatory responses.

FIGURE 1

Network pharmacological analysis of the potential mechanisms of action of TR in acute pharyngitis. (A) PPI analysis; (B) GO enrichment analysis; (C) KEGG pathway analysis; (D) compound-target-pathway network. (MOL1, tetrahydropalmatine; MOL2, columbamine; MOL3, menisperine; MOL4, magnoflorine; MOL5, fibleucin; MOL6, palmatine; MOL7, jatrorrhizine; MOL8, reticuline; MOL9, neoechinulin A; MOL10, columbin; MOL11, ecdysterone; MOL12, tinoside; MOL13, 2-deoxy-20-hydroxyecdysone-3-O-glucopyranoside; MOL14, stearic acid; MOL15, palmitic acid; MOL16, 2-deoxy-20-hydroxyecdysone; MOL17, tinophylloloside. hsa04933, AGE-RAGE signaling pathway in diabetic complications; hsa04657, IL-17 signaling pathway; hsa04668, TNF signaling pathway; hsa05167, Kaposi sarcoma-associated herpesvirus infection; hsa05161, Hepatitis B; hsa04064, NF-kappa B signaling pathway; hsa05163, Human cytomegalovirus infection; hsa05166, Human T-cell leukemia virus 1 infection; hsa05164, Influenza A; hsa04151, PI3K-Akt signaling pathway).

FIGURE 2

Therapeutic effects of TR on the AP rat model. (A) The epigenetic status of each group’s pharynx; (B) histological evaluation was performed by HE staining (original magnification ×10); (C) immunohistochemical staining (magnification, ×40) of IL-6 in the pharynx sections; (D) immunohistochemical staining (magnification, ×40) of COX-2 in the pharynx sections; (E) The weight changes of rats during modeling and dosing (n = 10); (F) expression level of IL-6 in the pharynx (n = 3); (G) expression level of COX-2 in the pharynx (n = 3). The data was expressed as the mean ± SD. ##p < 0.01 versus the Control group, *p < 0.05, **p < 0.01 versus the Model group.

FIGURE 3

Serum metabolomics analysis of rats with acute pharyngitis. (A–D) PCA and OPLS-DA analysis for discriminating the metabolic signatures. (A) PCA analysis for three groups. (B) OPLS-DA analysis for the control group and model group. (C) OPLS-DA analysis for the TR group and model group. (D) differential metabolite volcano plot for Normal versus Model groups. (E) differential metabolite volcano plot for TR versus Model groups. (F) heatmap of the 25 most discriminating metabolites. (G) Metabolic pathway analysis of potential biomarkers.

FIGURE 4

Common mechanistic analysis integrating network pharmacology analysis (NPA) and metabolomics analysis (MA). (A) A total of 114 pathways were enriched in KEGG analysis of network pharmacology, 71 metabolic pathways were selected in metabolomics, and 11 signaling pathways coincided. (B) The rank of 11 intersection pathways in network pharmacology. (C) The rank of 11 intersection pathways in metabolomics.

FIGURE 5

TR regulated inflammatory cytokines in AP rats. (A) Serum levels of TNF-α were detected by ELISA (n = 10). (B) Serum levels of IL-6 (n = 10). (C) The mRNA levels of IL-6 in the pharynx were detected by RT-PCR (n = 3). (D) The mRNA levels of TNF-α in the pharynx (n = 3). (E) The mRNA levels of COX-2 in the pharynx (n = 3). Data was presented as mean ± SD. Statistic difference is indicated as ##p < 0.01 versus the Control group, *p < 0.05, **p < 0.01 versus the Model group.

FIGURE 6

TR regulating PI3K-Akt signaling pathways in AP rats. (A) The mRNA levels of PI3K in pharynx were detected by RT-PCR (n = 3). (B) The mRNA levels of Akt in pharynx (n = 3) (C) The mRNA levels of NF-κB in pharynx (n = 3) (D–F) Phosphorylated PI3K and phosphorylated Akt protein levels in pharynx were detected by Western blotting. The data was expressed as the mean ± SD. ##p < 0.01 versus the Control group, *p < 0.05, **p < 0.01 versus the Model group.

FIGURE 7

Effects of the TR extract on inflammatory factors and PI3K-Akt signaling pathway in LPS-induced RAW264.7 macrophages. (A) Effect of TR on LPS-induced macrophage cell activity for 24 h. (B) Effect of TR on LPS-induced macrophage cell activity for 72 h. (C, D) Effects of TR on the secretion of IL-6 and TNF-α in RAW264.7 cells induced by LPS (n = 3). (E–G) The mRNA levels of IL-6, TNF-α, and COX-2 (n = 3). (H–J) The phosphorylation of PI3K and Akt was detected via Western blotting. The data was expressed as the mean ± SD of three independent experiments. ##p < 0.01 versus the Blank group, *p < 0.05, **p < 0.01 versus the LPS group.

FIGURE 8

Mechanism of TR in combating acute pharyngitis.