Steamed panax notoginseng mitigates CA-MRSA USA300-induced necroptosis in human neutrophils

Abstract

Background: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) disrupts innate immunity by inducing necroptosis in polymorphonuclear neutrophils (PMNs), a process linked to excessive inflammation and tissue damage. CA-MRSA releases virulence factors that enhance its pathogenicity by disrupting the host's innate immune response, particularly impairing the phagocytic function of PMNs. Steamed Panax notoginseng (S-PN), a traditional Chinese medicine (TCM), has demonstrated immune-regulatory and anti-inflammatory properties, showing promising therapeutic effects in alleviating the severe inflammatory responses induced by pathogenic microbial infections.

Objective: This study aims to investigate the pharmacological effects and mechanisms of S-PN alleviating CA-MRSA-induced PMN necroptosis by suppressing MRSA virulence factors and inhibiting the RIPK1/RIPK3/MLKL signaling pathway, thereby attenuating inflammatory damage.

Methods: A co-culture model of MRSA USA300 strain and PMNs isolated from healthy human blood was established to observe the changes in necroptosis marker HMGB1, PMNs counts, ROS, chemokine MCP-1 and pro-inflammatory cytokines IL-1β, IL-8, TNF-α. RNA-seq was employed to analyze the effects of S-PN on the transcriptional expression of pathogenesis-related genes of MRSA. RT-PCR was utilized to validate the expression of S-PN on MRSA virulence factors and PMNs necroptosis related genes.

Results: S-PN significantly inhibited HMGB1, ROS, MCP-1, IL-1β and IL-8 in MRSA-PMN co-cultures, the PMN count in the S-PN group was higher than that in the model group. S-PN downregulated MRSA pathogenic-associated S. aureus infection and quorum sensing signaling pathways, and significantly reduced the virulence factors PSM and PVL. S-PN suppressed the expression of genes associated with necroptosis ripk1, ripk3, and mlkl in PMNs.

Conclusion: S-PN alleviates CA-MRSA infection-induced immune damage through dual mechanisms: suppression of bacterial virulence factors (PSM and PVL) and inhibition of PMNs necroptosis. These findings underscore its potential as a complementary therapeutic strategy against CA-MRSA infections, providing a theoretical foundation for integrating TCM into adjuvant treatments for drug-resistant bacterial infections.

Keywords: CA-MRSA; necroptosis; polymorphonuclear neutrophils; steamed Panax notoginseng; virulence factors.

FIGURE 1

Effect of S-PN on PMNs. (A) Cytotoxicity of S-PN on PMNs. (B) Impact of S-PN on HMGB1 level released by PMNs in the MRSA-PMN model. (C) Expression levels of inflammatory cytokines in the MRSA-PMN model. (D,E) Flow cytometry analysis of the proportion of total PMNs in each group (n = 3). (F,G). Flow cytometry analysis of ROS levels in each group (n = 3).

FIGURE 2

Effect of S-PN on MRSA Gene Transcription. (A) Principal Component Analysis (PCA) showing significant differences in gene transcription between MRSA with S-PN intervention (S-PN group) and MRSA without intervention (Control group). (B) The volcano plot. S-PN vs. Control, with red indicating significantly upregulated genes and blue indicating significantly downregulated genes (P < 0.05, |log₂ FC| > 1). The top 10 genes with the most significant changes are labeled on the plot. (C,D). KEGG pathway analysis showing significantly upregulated and downregulated pathways (E,F). The difference expression levels of hub gene in each group in Staphylococcus aureus infection pathway and Quorum sensing pathway using STAMP.

FIGURE 3

Effect of S-PN on MRSA virulence factors PSM and PVL expression. (A) The mRNA expression levels of MRSA virulence factors psm α1, psm α2, psm α3, psm α4 and pvl. (B) The protein expression levels of MRSA virulence factors PSM and PVL.

FIGURE 4

Effects of S-PN on the Necroptotic Apoptosis Pathway in MRSA-PMNs. (A) Expression levels of HMGB1 in MRSA-PMNs. (B) Expression levels of genes associated with the necroptotic apoptosis pathway in PMNs.

FIGURE 5

Schematic of the mechanism of S-PN inhibits CA-MRSA-induced necroptosis in PMNs. (Left panel) Upon MRSA phagocytosis by PMNs, robust ROS production facilitates bacterial clearance; however, MRSA counteracts this defense through secretion of virulence factors PVL and PSMs, which activate PSM-mediated necroptosis pathways in PMNs, ultimately triggering neutrophil lysis and exacerbating inflammatory responses. (Right panel) S-PN intervention suppresses MRSA virulence factor release, thereby abrogating PMN necroptosis and mitigating pathological inflammation. PVL, Panton-Valentine Leukocidin; PSM, Phenol Soluble Modulin; ROS, Reactive Oxygen Species; RIPK1, Receptor-Interacting Protein Kinase 1; RIPK3, Receptor-Interacting Protein Kinase 3; MLKL, Mixed-Lineage Kinase Domain-Like; SMAC, Second Mitochondria-Derived Activator of Caspases; SPATA2, Spermatogenesis Associated 2; IL-8, Interleukin-8; IL-1β, Interleukin-1 Beta; HMGB1, High Mobility Group Box 1; MCP-1, Monocyte Chemoattractant Protein-1. Image created by Figdraw.