Network pharmacology integrated with experimental validation reveals the mechanism of Xanthii Fructus against allergic rhinitis via JAK2/STAT3/HIF-1α signaling pathway

Ethnopharmacological relevance: As a natural medicine, Xanthii Fructus (XF) is widely used in traditional Chinese medicine (TCM) and Tibetan medicine. It has been demonstrated to alleviate allergic rhinitis (AR) in modern research. However, the specific molecular mechanism underlying its treatment of AR is still unclear.

Aim of study: To elucidate the effect and mechanism of XF in treating AR through network pharmacology and experimental validation.

Methods: In the present study, blood-entry components of XF were analyzed using UPLC-Orbitrap-HRMS. Then, we conducted pharmacodynamic studies in vitro and vivo. In vitro study, Human IL-4 was used to treat HNEpCs cells to establish a vitro model. Subsequently, HNEpCs cells were administrated with XF extracts (0.5, 1, 2 mg/ml). And ovalbumin (OVA) was employed to establish an allergic rhinitis model, and different doses of XF (8, 16, 32 mg/kg) were administered by gavage to BABL/c mice for in vivo experiments. Next, the Swiss Target Prediction database was employed to acquire blood-entry components targets. Meanwhile, from OMIM and GeneCards databases, AR-related targets were obtained. A protein-protein interaction (PPI) network was established through the STRING database, and potential pathways of XF were identified through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. In the end, the results of network pharmacology were experimental validation in vivo and vitro experiments.

Results: Fifteen compounds were identified, most of which were phenolic acids. In pharmacodynamic studies, the vitro study revealed that XF-treated gave rise to a significant decline of iNOS and COX2 protein expression in inflammatory conditions, as evidenced by Western blot results, and there was a sharp decline in the mRNA levels of TNF-α, IL-1β and IL-6. Meanwhile, the vivo studies demonstrated that XF exhibited favorable therapeutic efficacy against AR, as evidenced by a decrease in IgE, TNF-α, IL-4, and IL-6 levels in mice serum, an improvement in nasal mucosal injury pathology. Based on these findings, through network pharmacology, we identified 14 core AR-related targets, including HIF-1α, STAT3, TLR4. Using KEGG pathway analysis, it has been revealed that XF can alleviate AR through JAK2/STAT3/HIF-1α signaling pathway. Therefore, further experiments were conducted to verify the molecular mechanism of the anti-AR effect of XF. A decline of the phosphorylation of JAK2, STAT3 and HIF-1α proteins was observed, which resulted in the suppression of JAK2/STAT3/HIF-1α signaling pathway. These findings were corroborated by the same results obtained through IF. The results were verified by RT-qPCR, which demonstrated that XF was capable of downregulating the mRNA levels of TSLP and CCL11. Then, the conclusions were further reinforced with the introduction of WP1066. It could be observed that XF inhibited the STAT3 nuclear translocation. Finally, a restoration of p-JAK2, p-STAT3, HIF-1α expression levels to normal levels in AR mice.

Conclusion: The combined findings led to the conclusion that XF play its therapeutic role in AR by suppressing the JAK2/STAT3/HIF-1α signaling pathway.

Allergic rhinitis; JAK2/STAT3/HIF-1α; Nasal inflammation; Network pharmacology; Xanthii Fructus.