Inflammation and oxidative stress are core pathological processes in the development of numerous chronic diseases, mutually reinforcing each other to create a vicious cycle. Consequently, identifying natural compounds capable of concurrently mitigating both processes, along with the elucidation of their mechanisms, is crucial for therapeutic development. This study aims to investigate the anti-inflammatory and antioxidant effects, along with their molecular mechanisms, of the novel sesquiterpene lactone compound 3-n-Propionyl Zaluzanin C (NPZC), isolated from Saussurea involucrata. Utilizing an integrated approach of network pharmacology, cellular assays, molecular docking, and zebrafish models, this study systematically evaluated the bioactivities of NPZC and elucidated its underlying mechanisms of action. Network pharmacology was utilized to screen potential targets and pathways. The effects of NPZC were then interrogated in cellular models of LPS-induced inflammation (RAW264.7) and H2O2-induced oxidative stress (HepG2), quantifying inflammatory mediators, ROS levels, antioxidant enzyme activity, and related protein expression. The interaction between NPZC and key targets was validated by molecular docking and cellular thermal shift assays (CETSA). Finally, the protective efficacy of NPZC was confirmed in a zebrafish model, ameliorating both AAPH-induced oxidative damage and LPS-triggered inflammatory phenotypes. NPZC effectively suppressed the release of LPS-induced inflammatory mediators (TNF-α, IL-6, NO and COX-2), scavenged H2O2-induced ROS, and enhanced the activity of antioxidant enzymes (CAT, SOD and GSH). Mechanistic studies indicate that NPZC does not directly inhibit the NF-κB pathway. Instead, it activates the NRF2 signaling pathway, promoting NRF2 nuclear translocation and upregulating downstream antioxidant genes (HO-1 and NQO1). Consequently, NPZC enhances cellular antioxidant capacity and indirectly suppresses NF-κB-mediated inflammation. The direct binding of NPZC to both NRF2 and KEAP1 was confirmed by molecular docking and CETSA. These actions were corroborated in vivo, where NPZC alleviated oxidative damage and inflammatory phenotypes in zebrafish, concomitant with the upregulation of NRF2 pathway. NPZC disrupts the vicious cycle of inflammation and oxidative stress by activating the NRF2 signaling pathway, thereby enhancing cellular antioxidant defenses and indirectly suppressing the NF-κB pathway. This study not only elucidates the modern scientific basis for the traditional medicinal effects of S.involucrata but also provides novel candidate compounds and theoretical foundations for developing natural anti-inflammatory and antioxidant drugs targeting NRF2. Future studies would clarify the relationship between the NPZC and NRF2 using point mutation analysis and surface plasmon resonance. Mechanistic validation will be further enhanced through multi-omics approaches, with confirmed conversion potential subsequently validated in mammalian models.
Sarcopenia, an age-related degenerative disease, corresponds to "Qi Xu" syndrome in traditional Chinese medicine. Juyuanjian (JYJ), a classical Qi-tonifying formula, has shown potential against muscle atrophy and functional decline, but its molecular mechanisms are not well understood. To investigate whether JYJ protects against sarcopenia and to elucidate its underlying mechanisms. Caenorhabditis elegans (C. elegans) RW1596, C2C12 myotube cells and senescence-accelerated mouse prone 8 (SAMP8) transgenic mice were used to explore the alleviative effect of JYJ on sarcopenia and the molecular mechanism in vivo and in vitro. This study systematically evaluated the therapeutic effects of JYJ using multiple biological models. Ultra-high performance liquid chromatography-high-resolution mass spectrometry (UPLC-MS) was employed to characterize its chemical constituents, followed by network pharmacology to predict potential targets and pathways. These mechanisms were further validated through molecular biology experiments. Additionally, molecular docking and molecular dynamics (MD) simulations were conducted to elucidate the interactions and binding stability between key bioactive components and target proteins. JYJ significantly alleviated muscle fiber damage in C. elegans RW1596 and mitigated the decline in skeletal muscle mass and strength in SAMP8 mice. Furthermore, JYJ inhibited chronic low-grade inflammation by reducing tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels, decreasing macrophage infiltration, and suppressing nuclear factor-kappa B (NF-κB) activation. Network pharmacology analysis indicated that mitochondrial biogenesis and proteasome-mediated ubiquitin-dependent processes were the main biological processes, with protein kinase B (Akt), forkhead box O1 (FoxO1), sirtuin 1 (SIRT1), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) as key targets. Consistent with this, JYJ enhanced the phosphorylation of Akt and FoxO1 both in vitro and in vivo, while downregulating the expression of muscle atrophy-related E3 ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx); it also upregulated the expression of SIRT1 and PGC-1α, promoting mitochondrial biogenesis and adenosine triphosphate (ATP) production. Molecular docking and 100-nanosecond MD simulations showed stable interactions between the bioactive components of JYJ and Akt/SIRT1, supported by favorable binding free energy and stable conformational dynamics. JYJ exerts anti-sarcopenic effects by dual regulation of protein degradation (Akt/FoxO1) and mitochondrial biogenesis (SIRT1/PGC-1α), providing a phytotherapeutic for sarcopenia.
The increased transcytosis in endothelial cells (EC) represents a significant secondary pathological event that results in BBB disruption following cerebral ischemia-reperfusion injury (CIRI). The Wnt/β-catenin pathway has been recognized for its vital role in maintaining BBB integrity, but its impact on transcytosis in brain ECs following CIRI remains unclear. Prior research has demonstrated that Fuzheng Jiedu Tongluo Granule (FZJDTL) was effective in treating post-stroke damage. Nonetheless, the precise mechanism underlying FZJDTL's protective effects on the BBB remains ambiguous and necessitates further exploration. This research sought to examine how FZJDTL protected the BBB in the aftermath of CIRI, and to clarify that the key mechanism pathway was via Wnt/ β-catenin/Mfsd2a/Cav-1 axis regulating endothelium transcytosis and maintaining BBB integrity. The chemical profile of FZJDTL was carried out through LC-MS. The potential protective effect mechanisms of FZJDTL on CIRI were first elucidated by an integrated approach combining transcriptomics analysis and network pharmacology. Molecular docking analysis was performed to investigate the interactions between the active ingredients of FZJDTL and the proteins Wnt3a, β-catenin, Mfsd2a, and Cav-1. Surface plasmon resonance (SPR) was further employed to validate the binding capacity. In vivo experiments were conducted using a MCAO/R rat model to assess the effects of FZJDTL on CIRI. TTC and HE staining were conducted to assess pathological injury. The function of BBB was evaluated employing Evans blue (EB) penetration, and the number of Cav-1 mediated transcytosis vesicles in murine cerebral capillary ECs was quantified by TEM. Immunofluorescent staining was employed to observe Cav-1 expression in brain. The mRNA and protein expression levels of Wnt3a, β-catenin, Mfsd2a, Cav-1, and claudin-5 were determined by RT-qPCR and Western Blotting analysis. Furthermore, a mouse brain microvascular endothelial cell (bEnd.3) model subjected to oxygen-glucose deprivation/ reoxygenation (OGD/R) was established in the in vitro experiment. The efficacy of FZJDTL for providing protection was assessed through the evaluation of cell viability using the CCK8 assay, measuring BBB integrity through transepithelial electrical resistance (TEER) value and sodium fluorescein (SF) leakage, and analyzing claudin-5 protein expression. Eighty-four components were identified in FZJDTL. Comprehensive network pharmacology and transcriptomics analysis hinted that FZJDTL alleviated CIRI by inhibiting transcytosis vesicles via triggering the Wnt/β-catenin signaling pathway. Given that activation of the vesicle inhibitory protein Mfsd2a downregulates the expression of the vesicle transport protein Cav-1 via the Wnt/β-catenin signaling pathway, it was prompted the hypothesis that FZJDTL inhibited transcytosis through Wnt/β-catenin modulation, thereby alleviating CIRI. The molecular docking analysis indicated that lithospermic acid B, luteolin, adenosine, hydroxysafflor yellow A, paeonol, and formononetin may serve as potential key active ingredients interacting with the proteins Wnt3a, β-catenin, Mfsd2a, and Cav-1. The SPR results confirmed that Wnt3a bound to lithospermic acid B, paeonol, formonoetin, luteolin, adenosine, and hydroxysafflor yellow A. In the in vivo experiments, treatment with FZJDTL dose-dependently reduced neurological function score and cerebral infarct volume, it also ameliorated cerebral histopathological injury. TEM results revealed that FZJDTL inhibited the increase of transcytosis vesicles in MCAO/R rats. The therapeutic effect of FZJDTL on post-CIRI by mitigating BBB dysfunction as demonstrated by a reduction in macromolecule (EB and albumin) leakage in MCAO/R rats, as well as proved by increased TEER value and reduced macromolecule SF leakage after OGD/R in bEend.3 cells. FZJDTL alleviated MCAO/R- or OGD/R-induced BBB damage via upregulation of the Wnt/β-catenin/Mfsd2a signaling pathway, which subsequently suppressed downstream Cav-1 expression, reduced Cav-1-mediated transcytosis vesicles, and increased expression of claudin-5, a tight junction protein critical to the structural integrity of the BBB. FZJDTL alleviated CIRI by modulating the Wnt/β-catenin signal pathway to promote Mfsd2a expression, inhibit the expression of Cav-1, further impeding vascular endothelial transcytosis, and ultimately protecting the function of the BBB.
The synergistic cardioprotective effects of monoester alkaloids and ginsenosides constitute the pharmacological basis of Shenfu decoction (SFD) for chronic heart failure. However, the underlying molecular interactions remain unclear. This study aimed to identify the key bioactive components of SFD, elucidate their molecular targets, and define the mechanistic pathways underlying their synergistic cardioprotective effects. We employed an ACE-dependent Ang I-stimulated cardiomyocyte hypertrophy model in vitro and a transverse aortic constriction (TAC) mouse model in vivo to evaluate pharmacological efficacy. Integrated transcriptomic-proteomic profiling identified downstream effectors, validated by genetic knockdown. The molecular targets were investigated using limited proteolysis-mass spectrometry (Lip-MS), molecular docking, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and enzyme activity assays. Benzoylaconitine (BAC) and ginsenoside Rb1 (Rb1) were identified as the dominant cardioprotective constituents of SFD. Pharmacological assessments demonstrated that BAC and Rb1 synergistically attenuated Ang Ⅰ-induced cardiomyocyte hypertrophy and fibrosis, and mitigated pathological cardiac remodeling in TAC mice. Integrated transcriptomic and proteomic analyses revealed that their combined treatment reduced mitochondrial reactive oxygen species (ROS) generation, suppressed mitochondrial fission, and restored mitochondrial homeostasis. Mechanistically, BAC promoted the transcription of Discs Large MAGUK Scaffold Protein 1 (DLG1), whereas Rb1 slowed its protein degradation, thereby synergistically upregulating DLG1 expression and improving mitochondrial function. Lip-MS further demonstrated that BAC specifically targeted angiotensin-converting enzyme (ACE) and inhibited its activity, whereas Rb1 targeted and activated ACE2, thereby rebalancing the renin-angiotensin-aldosterone system (RAAS) between the AT1R and MAS axes. This dual-target modulation ultimately contributed to the upregulation of DLG1, preserved mitochondrial integrity, and ameliorated maladaptive ventricular remodeling. BAC and Rb1 exert synergistic cardioprotection by targeting ACE and ACE2, restoring RAAS homeostasis, enhancing DLG1 expression, and sustaining mitochondrial stability, thereby attenuating pathological cardiac remodeling. These findings highlight the potential of BAC and Rb1 as a dual-target synergistic therapy for heart failure.
Spinal cord injury (SCI) causes severe neurological deficits due to extensive neurodegeneration and secondary pathological events, including disruption of the blood-spinal cord barrier (BSCB). Restoration of endothelial integrity is critical for limiting inflammation and promoting recovery. Rhynchophylline (Rhy), a tetracyclic oxindole naturally occurring alkaloid with known neuroprotective effects, has not been fully investigated for its role in maintaining BSCB integrity and endothelial function after SCI. This study aimed to evaluate the therapeutic potential of Rhy in preserving endothelial function and promoting neurovascular repair following SCI, and to elucidate its underlying molecular mechanisms, particularly the role of cluster of differentiation (CD36) signaling in vascular pathology. The therapeutic potential of Rhy was evaluated using a mouse model of SCI and complementary in vitro assays. Behavioral assessments and histological analyses were performed to examine functional recovery, vascular integrity, fibrosis, and remyelination. In vitro, bEnd.3 endothelial cells were exposed to IL-1β to induce inflammatory stress, and the effects of Rhy on CD36 expression, tight junction proteins, and trans-endothelial permeability were analyzed. Neuronal survival and neurite outgrowth assays were conducted to assess Rhy's neuroprotective actions. Rhy treatment significantly reduced vascular damage and inflammation following SCI via downregulating CD36 expression in endothelial cells, supported by molecular docking, qPCR, and immunohistochemistry. In vitro, Rhy preserved tight junction integrity and reduced trans-endothelial permeability under inflammatory conditions. Furthermore, Rhy promoted neuronal survival, enhanced neurite outgrowth, reduced fibrotic scar formation, supported remyelination, and improved endothelial migration. These effects collectively contributed to enhanced functional recovery in vivo. Rhy exerts neurovascular protective effects after SCI by preserving endothelial integrity, inhibiting CD36-mediated vascular damage, and promoting neural repair. These findings identify Rhy as a promising therapeutic candidate for mitigating vascular and neuronal dysfunction following SCI.
Carbapenem-resistant Enterobacterales (CRE), particularly strains producing New Delhi metallo-β-lactamase (NDM), represent a critical global health threat and underscore an urgent need for effective NDM inhibitors. Allicin, a major bioactive compound in freshly crushed garlic, shows broad-spectrum antibacterial activity, but its detailed mechanisms and its capacity to reverse NDM-mediated carbapenem resistance remain to be fully elucidated. This study aimed to evaluate the antibacterial effects of allicin alone and in combination with meropenem both in vitro and in vivo to uncover its mechanisms of action, thereby providing new mechanistic insights and a potential therapeutic strategy against NDM-mediated resistance. The antibacterial activity of allicin and its mechanisms were assessed using microbroth dilution, membrane-permeability assays, ROS and H₂S detection, and comet assays. The in vivo efficacy was evaluated using a Galleria mellonella infection model and a rat Pasteurella pneumonia model. Synergy was assessed in vitro via checkerboard assays and in vivo using a rat wound infection model. The interaction with NDM-5 was studied through molecular docking, site-directed mutagenesis, enzyme kinetics, and microscale thermophoresis. Allicin exhibited potent activity against clinical isolates of Pasteurella and NDM‑positive Escherichia coli (MICs 8-64 μg/ml, MBCs 16-128 μg/ml) both in vitro and in vivo. It disrupted bacterial redox homeostasis by inducing ROS accumulation and suppressing endogenous H₂S production. Moreover, allicin restored meropenem activity both in vitro and in vivo and acted as a non‑competitive inhibitor of NDM‑5. Its disulfide bond covalently bound to key zinc‑coordinating residues in the active site, leading to Zn²⁺ release and the loss of NDM enzymatic activity. Allicin possesses a dual pharmacological role: exerting direct antibacterial activity through oxidative stress induction and reversing carbapenem resistance via the non‑competitive inhibition of NDM. These findings provide a promising lead compound for developing natural product-derived antibiotic adjuvants to address the global challenge of antimicrobial resistance (AMR).
Atopic dermatitis (AD) is a recurrent inflammatory disease that significantly impacts patients' quality of life. Jingfang Granules (JFG) have been employed in the therapeutic management of AD and psoriasis (PSO), in contemporary clinical practice. However, further research is required to elucidate the molecular mechanisms of JFG in AD therapy. The present study aims to explore the potential mechanisms by which JFG alleviate AD. Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) and metabolomics were used to identify the absorbed components in the blood of JFG. BALB/c mice were continuously topically treated with 1-Chloro-2,4-dinitrobenzene (DNCB) on the dorsal skin to establish AD-mice model. The skin lesions, trans epidermal water loss (TEWL), scoring of atopic dermatitis (SCORAD), hematoxylin and eosin (H&E) staining, and inflammatory cytokines in serum were analyzed to elucidate the effects of JFG on AD. Furthermore, network pharmacology was employed to predict the potential active components and JFG targets of AD therapy. In vitro, the potential pharmacological effects of JFG toward AD were further elucidated using flow cytometry, western blot, reverse transcription-PCR (RT-PCR), immunofluorescence and enzyme linked immunosorbent assay (ELISA). Based on UPLC-MC/MS and metabolomics, a total of 901 components were identified in serum, with 365 confirmed as JFG-derived absorbed components. These components were predominantly enriched in amino acid metabolic pathways. In vivo, JFG significantly alleviated the pathological changes, including pruritus and impairment of skin barrier function damage in AD mice, while significantly reducing TEWL, SCORAD, inflammatory cytokines release and splenic index. Network pharmacology and molecular docking analyses indicated that these potential therapeutic targets were predominantly enriched in the inflammatory response and the tumor necrosis factor (TNF) signaling pathway, which primarily involved ALB, BCL2, EGFR, GAPDH, IL6, NFKB1, STAT3 and TNF targets. Mechanistically, JFG reduced inflammatory response through NF-κB/NLRP3 axis and JAK/STAT3 axis, and restored the expression of tight junction proteins (TJPs) Occludin, Zonula occludens-1 (ZO-1) and Claudin-1, thus restoring skin barrier function and improving AD. The present study confirmed that JFG alleviate atopic dermatitis by restoring skin barrier function and inhibiting NF-κB/NLRP3 axis and JAK/STAT3 axis. JFGs demonstrate potential therapeutic efficacy for treating AD.
Sepsis-associated liver injury (SALI) as a critical factor contributing to high mortality rates in patients with sepsis is characterized by significant coagulation disorders. Vascular endothelial cells (ECs) play a pivotal role in the coagulopathy in sepsis. Shenfu Injection (SFI), a traditional Chinese medicine, is widely used in sepsis management, yet its impact on EC activation and coagulopathy in SALI remains inadequately explored. This study aims to elucidate the mechanisms by which SFI alleviates EC activation and coagulopathy associated with SALI and to dissect the contribution of individual compounds in SFI in regulating EC coagulation activation. Plasma levels of coagulation- and liver injury-related biomarkers in septic patients treated with or without SFI were analyzed. A murine model of sepsis was built via intraperitoneal LPS injection for the study of SFI pharmacological effects in vivo. Human umbilical vein endothelial cells (HUVEC) stimulated with LPS were used as the in vitro model of sepsis-induced EC activation. Transcriptome sequencing was performed to investigate the molecular mechanisms of SFI in regulating EC activation. Transmission electron microscopy and confocal laser microscopy were applied for the evaluation of pyroptosis and LPS internalization in HUVEC. The clinical data showed that the coagulation markers and liver injury-related indicators in septic patients were significantly lower in SFI treated septic patients compared to that in non-SFI treated group. In the murine model, SFI administration alleviated liver damage and the inflammatory responses. The plasma levels of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) as well as the accumulation of fibrinogen in liver tissues were also reduced in septic mice treated with SFI. Mechanistically, SFI induced phosphorylation-dependent activation of AMPK, thereby leading to the dephosphorylation of RIPK2, which blocked NLRP3-mediated canonical pyroptosis. Additionally, SFI inhibited LPS internalization by blocking HMGB1/RAGE axis and suppressing the expression of LPS-binding protein (LBP), resulting in the inhibition of non-canonical pyroptosis. Furthermore, network pharmacology and molecular docking analysis highlighted ginsenoside Rb1 and ginsenoside Re in SFI for their strong binding affinity to AMPK and LBP, respectively, supporting the dual inhibitory effects of SFI on pyroptosis. Our findings revealed the significance of SFI for treating sepsis-associated coagulopathy and liver injury via dual inhibition of pyroptosis, providing theoretical basis for clinical application of SFI.
An arms race between cancer cells and therapeutic pressure fuels drug resistance, immune evasion, and tumor heterogeneity, hindering therapy. Ganoderma lucidum (G. lucidum), a traditional Chinese medicine (TCM) with multitarget pharmacology, offers potential to address this complexity. However, the mechanistic basis of the emergent effects of its meroterpenoids remains unexplored. This study used G. lucidum meroterpenoids as a model to elucidate the mechanisms underlying emergent effects in TCM and to evaluate their therapeutic potential for overcoming tumor resistance and heterogeneity. This study employed an integrative design combining phenotypic screening, in vitro cell-based assays, in vivo tumor xenograft models, and multiomics analyses to investigate the emergent antitumor mechanisms of G. lucidum meroterpenoids. Meroterpenoid and polysaccharide extracts were evaluated in vitro and in vivo for their effects on TNBC growth. Transcriptomic, metabolomic, and microbiome analyses were performed to explore systemic mechanisms. Phenotypic screening and transcriptome analyses were performed to characterize the synergistic (0 + 0 + 0…>0) and potentiation (0 + 0 + 0…+1 > 1) effects of the meroterpenoid combinations and to elucidate the underlying molecular mechanisms. In MDA-MB-231 cells, the meroterpenoid extracts inhibited proliferation and migration by inducing ferroptosis and reprogramming inflammatory signaling. In a mouse tumor model, total meroterpenoid extracts suppressed tumor growth, enriched beneficial gut microbiota, and partially restored tumor-depleted serum lipids and amino acids. Cotreatment with G. lucidum polysaccharides accelerated the restoration of the microbiota and achieved more extensive metabolomic correction. Notably, a set of inert meroterpenoids, defined as those lacking phenotypic effects, exhibited emergent anticancer activity when combined with one another or with active compounds. These combinations acted through 0 + 0 + 0…>0 synergy or 0 + 0 + 0…+1 > 1 potentiation to inhibit proliferation and migration by overcoming drug resistance, reprogramming metabolism, and inducing ferroptosis. They also triggered cell cycle collapse and necroptosis. This case study provides insights into how meroterpenoids, particularly combinations of inert meroterpenoids, drive the emergent pharmacological effects of G. lucidum and suggests a strategy to overcome drug resistance and tumor heterogeneity.
Despite existing reports on the hepatoprotective effects of limonin (Lim), its specific impact on hepatic fibrosis and cellular senescence in metabolic dysfunction-associated steatohepatitis (MASH) remains unclear. The precise molecular mechanisms and direct targets underpinning its pharmacological activity are also poorly defined. This research aimed to investigate the therapeutic potential of Lim against MASH-related hepatic fibrosis and senescence, and to delineate the underlying molecular pathways. A murine MASH model was generated by feeding a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). To elucidate the mechanism of action of Lim, an integrated strategy was adopted, comprising AAV8-mediated gene manipulation, RNA sequencing (RNA-seq), and pharmacological interventions with agonists or inhibitors. The direct protein targets of Lim were identified using human proteome microarrays and validated through molecular docking, pull-down assays, and site-directed mutagenesis. Our findings indicate that Lim effectively alleviates hepatic fibrosis and senescence in MASH mice, while suppressing galectin-3 expression and mechanistic target of rapamycin complex 1 (mTORC1) activation. Notably, targeted knockdown of galectin-3 in the liver hindered aging-related changes in MASH mice, with mTORC1 functioning as a downstream effector. Further results revealed that mTORC1 acts as a key mediator of Lim's protective effects against hepatic fibrosis and senescence in MASH mice. Mechanistically, Lim binds to the Src homology 2 (SH2) domain of signal transducer and activator of transcription 3 (STAT3), inhibiting its activity and leading to reduced galectin-3 expression and mTORC1 activation. Moreover, the application of STAT3 inhibitor has been shown to alleviate hepatic fibrosis and senescence in MASH mice, further corroborating the suppression of galectin-3 and mTORC1 activity. In conclusion, our study provides compelling evidence for the efficacy of Lim in ameliorating hepatic senescence and fibrosis in MASH mice, elucidating the involvement of the STAT3/galectin-3/mTORC1 signaling in these processes.
Medicinal plants are widely used for applications in agriculture, food, medicine, and cosmetics due to their abundant bioactive secondary metabolites (SMs) such as terpenoids, phenylpropanoids, and alkaloids. The biosynthesis and accumulation of SMs are highly associated with multiple environmental factors. Among these abiotic stresses, drought plays a pivotal role in regulating the quality of medicinal plants. Understanding the regulatory mechanisms of medicinal plants in response to drought is beneficial for (i) cultivating high-quality traditional Chinese medicinal plants via targeted water management strategies; (ii) screening candidate marker genes to breed high-quality novel cultivars with enhanced bioactive compound accumulation under drought conditions, thereby addressing the adverse impacts of drought induced by global climate change; (iii) mining dual-functional genes that confer drought tolerance while maintaining high bioactive compound content, thus ensuring both the yield and quality of medicinal plants. To summarize the latest advances in the transcriptional regulation of SM biosynthesis with a focus on terpenoids, phenylpropanoids, and alkaloids in medicinal plants under drought conditions. A comprehensive literature search was conducted in three electronic databases including PubMed, Scopus, and Web of Science using the search terms "regulatory mechanism", "secondary metabolites", "medicinal plants", "drought stress", "transcription factor", "bioactive compound", "synthetic biology", "smart irrigation", "terpenoid biosynthesis", "phenylpropanoid biosynthesis", "phenolic biosynthesis" and "alkaloid biosynthesis". All the retrieved data were then critically reviewed and summarized. Drought affects secondary metabolite biosynthesis via a complex molecular regulatory network, including shifts in microbial community composition, epigenetic remodeling, changes in global gene expression profiles, altered catalytic activity of core biosynthetic enzymes, as well as modifications of transcription factors. This review offers novel insights into unraveling the underlying transcriptional regulatory networks, and practical implications for researchers in the fields of medicinal plant biology, natural product chemistry, and crop stress physiology.
Mitochondrial dysfunction and the dysregulation of lipid metabolism are significant contributors to vascular aging, which in turn raises the risk of age-related cardiovascular diseases (CVDs). Buyang Huanwu decoction (BHD), a traditional formula widely used for treating CVDs, has not been thoroughly investigated in terms of its active components and the molecular mechanisms by which it may delay vascular aging. This study aims to investigate the effects and mechanisms of BHD and its primary active component, Astragaloside IV (AS-IV), in mitigating vascular aging, focusing on mitochondrial function and lipid metabolism. Experiments were conducted using naturally aged mice and a D-galactose-induced model of vascular smooth muscle cells (VSMCs) senescence. Vascular aging was evaluated through aortic histopathology, assessment of senescence markers and senescence-associated secretory phenotype (SASP) factors. Transcriptomics and lipidomics analyses were employed to uncover critical pathways and identify differential lipid profiles. Mitochondrial structure and function were examined using transmission electron microscopy (TEM), Mitochondrial DNA (mtDNA) copy number, and mitochondrial membrane potential (MMP). The molecular mechanisms were further validated through molecular biology and MLX Interacting Protein-Like (MLXIPL) knockdown and overexpression techniques. Both BHD and AS-IV improved age-related vascular morphological changes, mitigated elastic fiber disruption, diminished collagen deposition, and downregulated senescence markers along with SASP factors. Transcriptomic analysis identified MLXIPL as a key transcription factor, emphasizing the involvement of the peroxisome proliferator-activated receptor (PPAR) signaling pathway and mitochondrial fatty acid β-oxidation as critical processes. Lipidomic profiling indicated reduced serum acylcarnitine levels in aged mice, suggesting compromised fatty acid β-oxidation. Both BHD and AS-IV were found to suppress MLXIPL, activate PPARα, increase mtDNA copy number, enhance MMP, improve mitochondrial ultrastructure, and upregulate the expression of key genes and proteins associated with mitochondrial biogenesis and fatty acid β-oxidation. Co-immunoprecipitation assays confirmed that AS-IV facilitated PPARα-PGC-1α interaction. Functional validation established that MLXIPL knockdown produced a stronger effect than AS-IV, whereas MLXIPL overexpression negated the beneficial effects of AS-IV on mitochondrial function, lipid accumulation, and cellular senescence. BHD and its active component AS-IV delay vascular aging by inhibiting MLXIPL, thereby activating the PPARα/PGC-1α signaling pathway to promote mitochondrial fatty acid β-oxidation and reduce lipid accumulation. This elucidates a mechanistic basis for BHD's traditional use and highlights AS-IV as a key active principle with therapeutic potential.
Dengue virus (DENV) remains a significant public health threat, yet no effective antiviral therapies are currently available. Based on TCM theory, the treatment of dengue emphasizes the principles of clearing heat and detoxifying, cooling blood and dissipating blood stasis. Yinqiao Powder (YQS), a famous clearing heat and detoxifying formula, has a good curative effect on the virus-induced diseases, and theoretically has potential value in the treatment of dengue. This study aims to investigate the antiviral mechanism of YQS against DENV. Time-of-drug-addition assay elucidated phase-specific antiviral target of YQS in dengue infection, while plaque, cytopathic effect (CPE), quantitative real-time PCR, Western blot, and immunofluorescence assays were employed to assess the antiviral efficacy of YQS. Network pharmacology analysis was performed to identify convergent molecular targets between YQS constituents and DENV pathophysiological cascades. The interactions between wogonin and HSP90AA1 were characterized using cellular thermal shift assay, drug affinity responsive target stability, molecular docking, and surface plasmon resonance. Immunofluorescence and co-immunoprecipitation assays were implemented to interrogate whether wogonin modulates E/HSP90AA1 interaction. Ultimately, the in vivo protective activity of YQS was assessed in DENV-2-infected AG129 mice. YQS inhibited DENV-2 infection with an IC50 value of 468.5 μg/ml. YQS reduced progeny virus by over 75% and CPE, suppressed expression of viral RNA and proteins during the adsorption phase across multiple cell lines. The active component wogonin was identified through network pharmacology. Similarly, wogonin exhibited potent inhibition of DENV adsorption, reducing plaque formation by over 45%. Further target study demonstrated that wogonin targeted HSP90AA1 and blocked its interaction with viral E protein. In vivo findings revealed YQS mitigated weight loss, extended survival, diminished serum viral load, and conferred hepatoprotective effects in AG129 mice. YQS potently inhibits DENV infection in vitro and in vivo. The antiviral activity of its component wogonin may contribute to this effect, potentially through binding to the host receptor HSP90AA1 and reducing DENV adsorption.
Patients with acute decompensated ischemic heart failure (ADIHF) often present with severe clinical symptoms and poor quality of life. In China, Yiqi Fumai lyophilized injection (YQFM) is widely used in the treatment of ADIHF. However, high-quality evidence is still needed to support its efficacy and safety. This study aims to assess the therapeutic efficacy and safety of YQFM in patients with ADIHF. By conducting a multicenter, open-label, blinded-outcome, randomized controlled trial, we recruited patients with ADIHF from 37 hospitals in 20 regions of China from October 2015 to October 2018. Patients were allocated in a 1:1 ratio to either the YQFM group or the control group. Both groups received guideline-directed medical therapy (GDMT), with the YQFM group additionally receiving YQFM for 7 days. The primary outcome included the proportion of patients with a decrease from baseline B-type natriuretic peptide (BNP) value ≥ 30% on day 8 post-randomization. We evaluated the left ventricular ejection fraction (LVEF), New York Heart Association (NYHA) functional class, Minnesota Living with Heart Failure Questionnaire (MLHFQ) score, and composite endpoints. A total of 666 patients with ADIHF (332 in the YQFM group and 334 in the control group) were enrolled. The full analysis set (FAS) analysis revealed that the proportion of patients in the YQFM group with a reduction of at least 30% in BNP value on day 8 was higher than that in the control group (175 [55.21%] vs. 135 [41.93%], one-sided p < 0.001, two-sided p < 0.001; RR, 1.32 [95% CI, 1.12-1.56]). The YQFM group showed statistically significant improvements in LVEF, NYHA functional class, and MLHFQ scores compared to controls. There was no statistically significant difference between the two groups regarding composite endpoint events and adverse events during the follow-up period. Based on GDMT, the combined use of YQFM was associated with further reductions in BNP levels, improve quality of life and cardiac function in patients with ADIHF, without increasing safety risks. However, no significant differences were observed in clinical events, such as mortality or hospitalization, during the follow-up period.
Luteal phase defect (LPD) is a prevalent endocrine disorder contributing significantly to female infertility and early pregnancy loss. Nuangong Tiaojing Formula (NTF), a traditional Chinese medicine formula, has demonstrated clinical efficacy in treating LPD, yet its underlying mechanisms remain incompletely elucidated. This study aimed to explore the curative effects and mechanisms of NTF in LPD-related ovarian endocrine dysfunction and endometrial receptivity defects. UPLC-Q-TOF-MS/MS and HPLC technologies were utilized to identify and quantify the chemical components in NTF, respectively. An LPD rat model was established using mifepristone. The therapeutic effects of NTF on estrous cyclicity, serum progesterone (P) and estradiol (E2) levels, and ovarian and uterine histopathology were evaluated. Network pharmacology analysis predicted the underlying biological mechanism modulated by NTF, which were subsequently validated using molecular biology methods, such as Western blotting, ELISA, and TUNEL staining. Component-effect correlation analysis, in silico simulations, and pharmacokinetic study were conducted to identify candidate efficacious ingredients of NTF and their in vivo kinetic characteristics. NTF treatment significantly ameliorated LPD-related pathologies in a dose-dependent manner, including restored estrous cyclicity, increased serum P and E2 levels, and improved ovarian morphology and endometrial receptivity. NTF also reduced the secretion of pro-inflammatory cytokines TNF-α and IL-1β, and modulated lipid peroxidation markers such as SOD and MDA. Mechanistically, the therapeutic effects of NTF on restoring ovarian endocrine function and its downstream endometrial receptivity in LPD was closely associated with the suppression of ovarian inflammatory-apoptotic cascade mediated by the TLR4/MyD88/NF-κB/Bcl-2/Bax/Caspase-3 pathway. Component-effect correlation analysis, in silico simulations, and pharmacokinetic data suggested that Paeoniflorin, Albiflorin, Morroniside, Loganin, Salvianolic acid B, Gallic acid, and Hyperoside were candidate efficacious components of NTF, exhibiting stable binding interactions with core targets of the aforementioned pathway and multi-level exposure characteristics, supporting their potential for synergistic therapeutic effects. This study innovatively demonstrates that NTF effectively restores ovarian-uterine axis function in LPD probably by multi-target regulation of the inflammatory-apoptotic cascade, primarily via the TLR4/MyD88/NF-κB/Bcl-2/Bax/Caspase-3 pathway. Our findings present novel mechanistic insights into the curative effect of NTF against LPD.
Understanding the morphogenetic processes and their spatial crosstalk with metabolism is essential for improving the quality of medicinal plants and constructing the regulation of the metabolite network. However, there is a paucity of detailed research on the spatiotemporal dynamics of morphology-metabolism interplay in medicinal plants. The roots of perennial Scutellaria baicalensis Georgi (S. baicalensis) have typical morphological characteristics (hollow root) and flavonoid metabolic heterogeneity, but the relationship between them at the spatiotemporal level has not been explored. We present an integration of anatomical analysis, mass spectrometry imaging (MSI) based spatial metabolomics, and molecular biological methods to precisely dissect the spatiotemporal interplay and underlying mechanisms between morphological dynamics and metabolic reprogramming during hollow root development. Morphological and anatomical features were analyzed to determine the four typical development stage of hollow root, and revealed that they originate from a programmed cell death (PCD) progress. Representative metabolites alterations are imaged at micron-scale spatial level, and morphological, metabolites are connected through colocalized in the heterogeneous root tissues. With the development hollow roots, the expression of root-specific flavonoid glycosides is specificity decreases in the pith, while that of their aglycones increases. Comprehensive analysis further reveals that high expression of beta-glucuronidase (GUS) in hollow root is the key molecular mechanism responsible for the alterations of flavonoids. This study provides a new perspective on a comprehensive understanding of the metabolic dynamics during organogenesis, and provides developmental and mechanistic insights into the pharmaceutical value formation of S. baicalensis hollow roots.
Neuroinflammation resulting from myelin debris impedes axonal regeneration. Pharmacological modulation of myelin clearance and inflammatory responses is a potential strategy to enhance nerve regeneration. Hyperoside, a flavonoid with established anti-inflammatory and neuroprotective properties, has not been explored in peripheral nerve injury or in the context of myelin clearance. This study aims to investigate the effects and underlying mechanisms of hyperoside in promoting nerve regeneration. An in vitro phagocytosis model was established in RAW264.7 macrophages with purified myelin debris. Cells were treated with hyperoside (10, 15, 30 μM). Direct hyperoside targets were identified through molecular docking and small-molecule interaction assays. Gain- and loss-of-function experiments using LPS and Stattic were performed to validate the involvement of the STAT3/ADAM17/TREM2 signaling axis. In vivo sciatic nerve crush injury models were used to examine the relationship among TREM2-mediated myelin clearance, sciatic nerve regeneration, and functional recovery. Hyperoside dose-dependently inhibited pro-inflammatory gene expression and apoptosis-related proteins. Mechanistically, hyperoside bound STAT3 and inhibited its phosphorylation, thereby downregulating ADAM17 and preserving TREM2-mediated myelin phagocytosis. In the sciatic nerve crush injury model, hyperoside exerted neuroprotective effects, accelerated degenerated myelin clearance, promoted nerve regeneration, and reduced muscle atrophy. Trem2 silencing impaired myelin clearance and nerve regeneration. These findings highlight hyperoside as a promising therapeutic candidate for peripheral nerve injury. By targeting the STAT3/ADAM17/TREM2 signaling axis to enhance myelin clearance, hyperoside promotes structural and functional nerve regeneration in a rodent nerve injury model.
Sepsis-induced cardiomyopathy (SICM) is characterized by mitochondrial dysfunction, impaired mitophagic flux, and overwhelming oxidative stress. Spermidine (SPD), a natural polyamine known to enhance autophagy and preserve cardiac function in aging and metabolic disorders, has not been systematically evaluated in the context of septic cardiomyopathy. To determine the therapeutic potential and mechanistic basis of SPD in septic cardiac dysfunction. Network pharmacology, RNA sequencing, a cecal ligation and puncture (CLP) mouse model, and multiple cellular assays were integrated to assess the protective actions of SPD. Mitochondrial function, mitophagy flux, and oxidative stress were evaluated using transmission electron microscopy (TEM), immunohistochemistry (IHC), Western blotting, structured illumination microscopy (SIM), mitochondrial membrane potential assays, oxygen consumption rate (OCR) analysis, and mitochondrial DNA (mtDNA) quantification. Transcriptomic clustering and pathway enrichment identified molecular targets, which were validated through siRNA-mediated gene silencing. SPD markedly attenuated SICM in vivo and in vitro by improving both mitochondrial quantity and quality. It restored sepsis-impaired mitophagy by upregulating LC3B and ATG7, promoting autophagosome maturation, and enhancing cellular ubiquitination. Transcriptomic profiling highlighted metallothionein-1 (MT1) as a key node in metal-ion response pathways. SPD activated the NRF2-MT1-SOD2 antioxidant axis, reduced mitochondrial reactive oxygen species (mtROS) under lipopolysaccharide (LPS) stimulation, and reversed sepsis-induced suppression of SOD2. MT1 knockdown abolished SPD-mediated SOD2 stabilization and mtROS clearance, confirming its essential role in SPD's cardioprotective effects. SPD mitigates SICM by orchestrating the restoration of mitochondrial quality control, normalization of mitophagic flux, and stabilization of cellular redox homeostasis. These findings support SPD as a promising therapeutic candidate for septic cardiomyopathy.
Atherosclerosis is the inflammatory consequence of lipid accumulation with plaque formation in the vascular intima and is a common condition to develop into various cardiovascular diseases. Current therapies do not always lead to satisfactory treatment outcomes. Enterolactone, a mammalian lignan produced by bacterial transformation from plant lignans, has a preventive effect against cardiovascular disease. However, its effect on atherosclerosis and the underlying mechanism of action remain unclear. To explore the therapeutic effect of ENL on atherosclerosis and elucidate the underlying mechanism. We established a model of atherosclerosis on ApoE-/- C57BL/6 mice by high fat diet. The aortic root was collected and sectioned to assess arterial plaque area, collagen fibrillar proliferation, and lipid content. RT-qPCR was used to determine the inflammatory response in the artery of mice. The serum from mice was isolated to measure lipid levels, and the fecal microbiota was analyzed by 16S rDNA. H2O2 was used to induce HUVEC injury and ferroptosis to mimic endothelial cell dysfunction in atherosclerosis, and the inhibitory effect of ENL on HUVEC ferroptosis was appraised by monitoring ferroptosis indexes and levels of iron-related proteins. In the animals, enterolactone significantly improved lipid metabolism, attenuated ferroptosis occurring in the intima, facilitated the antioxidant mechanisms, and promoted healing of the endothelial lesions, by interacting with Nrf2. Of great importance, enterolactone massively altered the gut microbiota toward a curative outcome by elevating the abundance of beneficial bacteria, such as the SCFA-producing taxa. Additionally, ENL suppresses lipid peroxidation and inflammatory activation in HUVECs by regulating the Keap1/Nrf2/GPX4 pathway, and knocking down Nrf2 attenuates the treatment effect of ENL. Enterolactone effectively resolves intimal inflammation and redresses atherosclerosis by ameliorating the gut microbiome and modulating lipid metabolism via the Keap1/Nrf2/GPX4 pathway.
To systematically evaluate the efficacy and safety of Breviscapine injection as an adjunct to conventional Western medicine therapy for treating unstable angina pectoris (UAP). Randomized controlled trials (RCTs) comparing Breviscapine combined with conventional therapy to conventional therapy alone in patients with UAP were searched and retrieved from Chinese and English databases up to June 2025. Cochrane tool was used to assess the risk of bias. Meta-analyses were performed using RevMan 5.3 and STATA 18 software, and the quality of evidence was rated using the GRADE system. A total of 46 RCTs were included. Compared with conventional therapy alone, the combined therapy significantly increased the clinical effective rate (RR = 1.24, 95% CI [1.20, 1.28], p < 0.00001) but lowered frequency of angina attacks (MD = -1.90, 95% CI [-2.97, -0.84], p = 0.0005). There were superior improvements in hemorheological parameters, including plasma viscosity (MD = -0.27, 95% CI [-0.44, -0.10], p = 0.002), with a favorable safety profile. As an adjunct to conventional therapy, Breviscapine injection demonstrates certain clinical benefits in patients with UAP, including relief of anginal symptoms and improvement in electrocardiographic outcomes, with an acceptable safety profile. Breviscapine injection hold potential in the clinical management of unstable angina. Therefore, rigorous, high-quality randomized controlled trials should be conducted before wide adoption.