Alcoholic liver disease (ALD), a pervasive global health concern, involves multifaceted pathological mechanisms including oxidative stress, lipid dysregulation and inflammatory responses, yet lacks effective multi-target therapies. Carnosic acid (CA), a natural diterpenoid with extensively characterized antioxidant and anti-inflammatory properties, remains underexplored for its systemic therapeutic potential in ALD. This study aimed to comprehensively investigate CA’s hepatoprotective efficacy and molecular mechanisms against ALD. Results demonstrated that CA significantly reduced serum total cholesterol, triglycerides, and transaminase levels in ALD mice, concurrently alleviating alcohol-induced histopathological damage. Network pharmacology identified 36 potential targets, with 10 core targets predominantly enriched in lipid metabolism, PPAR signaling and inflammatory response. Molecular docking revealed superior binding affinities of CA to core targets compared to silibinin, a reference hepatoprotectant. RT-qPCR validation further confirmed CA’s regulatory effects on apoptosis-related TP53/BCL2 axis, TNF-mediated inflammatory pathways and cytochrome P450 (CYP450) metabolic genes. Through a tripartite strategy integrating phenotypic validation, mechanistic prediction, and experimental confirmation, this study elucidates CA’s multi-target therapeutic actions against ALD via coordinated modulation of lipid homeostasis, oxidative stress and inflammatory pathways, providing the groundwork for future mechanism exploration.

Macrophage polarization is a pivotal determinant in the modulation of immune responses and the formation of the tumor-microenvironment (TME), therefore represents a promising avenue for the development of cancer immunotherapy. Recent research highlights the potential of edible mushroom polysaccharides in inducing M1/M2 polarization of macrophages. Here, we report that oral administration of Flammulina (F.) velutipes polysaccharide (FVP) reduced the tumor volume of grafted 4T1 mammary and lewis lung carcinoma (LLC), and increased the overall survival rate of mice. Moreover, this polysaccharide induced the generation of CD11b⁺Ly6C^highF4/80⁺ macrophage cells in LLC and 4T1 tumor-bearing mice. Further investigation found that FVP decreased the ratio of M2-macrophages and regulatory T cells in TME, which is known to support tumor growth and metastasis. Mechanistically, FVP reversed the IL-4 induced M2-polarization by activating the TLR4/ NF-κB axis, and in vitro assays confirmed the expression of pro-inflammatory genes and the antitumor effects of the polysaccharide. Additionally, the expression of Th1 response relative genes also enhanced by FVP polarized macrophage secretions. In summary, these results suggest that FVP may represent a promising immunomodulatory agent for the reversal of macrophage polarization.

Vascular oxidative stress serves as a pathological foundation for various vascular injury-related diseases, including atherosclerosis, hypertension, restenosis, and abdominal aortic aneurysms. Recent studies have indicated that intestinal flora-derived metabolites, especially phenylacetylglutamine (PAGln) and phenylacetylglycine (PAGly), may contribute to the promotion of thrombosis, heart failure, and other related conditions. Aucubin (AU), an iridoid glycoside, has been shown to exhibit anti-cardiovascular properties. Nevertheless, the precise role and underlying mechanisms by which AU mitigates PAGly-induced vascular injury remain poorly understood. Our results indicated that PAGln/PAGly promoted oxidative stress in vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in vitro and in vivo. Network pharmacology suggest that AU may possess the capacity to regulate lipid and atherosclerosis, and reactive oxygen species (ROS) processes. We found that AU penetrated the blood vessels and mitigated oxidative stress induced by PAGln/PAGly. Mechanistically, combining the results from intersection analysis between the targets of AU and vascular diseases and molecular docking, we found that tumor necrosis factor (TNF) may be the potential target of AU. Further DARTS and molecular docking analysis demonstrated that AU bound to recombinant TNF-α, and AU could interact with multiple amino acid residues of TNF-α, including Asn-92 and Phe-144. Additionally, PAGly upregulated the level of soluble TNF-α (sTNF-α) in mouse VSMCs and plasma, and promoted the interaction between sTNF-α and TNF receptor 1 (TNFR1), whereas AU inhibited this interaction. Both AU and Infliximab, a specific monoclonal antibody of TNF-α, inhibit TNF-α-induced ROS production. In summary, our results revealed that TNF-α is a cellular target of AU, and the interaction between AU and sTNF-α may mitigate PAGln/PAGly-induced vascular oxidative stress by inhibiting the interaction of TNF-α-TNFR1.

Intermittent fasting can benefit breast cancer patients undergoing chemotherapy or immunotherapy. However, it is still uncertain how to select immunotherapy drugs to combine with intermittent fasting. Herein we observed that two cycles of fasting treatment significantly inhibited breast tumor growth and lung tissue metastasis, as well as prolonged overall survival in mice bearing 4T1 and 4T07 breast cancer. During this process, both the immunosuppressive monocytic- (M-) and granulocytic- (G-) myeloid-derived suppressor cell (MDSC) decreased, accompanied by an increase in interleukin (IL) 7R⁺ and granzyme B⁺ T cells in the tumor microenvironment. Interestingly, we observed that Ly6G^low G-MDSC sharply decreased after fasting treatment, and the cell surface markers and protein mass spectrometry data showed potential therapeutic targets. Mechanistic investigation revealed that glucose metabolism restriction suppressed the splenic granulocyte-monocyte progenitor and the generation of colony-stimulating factors and IL-6, which both contributed to the accumulation of G-MDSC. On the other hand, glucose metabolism restriction can directly induce the apoptosis of Ly6G^low G-MDSC, but not Ly6G^high subsets. In summary, these results suggest that glucose metabolism restriction induced by fasting treatment attenuates the immune-suppressive milieu and enhances the activation of CD3⁺ T cells, providing potential solutions for enhancing immune-based cancer interventions.

Benzo[a]pyrene (B[a]P) is a food contaminant toxic for cardiovascular diseases. The nuclear translocation of Arylhydrocarbon receptor (AhR) plays an important role in B[a]P-induced oxidative stress and vascular diseases. We confirmed that B[a]P promoted ROS production in vascular smooth muscle cells (VSMCs) in vitro and in vivo, associated with the nuclear translocation of AhR. It is known that phosphorylation inhibits while dephosphorylation of AhR promotes nuclear translocation of AhR. However, from the posttranslational modification level, the mechanism by which B[a]P activates and regulates the nuclear translocation of AhR is unclear. Co-immunoprecipitation results showed that cytoplasmic AhR was phosphorylated before B[a]P stimulation, and switched to O-GlcNAcylation upon B[a]P 1-h stimulation in VSMCs, suggesting there may be a competitively inhibitory relationship between O-GlcNAcylation and phosphorylation of AhR. Next, siRNAs of O-linked N-acetylglucosamine transferase (OGT), O-GlcNAcase (OGA) and OGA inhibitor PUGNAc were used. SiOGT blocks but siOGA and PUGNAc promote B[a]P -dependent AhR nuclear translocation and oxidative stress. Ser11 may be the competitive binding site for phosphorylation and O-GlcNAcylation of AhR. Phosphorylation-mimic variant inhibits but O-GlcNAcylation of AhR promotes AhR nuclear translocation and oxidative stress. Our findings highlight a new perspective for AhR nuclear translocation regulated by the competitive modification between phosphorylation and O-GlcNAcylation.

The increased vascular inflammation is a key event in the development of atherosclerotic lesions. Antrodia cinnamomea has been shown to promote anticancerogenic activity through decreasing inflammation. However, the potential role of A. cinnamomea in cardiovascular diseases remains unexplored. Herein, using carotid arterial ligation models, we found that ethanol extract from A. cinnamomea (EEAC) significantly inhibited neointimal hyperplasia in a dose-dependent manner, accompanied with the reduced expression of activated p65 and inflammatory cytokines. We also show that EEAC ameliorated TNF-α-induced phosphorylation of p65 and pro-inflammatory cytokine expression in both vascular smooth muscle cells (VSMCs) and macrophages in vitro. Mechanistically, EEAC suppressed expression levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1) in VSMCs, which attenuates the ability of monocytes/macrophages adhesion to VSMCs. Furthermore, the expression level of these adhesion molecules and infiltration of monocytes/macrophages were also decreased in neointimal VSMCs of arteries pretreated with EEAC. Altogether, our results reveal a novel function of A. cinnamomea in suppressing vascular inflammation upon ligation injury during neointimal formation, likely through inhibition of inflammatory cell infiltration via downregulating the adhesion molecules in VSMCs. Thus, A. cinnamomea may offer a pharmacological therapy to slow down disease progression in patients with vascular injury.