Safflower Alleviates Pulmonary Arterial Hypertension by Inactivating NLRP3: A Combined Approach of Network Pharmacology and Experimental Verification

Abstract

Introduction: Traditional Chinese medicinal plant, safflower, shows effective for treating pulmonary arterial hypertension (PAH), yet the underlying mechanisms remain largely unexplored. This study is aimed at exploring the potential molecular mechanisms of safflower in the treatment of PAH.

Methods: Network pharmacology approach and molecular docking were applied to identify the core active compounds, therapeutic targets, and potential signaling pathways of safflower against PAH. Meanwhile, high-performance liquid chromatography (HPLC) assay was performed to determine the core compounds from safflower. Further, the mechanism of action of safflower on PAH was verified by in vivo and in vitro experiments.

Results: A total of 15 active compounds and 177 targets were screened from safflower against PAH. Enrichment analysis indicated that these therapeutic targets were mainly involved in multiple key pathways, such as TNF signaling pathway and Th17 cell differentiation. Notably, molecular docking revealed that quercetin (core compound in safflower) displayed highest binding capacity with NLRP3. In vivo, safflower exerted therapeutic effects on PAH by inhibiting right ventricular hypertrophy, inflammatory factor release, and pulmonary vascular remodeling. Mechanistically, it significantly reduced the expression of proangiogenesis-related factors (MMP-2, MMP-9, Collagen 1, and Collagen 3) and NLRP3 inflammasome components (NLRP3, ASC, and Caspase-1) in PAH model. Similarly, these results were observed in vitro. Besides, we further confirmed that NLRP3 inhibitor had the same therapeutic effect as safflower in vitro.

Conclusion: Our findings suggest that safflower mitigates PAH primarily by inhibiting NLRP3 inflammasome activation. This provides novel insights into the potential use of safflower as an alternative therapeutic approach for PAH.

Keywords: NLRP3 inflammation; network pharmacology; pulmonary arterial hypertension; quercetin; safflower.

FIGURE 1

Identification of active compounds and potential targets of safflower for pulmonary arterial hypertension (PAH) management. (A) A Venn diagram showcasing the intersection of 177 shared targets between PAH and safflower‐related targets. (B) The drug‐compound‐common target network. Active compounds are denoted in blue, drugs in purple, and common targets in green.

FIGURE 2

Protein–protein interaction (PPI) network. The network visualizes interactions among the 177 shared targets.

FIGURE 3

Biological function enrichment analysis. (A) The Top 10 Gene Ontology (GO) biological processes highlight the primary biological activities influenced by safflower in PAH management. (B) The Top 10 KEGG pathways underscore the essential pathways that safflower engages to combat PAH.

FIGURE 4

Core compounds and target identification and network construction. (A) PPI network depicting NLRP3 interactions. (B) The target‐pathway network, highlighting the primary pathways involving the core targets.

FIGURE 5

Molecular docking analysis illustrates potential binding modes and the reliability of interactions between core compounds and targets of safflower for PAH treatment.

FIGURE 6

Experimental validation using a monocrotaline (MCT)‐induced PAH rat model. (A) The right ventricular hypertrophy index in the PAH rat model following safflower injection (SI) treatment. (B) Expression levels of inflammatory markers (IL‐1β and IL‐18) in lung tissues post‐SI treatment. (C) Hematoxylin and eosin staining of lung tissue across different experimental groups; scale bar: 50 μm. (D, E) Immunohistochemical analysis of α‐SMA and matrix metalloproteinase‐2 and matrix metalloproteinase‐9 (MMP2 and MMP9) post‐SI treatment; scale bar: 50 μm. (F) Expression levels of Collagen 1 and Collagen 3 post‐SI treatment. (G) Western blot analysis of NLRP3 inflammasome components (NLRP3, ASC, and Caspase‐1) post‐SI treatment. **p < 0.01 versus the control group; ^# p < 0.05 and ^## p < 0.01 versus the PAH group.

FIGURE 7

Validation of the inhibitory effects of safflower on NLRP3 inflammasome activation in primary pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial cells (PAECs). (A) ELISA results indicating the expression levels of IL‐1β and IL‐18 posttreatment with SI or the NLRP3 inhibitor MCC950. (B) Quantitative real‐time PCR (qRT‐PCR) results showcasing expression levels of MMP2 and MMP9 posttreatment with SI or MCC950. (C) Western blot analysis demonstrating expression levels of NLRP3 inflammasome components (NLRP3, ASC, and Caspase‐1) posttreatment with SI or MCC950. **p < 0.01 versus the control group; ^# p < 0.05 and ^## p < 0.01 versus the MCT group.