Huanglian Jiedu Decoction Treats Ischemic Stroke by Regulating Pyroptosis: Insights from Multi-Omics and Drug-Target Relationship Analysis

Abstract

Background: Ischemic stroke (IS) is a severe condition with limited therapeutic options. Pyroptosis, a type of programmed cell death linked to inflammation, is closely associated with IS-related damage. Studies suggest inflammation aligns with the traditional Chinese medicine (TCM) concept of "fire-heat syndrome". Huanglian Jiedu Decoction (HLJD), a TCM formula known for clearing heat and purging fire, has shown therapeutic effects on IS, potentially by regulating pyroptosis. Study design: Eight-week-old male mice were divided into six groups: sham operation, model, positive drug, and low-, medium-, and high-dose HLJD groups. After a week of adaptive feeding, mice received respective treatments for five days, followed by modeling on the sixth day, with samples collected 23 h post-perfusion. Analyses included multi-omics, physiology, histopathology, virtual drug screening, target affinity assessment, and molecular biology techniques to measure relevant indicators. Results: HLJD effectively mitigated IS-related damage, maintaining neurological function, reducing ischemic levels, protecting cellular morphology, inhibiting neuronal apoptosis, and preserving blood-brain barrier integrity. Bioinformatics of high-throughput omics data revealed significant activation of pyroptosis and related inflammatory pathways in IS. ScRNA-seq identified neutrophils, macrophages, and microglia as key pyroptotic cell types, suggesting potential therapeutic targets. Network pharmacology and molecular docking identified NLRP3 as a critical target, with 6819 ligand-receptor docking results. SPR molecular fishing, LC-MS, molecular dynamics, and affinity measurements identified small molecules with high affinity for NLRP3. Molecular biology techniques confirmed that HLJD regulates pyroptosis via the classical inflammasome signaling pathway and modulates the inflammatory microenvironment. Conclusions: Following IS, pyroptosis in myeloid cells triggers an inflammatory cascade, leading to neural damage. HLJD may inhibit NLRP3 activity, reducing pyroptosis and associated inflammation, and ultimately mitigating damage.

Keywords: HLJD; drug target relationship; ischemic stroke; multiomics; myeloid cell; pyroptosis.

Figure 1

Protective effect of HLJD on cerebral infarction volume, cerebral blood flow, and nerve function defects in tMCAO mice. (A) Neurological deficit score analysis (Longa Score). (B) Detection of cerebral infarct volume. (C) The volume of cerebral infarction in mice by TTC Staining. (D) Healthy side CBF measurement. (E) CBF on the ischemic side was measured. (F) The CBF was measured by laser scattering. Results are presented as means ± SD. n = 6, one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Effect of HLJD on micropathology and BBB permeability in tMCAO mice. (A) HE in the cortex and hippocampus of tMCAO mice. (B) Nissl staining in the cortex and hippocampus of tMCAO mice. (C) BBB was tested by EB. (D) EB absorbance measurement. Results are presented as means ± SD. n = 6, one-way ANOVA, * p < 0.05.

Figure 3

Analysis of bulk RNA-seq in tMCAO mice. (A) Observe the differences in pyroptosis gene set scores between the CT group and the IS group using the ssGSEA method. (B) Heatmap presentation of differential genes. (C) Volcano map presentation of differential genes. (D) PPI network formed by 14 differential genes. (E) The core pyroptosis gene network in IS mice. (F) GO analysis of differential genes. (G) KEGG analysis of differential genes. (H) IPA analysis of differential genes.

Figure 4

Analysis of scRNA-seq in tMCAO mice. (A) UMAP plot after dimensionality reduction and clustering. (B) T-SNE plot after dimensionality reduction and clustering. (C) UMAP plot showing cell type annotations after clustering. (D) MARKER genes of each cell type. (E) Differential genes of each cell type. (F) Proportional changes in each cell type.

Figure 5

Subpopulation analysis of microglia, macrophages, and neutrophils. (A) Scores of the pyroptosis gene set in various cell types. (B) GO analysis of different genes in various cells. (C) KEGG analysis of different genes in various cells. (D) Metabolic analysis of microglia, macrophages and neutrophils. (E) The number of cell communications. (F) Cell communication in sham group. (G) Cell communication in m group. (H) Proportion of the number of microglia subsets in each group. (I) Pyroptosis gene set scores in subtypes of microglial cells. (J) The ratio of P- microglia and P+ microglia in sham group and mcao group. (K) Distribution of microglia in sham group and mcao group on the pseudo-time axis.

Figure 6

Molecular docking mode, SPR fishing diagram, and LC-MS diagram. (A) NLRP3-Kihadanin A docking diagram. (B) NLRP3-Kihadanin B docking diagram. (C) NLRP3-Obacunone docking diagram. (D) SPR fishing combined resonance unit performance chart with blank chip control. (E) NLRP3 protein binding chip control SPR fishing binding resonance unit performance map. (F) LC-MS cationic material diagram. (G) LC-MS anionic material diagram.

Figure 7

NLRP3 molecular dynamic binding of nine small molecules to RMSD determined by LC-MS. (A) RMSD of NLRP3-Chrysin-7-O-Glucuronide. (B) RMSD of NLRP3-Dictamine. (C) RMSD of NLRP3-Isomartynoside. (D) RMSD of NLRP3-Matrine. (E) RMSD of NLRP3-Neochlorogenic Acid. (F) RMSD of NLRP3-Puerarin. (G) RMSD of NLRP3-Rutin. (H) RMSD of NLRP3-Skullcapflavone II. (I) RMSD of NLRP3-Wogonoside.

Figure 8

The affinity of NLRP3 to eight small molecules (available) derived from LC-MS detection was determined using SPR. (A) NLRP3-Chrysin-7-O-Glucuronide. (B) NLRP3-Dictamine. (C) NLRP3-Matrine. (D) NLRP3-Neochlorogenic Acid. (E) NLRP3-Puerarin. (F) NLRP3-Rutin. (G) NLRP3-Skullcapflavone II. (H) NLRP3-Wogonoside.

Figure 9

Pyroptosis-related mRNA and protein expression levels. (A) Pyroptosis-related mRNA expression levels. (B) WB plot and protein expression levels. (C) IL-1β protein expression levels. (D) IL-18 protein expression levels. Results are presented as means ± SD. n = 6, one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 10

Expression levels of various inflammatory factors in blood and brain. (A) The expression level of a variety of inflammatory factors in blood. (B) The expression level of a variety of inflammatory factors in the brain. Results are presented as means ± SD. n = 3, one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 11

After IS occurs, microglia, macrophages, and neutrophils become activated. Macrophages and neutrophils infiltrate the brain and, along with microglia, polarize into M1 and N1 pro-inflammatory phenotypes. These cells undergo pyroptosis, releasing inflammatory factors, cellular contents, and chemokines, which ultimately damage neurons and endothelial cells, impair neural function, disrupt the BBB, and further activate and recruit immune cells, exacerbating cerebral ischemia. The small molecules in HLJD can bind to NLRP3, inhibiting its function, thus disrupting the self-amplifying cycle of pyroptosis, reducing the production of GSDMD-N and mature IL-1β and IL-18, mitigating the progression of pyroptosis, and providing neuroprotective effects.