NOSs x Reactive oxygen species

The tumor microenvironment plays a critical role in cancer progression, with tumor-associated macrophages regulating immune responses. These macrophages can adopt a pro-inflammatory M1 phenotype that suppresses tumor growth or an anti-inflammatory M2 phenotype that promotes progression. Reprogramming macrophages toward the M1 phenotype is a therapeutic strategy. Previous studies showed that 4-Fluorophenylacetamide-acetyl coumarin (4-FPAC), a synthetic coumarin derivative, exhibits cytostatic activity in A549 lung carcinoma cells by modulating reactive oxygen species (ROS), nitric oxide synthase, and signaling pathways, including PI3K/AKT/NF-κB. This study evaluates the impact of 4-FPAC on macrophage polarization.

We hypothesized that 4-FPAC promotes M1 macrophage polarization while suppressing M2 markers through modulation of signaling pathways, thus serving as an immunomodulatory agent.

Treatment with 4-FPAC induced M1 polarization in THP1-derived macrophages, evident from morphological elongation, elevated ROS and NO production, and increased IL-12 levels. IL-10 levels and M2 markers (CD163, STAT3, AKT1) were downregulated, while M1 markers (CD80, STAT1, AKT2) were upregulated. Gene expression and western blot analyses revealed activation of P38 and NF-κB pathways and reduced phosphorylated AKT1 levels. In silico docking showed strong interactions of 4-FPAC with regulatory proteins like P38, NF-κB, and AKT1, suggesting pathway modulation.

4-FPAC facilitates M1 macrophage polarization and inhibits M2 signaling, demonstrating its potential as an immunomodulatory agent. Coupled with its cytostatic effects on A549 cells, these findings position 4-FPAC as a promising candidate for cancer therapy. Further in vivo studies are warranted to validate its therapeutic potential and explore applications in immunotherapy and inflammation-associated diseases.

Breast cancer (BC) is a prevalent malignancy among women with strong heterogeneity. This study is designed to investigate the therapeutic effects of Huanglian Jiedu Tang (HLJDT) on BC progression and reveal its potential mechanism.

The effects of different concentrates of HLJDT on the viability, proliferation, migration, and invasion of BC cells were measured by CCK-8 assay, EdU staining, scratch test, and transwell assay, respectively. The levels of inflammatory factors and oxidative stress indicators were detected by ELISA. RhoA/ROCK pathway related protein was measured by western blot. A xenograft tumor model was constructed to explore the effects of HLJDT in vivo. Hematoxylin eosin staining was exploited to observe the pathological changes. Tumor proliferation was detected by immunohistochemistry (Ki67). Macrophage markers were detected by flow cytometry and RT-PCR. Furthermore, the ROCK agonist was used for feedback functional experiments.

There was a concentration-dependent increase in the inhibitory impact of HLJDT on the viability, proliferation, migration, and invasion of BC cells (MCF-7 and MDA-MB-23). Moreover, HLJDT inhibit inflammation (TNF-α, IL-6 and IL-1β), oxidative stress (ROS, MDA and GSH) and RhoA/ROCK pathway. HLJDT regulated inflammatory microenvironment in BC, with increased IL-10, IL-4 levels and reduced TGF-β. In vivo, HLJDT restrained the tumor growth and proliferation, diminishing inflammatory infiltration and reducing malignancy. Additionally, HLJDT significantly increased F4/80 ⁺CD86 and iNOS levels, as well as the decreased F4/80 ⁺CD163 ⁺ and Arg1 levels. The treatment of ROCK agonist weakened the inhibitory effects of HLJDT on inflammation and BC progression.

HLJDT may regulate inflammatory microenvironment to suppress BC progression through inhibiting the RhoA/ROCK pathway.

Osteoporosis (OP) represents a systemic disease causing reduced bone mass and fragility fractures. Qigu Capsule (QGC), a traditional Chinese medicine, shows potential in alleviating human OP, but its precise mechanisms remain unclear, limiting clinical application.

The bioactive components of QGC were analyzed using high-performance liquid chromatography (HPLC). An ovariectomy (OVX)-provoked OP rat model was established to evaluate QGC's effects on bone mass, trabecular architecture, and mechanical strength using micro-CT, histological staining, and biomechanical testing. RNA-seq analysis of human OP-derived mesenchymal stem cell (MSC) samples was performed to identify oxidative stress (OxS)- and senescence-associated gene changes. OxS-induced MSC senescence was modeled in vitro using H₂O₂, and QGC's effects on MSC proliferation, migration, and osteogenic differentiation were assessed. Network pharmacology (NP) was deployed to predict the key mechanisms behind the QGC treatment of OP. Further mechanistic studies utilized pharmacological inhibitors and siRNA-mediated gene knockdown to confirm the involvement of critical signaling pathways.

HPLC-MS analysis identified 505 unique bioactive compounds in QGC. In vivo, QGC significantly improved BMD, enhanced trabecular microarchitecture, and restored mechanical properties in OVX rats. ELISA, histological, and immunohistochemical analyses confirmed that QGC primarily enhanced osteoblast activity. RNA-seq analysis of GEO datasets revealed upregulation of senescence and OxS markers (P53, CDKN1A, and INOS) in human OP-derived MSCs. Both in vivo and in vitro QGC alleviated OxS-induced MSC senescence, reduced reactive oxygen species (ROS) levels, suppressed senescence and OxS marker, and promoted MSC proliferation, migration, and osteogenic differentiation. Moreover, NP predicted HIF-1α signaling as critical in QGC's regulation of MSC function during OP. Mechanistic studies demonstrated that QGC activated the HIF-1α/AMPK axis, and inhibition of either HIF-1α or AMPK abolished its therapeutic effects.

QGC mitigates OxS-induced MSC senescence and promotes osteogenesis through the HIF-1α/AMPK axis, highlighting its mechanistic basis in treating OP. These findings show QGC's potential as a therapeutic agent, not only by promoting osteogenesis but also by complementing or serving as an alternative to current OP treatments, offering valuable prospects for enhanced clinical management.