Astragali Radix (Huangqi) and Salvia miltiorrhiza (Danshen) represent a frequently paired herbal combination in traditional Chinese medicine for tonifying Qi and promoting blood circulation. Previous pharmacological investigations have shown that phytochemicals derived from these herbs exhibit neuroprotective properties against cerebral ischemia-reperfusion (CI/R) injury. Nevertheless, the underlying principles governing the combined application of Huangqi and Danshen (QD) and the molecular pathways involved in their therapeutic efficacy remain unexplored in ischemic stroke management. This study sought to systematically investigate the bioactive components in the QD formulation and elucidate their mechanisms of action against cerebral ischemic injury. The therapeutic effects of QD were evaluated in a mouse model of middle cerebral artery occlusion (MCAO). UHPLCHRMS was used to identify QD-derived components in blood and brain tissues. An SSA-BP neural network was constructed to predict the optimal combination of active ingredients. 4D label-free proteomics, coupled with GO and KEGG enrichment analyses, was performed to identify key pathways. Molecular docking and molecular dynamics simulations were used to validate candidate targets. Mitochondrial function and iron homeostasis were assessed by measuring ROS, mitochondrial membrane potential (ΔΨm), ATP, complex I activity, Fe2+, and the GSH/GSSG ratio. Western blotting and immunofluorescence were used to detect FUNDC1, Nrf2, SLC7A11, NCOA4, p62, FTH1, UQCRC2, and GPX4. The autophagy inhibitor 3-MA was used for mechanistic validation. For cellular studies, HT22 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), and QD was added during reoxygenation. Cell viability and cytotoxicity were assessed using CCK-8 and LDH assays, respectively. ROS, ΔΨm, Fe2+, and GSH were measured using commercial kits. Western blotting was used to analyze FUNDC1, UQCRC2, LC3-II/I, p62, GPX4, SLC7A11, NCOA4, Nrf2, and FTH1. siFUNDC1 was transfected 48 h before OGD/R to assess FUNDC1 dependency. Among the tested ratios (1:1, 2:1, 3:1, 3:2, and 2:3), the 3:2 QD combination was the most effective, significantly improving neurological function, reducing infarct size, and alleviating neuronal damage in MCAO mice. UHPLCHRMS identified 21 compounds absorbed into the bloodstream, seven of which were detected in brain tissue. Astragaloside IV, astragaloside II, lithospermic acid, tanshinone IIA, and calycosin were identified as key active components. Their combination, predicted by the SSA-BP neural network, exerted significant neuroprotection in vivo. Proteomics revealed 124 differentially expressed proteins, with GO and KEGG enrichment analyses identifying mitophagy and ferroptosis as the primary therapeutic pathways. Molecular docking and dynamics analyses revealed high-affinity binding interactions between QD constituents and key targets, including FUNDC1, UQCRC2, GPX4, and SLC7A11. QD upregulated FUNDC1, UQCRC2, GPX4, SLC7A11, Nrf2, and FTH1 and downregulated p62 and NCOA4; these effects were partially reversed by 3-MA. In HT22 cells, QD improved cell viability, reduced LDH release, restored ΔΨm and GSH levels, and attenuated ROS and Fe²⁺ accumulation following OGD/R. The protein expression changes were consistent with those observed in vivo. Knockdown of FUNDC1 largely blocked the protective effects of QD, confirming that FUNDC1 is essential for QD-mediated neuroprotection. QD ameliorates MCAO-induced cerebral ischemic injury via mitophagy and ferroptosis pathways, offering a novel therapeutic perspective for treating ischemic stroke with traditional Chinese medicine that tonifies Qi and promotes blood circulation.
An essential uncontrolled process-liver fibrosis-is vital to cirrhosis and liver cancer onset. There are currently no approved antifibrotic drugs in clinical application. Saponins are a class of major bioactive components of Bupleuri radix (Chaihu) that play important roles against liver fibrosis. Glycolysis inhibition may control the activation of hepatic stellate cells (HSCs), serving as an effective anti-fibrotic strategy. However, the mechanism by which saikosaponins exert anti-liver fibrosis effects by regulating glycolysis through HIF-1α is unclear. To explore the mechanism by which Bupleuri radix total saikosaponins (BRTS) inhibit HSC activation and alleviate liver fibrosis. Purified BRTS were obtained by extraction with saturated aqueous n-butanol solution, followed by adsorption and purification with AB-8 macroporous resin. After removing impurities using 40% ethanol, the product was washed with 90% ethanol. LC-MS/MS and HPLC were used to characterize the BRTS components and determine their contents. We used Network pharmacology to predict the possible mechanism underlying BRTS. CCl4 was intraperitoneally injected to create a liver fibrosis rat model, and we added TGF-β1 to LX-2 cells to create a HSC activation model. Haematoxylin and eosin (H&E), Masson, and Sirius Red staining of liver tissue, along with serum and liver biochemical indicator measurement analysed the extent of liver injury and fibrosis. Western blotting, qRT-PCR, immunofluorescence, immunohistochemistry, transcriptomics, and metabolomics practices analysed liver fibrosis- or HSC activation-related protein, gene, and metabolite expression. ELISA kits were used to detect glycolytic activity, SIRT3 deacetylase activity, lactate secretion, and the glucose uptake rate. Moreover, the mechanism by which BRTS improves liver fibrosis was validated using a glycolysis inhibitor, i.e., 2-deoxy-D-glucose (2-DG), 2-methoxyestradiol (2-ME, a kind of HIF-1α inhibitor), dimethyl oxoglutarate (DMOG, a kind of HIF-1α stabilizer), and SIRT3 inhibitor (3-TYP). The overall yield of BRTS was 6.56 ± 0.41%, and the main components were saikosaponin A (176.1 ± 10.5 mg/g) and saikosaponin D (132.7 ± 7.7 mg/g). Network pharmacology and LC-MS analyses revealed that the core anti-liver fibrosis target was HIF-1α. BRTS may show anti-liver fibrosis properties by regulating the HIF-1 signalling pathway, per the KEGG pathway enrichment analysis. Molecular docking suggested that both saikosaponin A and saikosaponin D can stably bind to SIRT3. In vivo, BRTS can improve the symptoms of CCl4-mediated liver fibrosis, alleviate liver injury, reduce collagen fibre content, and decrease collagen deposition, including significantly reduced serum AST and ALT activity, serum LN, HA, PC III, and COL IV levels, and liver lactate levels. BRTS significantly reversed the increased α-SMA, COL1, HIF-1α, and p-SMAD3 protein expression and the decreased SIRT3 protein expression (p<0.05). In addition, BRTS downregulated GLUT1, PKM2, HK2, and LDHA mRNA expression (all glycolytic genes) than the MOD group (p<0.05). The in vitro experimental results revealed that BRTS significantly inhibited LX-2 cell activation caused by TGF-β1, and reduced glucose uptake rates and level of lactate released (p<0.05). BRTS administration raised SIRT3 protein expression and activation, reduced HIF-1α and p-SMAD3 nuclear translocation, downregulated COL1, HIF-1α, α-SMA, and p-SMAD3 protein expression, and reduced glycolytic gene mRNA expression (p<0.05). In LX-2 cells, 2-DG and 2-ME further promoted the suppressive impact of BRTS on glycolysis and activation. Compared with BRTS treatment, DMOG resulted in significant reversal of the suppressive impact of BRTS on glycolysis as well as activation and COL1 and HIF-1α protein expression. In addition, BRTS promoted the deacetylase activity of SIRT3. In LX-2 activated cells where SIRT3 is inhibited, the inhibitory effect of BRTS on HIF-1α and p-SMAD3 protein expression is eliminated. BRTS regulates the SMAD3/HIF-1α signalling axis through SIRT3 to mediate glycolysis in HSCs, which suppresses HSC activation and improves liver fibrosis.
Diabetic microvascular complications (DMCs) mainly consist of diabetic kidney disease (DKD), diabetic retinopathy (DR), and diabetic peripheral neuropathy (DPN). Current therapeutic options for DMCs are often suboptimal and frequently accompanied by a range of adverse effects. By contrast, traditional Chinese medicine (TCM) offers a promising comprehensive approach for DMC management, characterized by multi‑target actions, fewer side effects, the ability to intervene in complications, potential for combined effect with western drugs, and cost advantages, thereby meriting further promotion and investigation. This review comprehensively assesses the pharmacological actions, potential mechanisms, and current clinical evidence of Astragalus membranaceus (AM) and its active components in DMCs, which could provide novel therapeutic insights for DMCs, and future research directions. We conducted precise searches in PubMed using the keywords ("Astragalus membranaceus" OR "Huangqi" OR "astragaloside") AND ("diabetic kidney disease" OR "diabetic nephropathy"), ("Astragalus membranaceus" OR "Huangqi" OR "astragaloside") AND ("diabetic peripheral neuropathy"), ("Astragalus" OR "Huangqi" OR "astragaloside") AND ("diabetic retinopathy"), "astragaloside IV (AS-IV)", "Astragalus polysaccharide (APS)", "flavonoids", "formononetin (FMN)" and "calycosin" to accurately retrieve research reports on AM, its active components, and AM-based preparations in DMCs. The retrieved data followed PRISMA criteria. AM has protective effects on the kidneys, retinal blood vessels, and peripheral nerve myelin sheaths. Its mechanisms of action mainly include anti-oxidation, anti-inflammatory effects, and the inhibition of ferroptosis. Especially, AS-IV, flavonoids, APS, FMN and calycosin, these active components of AM have therapeutic potentials for preventing and treating DMCs. The existing clinical data initially indicate that AM can reduce proteinuria, improve metabolic disorders and regulate blood glucose, thereby playing a role in the prevention and treatment of DMCs. AM is a promising candidate of TCM for DMCs. Most researches focus on the protective effect of AM on DKD, with the lack of the studies of AM against DPN and DR. Moreover, clinical research of AM in DMCs remains markedly inadequate compared to its basic research. Therefore, future research efforts should prioritize conducting high-quality basic and clinical trials to verify the therapeutic effects of AM against DMCs.
Chemotherapy resistance remains a major clinical challenge in the treatment of gastrointestinal (GI) cancers. Mitochondrial remodeling contributes to chemoresistance through several interconnected functional modules, including metabolic reprogramming, elevation of the apoptotic threshold, adjustment of the reactive oxygen species setpoint, and mitophagy-mediated mitochondrial quality control. Polysaccharides and polysaccharide-based systems may represent promising candidates for modulating mitochondria-associated dysfunctions because of their structural diversity, biological activity, and generally favorable biocompatibility in specific experimental settings. This review aimed to systematically summarize the molecular mechanisms by which mitochondrial remodeling contributes to chemotherapy resistance in GI tumors and to evaluate the potential of polysaccharides and polysaccharide-based systems as therapeutic candidates for modulating these mitochondrial abnormalities. This PRISMA-guided systematic review with qualitative evidence synthesis searched PubMed and Web of Science from database inception to July 4, 2026. The search strategy covered terms related to polysaccharides and polysaccharide-based delivery systems, GI tumors, chemoresistance or chemosensitization, and mitochondria-related mechanisms. Records were deduplicated and screened according to predefined eligibility criteria. Core eligible studies were original experimental studies that investigated polysaccharide or polysaccharide-based interventions in GI tumor models, reported chemotherapy-related outcomes, and included endpoints related to mitochondria, redox regulation, apoptosis, autophagy, metabolism, or the tumor microenvironment. Because of substantial heterogeneity in polysaccharide sources, tumor models, chemotherapeutic agents, intervention formats, and mechanistic endpoints, the evidence was synthesized qualitatively rather than by meta-analysis. The search identified 652 records, including 322 from PubMed and 330 from Web of Science. After removal of 174 duplicate records, 478 records were screened. Full texts were sought for 75 reports, of which 20 could not be retrieved. Ultimately, 55 reports were assessed for eligibility; one retracted article and 34 reports that did not meet the core eligibility criteria were excluded. In total, 20 studies were included in the core evidence synthesis. These studies supported the involvement of polysaccharides or polysaccharide-based systems in modulating chemotherapy responses in GI tumors through several mitochondria-related mechanisms, including metabolic remodeling, regulation of the apoptotic threshold, modulation of redox status, and autophagy/mitophagy-associated adaptation. Additional mechanistic and contextual evidence suggested that polysaccharide-based delivery platforms and gut microenvironment modulation may influence drug accumulation, chemotherapy-related toxicity, and therapeutic sensitivity. Current evidence, mainly from preclinical studies, suggests that polysaccharides and polysaccharide-based systems may help modulate chemotherapy resistance in GI cancers by targeting mitochondria-related pathways. However, clinical translation remains limited by heterogeneity in polysaccharide structure, incomplete pharmacokinetic characterization, limited human evidence, and insufficient standardization of intervention models. Future studies should prioritize structural characterization, mechanism-guided validation, pharmacokinetic evaluation, and well-designed clinical studies to clarify their therapeutic value.
The pathogenic mechanisms underlying metabolic dysfunction-associated steatohepatitis (MASH) are highly complex and multifactorial, and there are limited pharmacological therapies proven effective for clinical use. Although the natural product wedelolactone (WED) demonstrates potential hepatoprotective effects, its therapeutic efficacy in MASH and the underlying mechanisms remain poorly understood. This study investigates the therapeutic efficacy of WED in ameliorating diet-induced MASH and aims to elucidate its direct molecular targets and underlying mechanisms. To investigate the therapeutic potential of WED in MASH, this study established an in vitro model of lipid accumulation and inflammatory response by treating HepG2 cells and primary mouse hepatocytes with palmitic acid (PA) and oleic acid (OA), thereby assessing the in vitro effectiveness of WED. Additionally, we employed the MCD diet- and HFHC diet-induced mouse MASH models to evaluate the therapeutic effects of WED. To identify its molecular targets, we synthesized biotin-labeled WED probes and screened potential direct binding proteins using a human proteome microarray. The binding affinity was validated by surface plasmon resonance (SPR) and cellular thermal shift assay (CETSA). Molecular dynamics simulation and molecular docking were performed to determine the key interaction sites between WED and its targets. Transcriptomic and bioinformatics analyses were integrated to elucidate the downstream signaling pathways regulated by WED through these targets. Finally, biological functional assays were performed to validate the underlying mechanism of WED. This study has demonstrated that WED exerts a significant anti-MASH effect by directly targeting SORBS1, with binding occurring at VAL32 and HIS47. The underlying mechanism involves the direct binding and activation of SORBS1 by WED, leading to multiple regulation of downstream signaling pathways. Specifically, WED regulates the expression of SORBS1 in liver tissue, improves glycolipid metabolism through the CBL/TC10/GLUT4 axis, and suppresses proinflammatory cytokines such as TNF-α and IL-6. This coordinated action results in effective alleviation of hepatic lipid accumulation, inflammation, and fibrosis. Furthermore, inhibiting SORBS1 exacerbates the MASH process and completely abolishes the efficacy of WED, thereby confirming that SORBS1 is the key target for its therapeutic effect. Here, this study reveals that WED ameliorates insulin resistance and suppresses hepatic inflammation and fibrosis by directly binding to SORBS1, thereby significantly attenuating the progression of MASH. These findings establish SORBS1 as a potential novel therapeutic target for MASH and provide a potential candidate compound for the advancement of therapies targeting MASH.
Aging disrupts choline metabolism, impairs mitochondrial function, and accelerates tissue degeneration. As gut microbes critically influence host nutrient handling, microbiota-targeted interventions may provide a promising strategy to delay mammary senescence. This study aimed to determine whether cinnamon polysaccharides (CP) and the commensal bacterium, Alloprevotella rava, could remodel host gut microbiota, enhance host choline utilisation, improve mitochondrial function, and thereby delay mammary senescence. An experimental study was conducted using mouse models, maternal intervention experiments, microbiota and metabolite analyses, and complementary cellular assays to evaluate the effects of CP and A. rava on choline metabolism, mitochondrial function, mammary senescence, and offspring development. Mice were treated with CP to assess its effects on mammary senescence and gut microbiota composition. The enrichment and supplementation of A. rava were further examined for their capacity to regulate serum and mammary choline levels, as well as the expression of host choline transporters, phosphatidylcholine biosynthetic enzymes, and microbial choline-trimethylamine (TMA) lyases (cutA and cutC). Cell-based experiments were performed to evaluate the direct effects of A. rava culture supernatant on epithelial choline uptake and biosynthesis. Metabolite analyses were used to identify the major microbial effector involved, and maternal intervention studies were conducted to assess systemic and offspring-related outcomes. CP alleviated mammary senescence in mice by reshaping the gut microbiota and enriching A. rava. Supplementation with A. rava increased choline levels in serum and mammary tissue, upregulated the expression of host choline transporters and phosphatidylcholine biosynthetic enzymes, and reduced microbial choline-TMA lyase expression, thereby improving host choline utilisation. The A. rava culture supernatant directly stimulated epithelial choline uptake and biosynthesis, and metabolite analyses identified acetate, rather than succinate, as the principal effector. These alterations reinforced choline-dependent phospholipid synthesis, improved mitochondrial function, and protected against cellular aging. In addition, cellular assays and maternal intervention experiments showed that A. rava enhanced epithelial metabolic resilience and supported offspring brain development. Supplementation with A. rava establishes a gut-mammary axis that promotes choline metabolism, strengthens mitochondrial function, and delays mammary senescence. Targeting specific gut microbes or their acetate-driven metabolites may represent a feasible strategy to improve maternal tissue health and support progeny neurodevelopment.
Mitophagy is vital for preventing cerebral ischemia-reperfusion (CI/R) injury. Naringin, a flavanone glycoside, reduces CI/R injury by blocking harmful mitophagy induced by peroxynitrite (ONOO⁻). Our earlier studies identified that mitochondrial calcium (mtCa2+) efflux stabilizes mitochondrial endosymbiosis and initiates protective mitophagy. However, it is unclear whether and how naringin inhibits harmful mitophagy in association with mtCa2+. This study aimed to examine the influence of the S-nitrosylated dynamin-related protein 1 (SNO-DRP1)/leucine-rich repeat kinase 2 (LRRK2)/mitochondrial calcium uniporter (MCU) pathway on harmful mitophagy, which is inhibited by naringin in CI/R injury. To validate the hypotheses, a transient middle cerebral artery occlusion/reperfusion (tMCAO/R) model in rats and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced PC12 cells were used to investigate the effects of naringin. Specifically, linsidomine hydrochloride (SIN-1) was used to enhance SNO-DRP1, whereas spermine (Spm) was used to activate MCU. Mitochondrial fragmentation was assessed by measuring the average mitochondrial diameter via transmission electron microscopy (TEM) and evaluating the protein levels of mitofusin 1 (MFN1) and dynamin-related protein 1 (DRP1) through western blotting (WB). The SNO-DRP1 formation was determined by a biotin switch assay. The extent of SNO-DRP1-mediated excessive mitophagy was examined by quantifying the number of autophagosomes using TEM and assessing the colocalization of translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20)-DRP1-3-NT and 3-NT-DRP1-LC3 via immunofluorescence (IF). The interaction between SNO-DRP1 and LRRK2 was confirmed using co-immunoprecipitation and IF colocalization. MtCa2+ overload induced by MCU was evaluated by analyzing MCU protein levels, measuring mtCa2+ fluorescence intensity following Rhod-2 AM treatment, and assessing the opening of the mitochondrial permeability transition pore. The mitochondrial reactive oxygen species and mitochondrial DNA release were used to evaluate the mitochondrial endosymbiosis failure. Naringin exhibited a dose-dependent protective effect against CI/R injury in rats subjected to the tMCAO/R model. It effectively reduced mitochondrial fragmentation and SNO-DRP1-mediated mitophagy, attenuated the interaction between SNO-DRP1 and LRRK2, and decreased the protein level of MCU in the penumbra region of the cortex. SIN-1 and Spm counteracted the neuronal protection of naringin in OGD/R-induced PC12 cells. They also mitigated the impact of naringin on mitochondrial fragmentation, harmful mitophagy, and MCU-mediated mtCa2+ overload and mitochondrial endosymbiosis disruption. Notably, only SIN-1 reversed naringin's suppression of SNO-DRP1 formation and its interaction with LRRK2 in vitro. This study demonstrates that naringin reduces ONOO⁻-caused harmful mitophagy in CI/R injury by blocking the SNO-DRP1/LRRK2/MCU pathway, which helps prevent mitochondrial fragmentation and preserve mitochondrial endosymbiosis. This pathway may be among the mechanisms by which naringin exerts its protective effects.
Given the limitations of the existing monoclonal antibody (mAb)-based therapies, more efficient and safer small-molecule-based checkpoint therapies targeting the programmed cell death-1 (PD-1) / programmed cell death ligand-1 (PD-L1) axis are gaining growing attention and urgently required. To identify a novel PD-L1 small-molecule inhibitor from natural products and systematically evaluate its antitumor activity, mechanism of action, and potential biomarkers. Based on the co-crystal structure of PD-L1 with BMS-202 (PDB ID: 5J89), molecular docking was employed for high-throughput virtual screening of 16,563 natural products. The binding affinity of candidate compounds to PD-L1 protein was validated through microscale thermophoresis (MST), cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assays and nuclear magnetic resonance (NMR). The blockade of PD-1/PD-L1 interaction was assessed using homogeneous time-resolved fluorescence (HTRF), NFAT-Luc luciferase reporter gene system, and cell membrane PD-1 binding assays. In vivo antitumor efficacy was systematically assessed in humanized PD-L1 knock-in B16F10 and MC38 tumor-bearing mouse models. The mechanism of action was investigated using RNA-seq transcriptomics, flow cytometry, and immunofluorescence staining. Finally, response mechanisms and potential biomarkers were systematically analyzed by comparing differential responses across distinct tumor models. Through high-throughput virtual screening, we identified rosavin as a small molecule with a novel scaffold that targets PD-L1, exhibiting the unusual small-molecule property of inhibiting PD-L1 without inducing its dimerization. Rosavin demonstrated significant antitumor activity in vivo by promoting antitumor immunity through enhancing CD8+ T cell activation, consistent with the effects of PD-L1/PD-1 blockade. Notably, rosavin was particularly effective for fighting against tumor progression in microsatellite instability-high (MSI-H) solid tumors and robustly strengthened the expression levels of CXCL9 and CXCL10 in tumors, which may serve as potential biomarkers for predicting responsiveness to rosavin-mediated PD-1/PD-L1 blockade. Rosavin serves as a privileged novel and unexpected scaffold for designing potent PD-1/PD-L1 modulators, offering promising candidates for cancer immunotherapy.
As natural bioactive macromolecules isolated from various berries, Berry polysaccharides (BPs) possess excellent biocompatibility, low toxicity and diverse health-promoting properties, which has garnered extensive interest in functional food and pharmaceutical research. Nevertheless, imperfect large-scale manufacturing processes, ambiguous structure-activity relationship (SAR) and limited metabolic research substantially impede their industrial transformation and practical application. This review systematically summarizes recent research advances on BPs, clarifies their preparation technologies, multifunctional bioactivities, molecular mechanisms, SAR rules, pharmacokinetic profiles and safety assessment, and highlights the cutting-edge technologies, so as to offer theoretical support for subsequent development and utilization of BPs. This comprehensive review was conducted to integrate and critically appraise the latest research progress on BPs, covering innovative preparation technologies, biological function exploration, structural-activity correlation analysis, pharmacokinetic profiles and cutting-edge interdisciplinary translational applications. Peer-reviewed literatures focusing on the extraction and purification, bioactivity evaluation, molecular mechanistic exploration, SAR analysis, pharmacokinetics and toxicological assessment of BPs were rigorously retrieved, screened and summarized for synthetic discussion. Novel eco-friendly extraction techniques, including ultrasonic-, microwave-, enzyme-assisted extraction and membrane separation are progressively replacing traditional extraction strategies. Berry polysaccharides display diverse bioactivities such as anti-inflammation, immunomodulation, hypoglycemia, hypolipidemia, neuroprotection and gut microbiota regulation via NF-κB, PI3K/Akt, MAPK, Keap1-Nrf2 signaling pathways and short-chain fatty acid metabolism. Meanwhile, SAR studies identify the molecular weight, glycosidic linkage, branching patterns and spatial conformation as critical factors dominating their bioactivity and bioavailability. BPs exhibit low oral bioavailability but favorable safety profiles, relying primarily on intestinal microbiota metabolism. Notably, artificial intelligence, multi-omics and nanotechnology greatly accelerate the comprehensive research on BPs. This review systematically summarizes current research progress and application prospects of berry polysaccharides (BPs). It deepens the understanding of their structure-function correlations and molecular mechanisms, offering valuable references for the rational development and translational application of BPs in functional foods, pharmaceuticals and health-benificial products.
Durable mucosal healing is the primary therapeutic goal in ulcerative colitis (UC). Intestinal epithelial stemness and microbiota homeostasis drive this healing process. Cystathionine-β-synthase (CBS) is a key enzyme in endogenous sulfur metabolism. Sulfur metabolism is essential for mitochondrial energy production and mucosal integrity. However, the precise role of CBS in UC pathogenesis remains unclear. Single plant-derived polyphenols, such as resveratrol or curcumin, show limited clinical efficacy due to low bioavailability. The combined effect of these botanical agents on CBS-mediated gut homeostasis requires further investigation. This study aimed to define the molecular function of CBS in colitis and to evaluate the therapeutic mechanism and clinical translational potential of a combined plant-derived resveratrol-curcumin treatment for UC. A comprehensive bench-to-bedside translational approach was employed. The study integrated human clinical sample analysis, in vivo animal experiments using transgenic models, in silico molecular docking, and a pilot clinical trial. CBS expression was quantified in colonic biopsies from UC patients and healthy controls. Dextran sulfate sodium (DSS) was used to induce colitis in wild-type (WT) and CBS knockout (Cbs-/-) mice. Clinical phenotypes, mucosal protein expression, microbiota composition, and short-chain fatty acid (SCFA) profiles were systematically assessed. Molecular docking evaluated the binding affinity between the CBS protein and the resveratrol-curcumin combination. The in vivo efficacy of this combination was tested in both WT and Cbs-/- mice. Finally, a pilot clinical trial assessed the combination therapy alongside mesalamine in UC patients. Colonic CBS expression was downregulated in both UC patients and colitis mice. CBS deficiency worsened DSS-induced clinical damage. It disrupted intestinal barrier homeostasis and severely impaired SCFA production. Molecular docking demonstrated strong binding affinity between the resveratrol-curcumin combination and the CBS protein. In vivo, this combined treatment effectively alleviated colitis symptoms. It reduced pro-inflammatory cytokines, upregulated mucosal proteins via CBS activation, and restored microbiota structure. Notably, these mucosal protective effects were completely abolished in Cbs-/- mice, confirming CBS as the essential target for this combined protective effect. In the pilot trial, the addition of resveratrol and curcumin to mesalamine improved clinical outcomes in UC patients. CBS acts as a crucial endogenous protector in the colon. It maintains epithelial stemness and microbiota metabolic homeostasis. The plant-derived resveratrol-curcumin combination specifically targets and restores CBS expression, promoting epithelial regeneration and microbiota remodeling. These findings highlight host-microbiome interactions in UC pathogenesis and offer a novel, targeted polyphenol strategy to achieve mucosal healing.
Baicalin is one of the main active components of Scutellaria baicalensis Georgi, a traditional herbal medicine. It exhibits a wide range of pharmacological activities, including antibacterial, anti-inflammatory, and antitumor effects. However, its potential side effects-particularly those related to cardiovascular function-remain incompletely understood. This study aimed to investigate the impact of baicalin on isoproterenol (ISO)-induced ventricular remodeling and to elucidate the underlying mechanisms. A mouse model of ventricular remodeling was established via continuous subcutaneous infusion of ISO. Cardiac function was evaluated using transthoracic echocardiography after baicalin treatment, while myocardial pathology and collagen deposition were assessed through histological staining. Neonatal rat cardiac fibroblasts (NRCFs) were isolated and subjected to CCK-8, scratch wound healing, and immunofluorescence assays to examine the effects of baicalin on proliferation, migration, and activation of NRCFs, both with and without ISO stimulation. To further explore the mechanism, NRCFs were either treated with an activator protein 1 (AP-1) inhibitor or transfected with a recombinant adenovirus encoding lysine-specific demethylase 1 (LSD1). Protein and mRNA expression levels of relevant targets were analyzed via western blot (WB) and real-time fluorescence quantitative PCR (RT-PCR), respectively. The data showed that baicalin acted as an independent fibrogenic factor, significantly aggravating ISO-induced ventricular remodeling, particularly myocardial fibrosis, both in vivo and in vitro. ISO pretreatment triggered a signaling cascade in mouse hearts and NRCFs, characterized by the sequential activation of extracellular signal-regulated kinase (ERK) and mitogen- and stress-activated protein kinase 2 (MSK2), resulting in histone H3 phosphorylation at Ser10 (p-H3Ser10) with a concomitant suppression of LSD1 activity. Furthermore, we have found that functioning as an LSD1 inhibitor, baicalin acted synergistically with ISO to further suppress LSD1 activity. This suppression led to the activation of AP-1, which in turn promoted the transcription of type I collagen. Conversely, adenovirus-mediated overexpression of LSD1 in NRCFs reduced the expression of AP-1 subunits (c-Jun and c-Fos) and reversed the pro-fibrotic effects of baicalin under ISO stimulation. ISO induces myocardial fibrosis by activating the ERK-MSK2-p-H3Ser10 signaling pathway. This leads to the suppression of LSD1 activity. As a natural LSD1 inhibitor, baicalin further suppresses LSD1 activity and exacerbates ISO-induced cardiac fibrosis. Mechanistically, LSD1 inhibition upregulates the transcription factor AP-1. This promotes the transcription of collagen I and drives fibrosis development.
Myocardial ischemia (MI) causes diverse pathological injuries, and pharmacological intervention remains a key therapeutic approach. Tanshinone IIA (Tan IIA), a principal active compound derived from the traditional Chinese medicine Danshen, is clinically used in the form of sodium tanshinone IIA sulfonate for the treatment of ischemic heart disease. However, its precise molecular mechanisms and targets are not fully understood. This study aimed to explore the targets and mechanisms of Tan IIA against MI in vitro and in vivo. In this study, mouse models of MI induced by left anterior descending artery ligation and isoproterenol were established to validate the therapeutic effects of Tan IIA on MI. We then reanalyzed the GSE1145 dataset of human cardiac tissues and integrated network pharmacology, molecular docking, and phosphoproteomics to screen potential target proteins of Tan IIA. Subsequently, the affinity of Tan IIA for its target was validated using gene knockdown models and multiple techniques, including cellular thermal shift assay, drug affinity responsive target stability assay, biolayer interferometry, and molecular dynamics simulations. Fibroblast growth factor receptor 1 (FGFR1) was identified as a novel target of Tan IIA, with binding assays showing a specific interaction and a dissociation constant of 13.8 μM. Site-directed mutagenesis further demonstrated that Tan IIA binds directly to the Gln284 residue of FGFR1 and promotes trans-autophosphorylation at Y653/Y654. Mechanistically, Tan IIA activates the downstream FRS2-MAPK pathway to inhibit autophagy, whereas in vitro enzyme assays indicate that it also limits the catalytic turnover of kinase domains. This unique modulatory profile defines Tan IIA as an extracellular domain-directed allosteric modulator that dissociates receptor phosphorylation from steady-state enzymatic flux to effectively provide cardioprotection. This study identifies FGFR1 as a direct molecular target of Tan IIA, establishing a target-specific mechanism underlying its cardioprotective effects and providing a mechanistic basis for the development of precision therapeutic strategies for MI.
. Despite advances in reperfusion therapy, the incidence and mortality risk of heart failure (HF) remain high among patients with acute myocardial infarction (AMI). Naoxintong (NXT), a well-characterized polyherbal formulation derived from traditional Chinese medicine, has shown potential benefits in preclinical and small-scale clinical studies, but its efficacy has not been tested in a large-scale randomized controlled trial. . To rigorously evaluate the efficacy and safety of NXT in reducing HF and major adverse cardiovascular events (MACEs) among AMI patients after reperfusion therapy. . A prospective, multicenter, randomized, double-blind, placebo-controlled, superiority trial. . Eligible post-reperfusion AMI patients from 21 hospitals in China were randomly assigned to receive either NXT (4 capsules, three times daily) or a matching placebo for 3 months, in addition to guideline-directed medical therapy (GDMT), with follow-up through 12 months. The primary endpoint was the incidence of new-onset HF within 12 months after randomization. Participants, clinicians, and outcome assessors were blinded to treatment allocation throughout the study period. . A total of 379 patients were included in the primary analysis (mean age 61.6 years; 78.1% male). The incidence of HF was significantly lower in the NXT group than in the placebo group (5.98%vs. 14.06%; HR 0.41, 95% CI 0.21-0.81; p = 0.01). The incidence of MACEs was also reduced with NXT (7.97%vs. 14.84%; HR 0.52, 95% CI 0.28-0.98; p = 0.04). The rates of serious adverse events (7.97%vs. 14.84%; p = 0.042) and any adverse events (7.97%vs. 21.09%; p < 0.001) were lower in the NXT group. No other significant between-group differences were observed for secondary outcomes or adverse event subtypes. . NXT, administered in addition to standard GDMT, significantly reduced the 12-month risk of HF and MACEs in reperfused AMI patients, with a favourable safety profile. These findings support NXT as a promising complementary strategy for secondary prevention after AMI.
Chronic obstructive pulmonary disease (COPD) is a prevalent respiratory disease characterized by airflow limitation, with genetic factors playing a crucial role in its pathogenesis. Effective-component compatibility of Bufei Yishen formula Ⅲ (ECC-BYF Ⅲ) has been shown to effectively ameliorate COPD symptoms; however, its underlying molecular mechanism remains elusive. To investigate the intervention mechanism of ECC-BYF Ⅲ on COPD and identify hub genes. Using the GSEA method, which retains all gene information, and exhibits robustness under small-sample conditions as well as high statistical power, COPD-related pathways were identified. Among the genes contained in those pathways, hub genes were identified by protein-protein interaction (PPI) network analysis, cytoHubba algorithm, etc. The reliability of the identified hub genes was validated by independent datasets, qRT-PCR in rats with COPD, HUVEC and MH-S cells, molecular docking, molecular dynamics simulations (MD), and surface plasmon resonance (SPR). Compared with COPD rats, ECC-BYF Ⅲ could ameliorate pathological changes and improve lung function. Then, utilizing the RNA-seq data from COPD rats, 106 disease related pathways were identified in the COPD group when contrasted with the control group (FDR < 0.05), of which 72 pathways were reversed after ECC-BYF Ⅲ intervention. Of the 3081 genes in those reversal pathways, 395 genes were differentially dysregulated in the COPD rat samples as opposed to the control samples (FDR < 0.05). Then, six genes (Gsk3b, Ctnnb1, Pten, Met, Pik3r3, and Erbb2) were identified by PPI network analysis, cytoHubba algorithm, and GeneCards database. Two hub genes (Ctnnb1 and Pik3r3) were well confirmed in independent datasets, the COPD rat model, HUVEC and MH-S cells, molecular docking, MD, and SPR. Two COPD hub genes were identified, and their regulation by ECC-BYF Ⅲ was demonstrated. Our results might provide a basis for the study of the mechanism of COPD, and its prevention and treatment guided by traditional Chinese medicine.
Excessive NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activity is an important driver of inflammatory tissue damage in acute lung injury (ALI), yet effective targeted therapies remain lacking. Neferine, a natural bisbenzylisoquinoline alkaloid extracted primarily from the mature seed embryo of Nelumbo nucifera, has shown anti-inflammatory potential, but its role in inflammasome regulation during ALI is still not well understood. This study was designed to examine whether neferine could attenuate lipopolysaccharide (LPS)-induced ALI through modulation of NLRP3 inflammasome signaling and to explore the mechanism involved. We carried out a preclinical investigation using an LPS-triggered mouse model of ALI together with a macrophage pyroptosis model induced by LPS+nigericin (Nig). Both preventive administration and delayed post-challenge treatment with neferine were evaluated in vivo. To define pathway dependence, we combined pharmacologic AMP-activated protein kinase (AMPK) inhibition, Prkaa1 deficiency, and blockade of autophagic flux. Neferine was examined in LPS-treated mice and in macrophages exposed to LPS plus Nig. Lung pathology, inflammatory injury, and pyroptosis-related changes were assessed. Additional experiments focused on AMPK signaling, autophagic flux, and NLRP3 degradation. Neferine attenuated inflammatory lung injury in mice, accompanied by reduced inflammatory cytokine production and decreased macrophage pyroptosis. Mechanistic analyses showed that neferine increased AMPK phosphorylation and downstream unc-51 like autophagy activating kinase 1 (ULK1) signaling, enhanced autophagic flux, and promoted K63-linked ubiquitination of NLRP3. These changes favored trafficking of NLRP3 into autophagic compartments and its subsequent lysosomal degradation, thereby limiting inflammasome activity. Pharmacological inhibition of AMPK signaling and genetic loss of Prkaa1 weakened the effects of neferine on ULK1-related autophagy and pyroptosis-associated readouts, supporting a functional contribution of AMPKα1 signaling to this process. Notably, neferine also remained effective when given after LPS exposure, reducing inflammatory injury and pyroptosis-associated readouts in the therapeutic setting. These findings indicate that neferine alleviates inflammasome-related lung injury at least partly through AMPKα1-dependent autophagy, accompanied by increased ULK1 phosphorylation and subsequent autophagy-lysosomal clearance of NLRP3.
Ulcerative colitis (UC) is an intractable inflammatory bowel disorder characterized by persistent intestinal inflammation and impaired gut barrier integrity. Its pathogenesis is multifactorial, involving gut microbiota dysbiosis, metabolic dysfunction, and cellular senescence. Current therapeutic regimens remain limited, underscoring an urgent need for innovative agents that target these interrelated pathological cascades. This study aimed to evaluate the pharmacological effects of poricoic acid A (PAA) on DSS-induced senescence in intestinal epithelial cells and in a murine model of ulcerative colitis (UC), as well as the underlying molecular mechanisms. We established a DSS-stimulated senescent intestinal epithelial cell model and a DSS-induced UC mouse model. Multi-omics and bioinformatics strategies, including network pharmacology, transcriptome profiling, gut metagenomics, and intestinal targeted metabolomics, were combined with molecular docking to predict candidate signaling axes. Subsequent pharmacological inhibition and siRNA-mediated silencing assays were performed to validate core pathways functionally. PAA robustly suppressed DSS-induced senescence and inflammatory responses in intestinal epithelial cells. In vivo assays verified that PAA alleviated UC-related manifestations, including body weight loss, rectal hemorrhage, and colonic histological injury. Joint network pharmacology and transcriptomic screening identified the AMPK/PPARγ as the core pathway mediating PAA's bioactivity. Mechanistic experiments confirmed that PAA directly bound and activated PPAR, further functionally triggering downstream AMPK/SirT1/PGC1α signaling. Blockade of AMPK via pharmaceutical antagonists or siRNA largely abolished PAA's anti-senescence and anti-inflammatory capacities; PPARγ suppression, in turn, secondary deactivated the AMPK and its downstream functional effectors. In mouse models, AMPK inhibition drastically compromised PAA's protective effects against UC. Moreover, PAA treatment of UC is closely associated with remodeling of the gut microbiome-metabolome axis and restoration of intestinal homeostasis. PAA exerts potent anti-senescence, anti-inflammatory, and colon-protective effects in UC via activating the AMPK/PPARγ signaling pathway. Such beneficial activity may be associated with the normalization of gut microbiota-metabolome homeostasis. This work identifies novel molecular targets and a promising lead compound for the intervention of ulcerative colitis.
Refined Changqin NO.1 (R-Cq1) has shown favorable therapeutic effects in traumatic brain injury (TBI), which triggers neuronal PANoptosis and involves dysregulation of gut microbiota and metabolites. This study investigates whether R-Cq1 improves TBI through gut-brain axis-mediated neuronal PANoptosis. A TBI mouse model and injured neurons were used to assess the effects of R-Cq1 on gut microbiota, short-chain fatty acid (SCFA) profiles, TNF-α signaling, and neuronal PANoptosis. R-Cq1 was administered to TBI mice, and their fecal samples were subjected to 16S rRNA sequencing and targeted metabolomics. Injured neurons were treated with 3-methylbutanoic acid (3-MA), R-Cq1, and TNF-α inhibitor 3,6'-dithiothalidomide, followed by assessment of neuronal PANoptosis. Anaerotruncus colihominis was transplanted into R-Cq1-treated TBI mice, which underwent neurological function assessment. R-Cq1 improved neurological outcomes in TBI mice and attenuated TBI-induced neuronal death. Immunofluorescence analysis of the gut revealed TBI-induced intestinal barrier disruption, which was mitigated by R-Cq1. Consistently, R-Cq1 reshaped the gut microbiota, reducing the abundance of the genus Anaerotruncus in TBI mice. Anaerotruncus showed a significant positive correlation with modified neurological severity scores. TBI induced alterations in SCFA profiles, with 3-MA significantly increased, which was suppressed by R-Cq1 treatment. 3-MA was positively correlated with modified neurological severity scores and genus Anaerotruncus. In vitro, 3-MA exacerbated PANoptosis, as indicated by increased levels of N-GSDMD, p-MLKL, and cleaved caspase-8, and reversed the inhibitory effect of R-Cq1 on PANoptosis. We also found that 3-MA upregulated TNF-α in scratched neurons. However, when TNF-α was inhibited, the pro-PANoptotic effect of 3-MA was attenuated. In TBI mice treated with R-Cq1, gavage with 3-MA-related Anaerotruncus colihominis abolished the neuroprotective effects of R-Cq1 and increased neuronal death. Collectively, R-Cq1 suppresses Anaerotruncus-associated 3-MA to hinder TNF-α upregulation, thereby alleviating neuronal PANoptosis and TBI. This study elucidates the underlying therapeutic mechanism of R-Cq1 in TBI.
Drug nephrotoxicity is a critical concern in drug safety assessment. Podophyllotoxin (PPT) has clear antitumor activity, but its clinical application is limited by nephrotoxicity, the underlying mechanisms of which remain unclear. Employing our innovative toxicological evidence chain (TEC) concept, this study aimed to elucidate the mechanisms underlying PPT-induced nephrotoxicity through a multi-omics approach. Rats were administered PPT at 10 or 20 mg/kg by gavage for 4 days. Behavioral and appearance changes were observed to obtain injury phenotype evidence (IPE). Renal injury was assessed by histopathology and serum biochemical parameters as adverse outcome evidence (AOE). Toxic event evidence (TEE) was obtained by integrating TMT-based proteomics, acetylated proteomics, and targeted metabolomics, combined with functional validation using Western blot, quantitative real-time PCR, and ferroptosis-specific interventions (Fer-1/Erastin). PPT caused renal injury and abnormalities in biochemical parameters in rats (AOE). PPT downregulated SIRT3, reduced LKB1 deacetylation, and inhibited AMPK activity, thereby decreasing the expression of p-ACC1, PPARα, and PGC-1α, leading to fatty acid oxidation (FAO) dysfunction and abnormal lipid accumulation (TEE). Concurrently, PPT suppressed Nrf2, HO-1, and GPX4, disrupting the antioxidant system; downregulated GCLM and GSS, interfering with GSH metabolism; and induced iron overload and lipid peroxidation, triggering ferroptosis (TEE). Functional validation experiments further demonstrated that the ferroptosis-specific inhibitor Fer-1 significantly reversed PPT-induced renal injury, whereas the ferroptosis inducer Erastin exacerbated the injury, confirming the key mediating role of ferroptosis in PPT-induced nephrotoxicity. This study initially revealed that PPT caused FAO dysfunction via the SIRT3-LKB1-AMPK-ACC-PPARα/PGC-1α axis, and triggered ferroptosis through the Nrf2-HO-1/GPX4 and GCLM-GSS-GSH/GPX4 axes, thereby leading to nephrotoxicity. These findings provide new targets for the prevention and treatment of PPT-induced nephrotoxicity.
Excessive neuroinflammation exacerbates secondary brain injury after intracerebral hemorrhage (ICH). Adenosine monophosphate deaminase 2 (AMPD2) has been linked to immune regulation, but its role and mechanism in ICH-related neuroinflammation remain unclear. Emerging evidence underscores the importance of epigenetic modifications in post-ICH inflammation. This study aimed to determine whether AMPD2 contributes to neuroinflammation and whether folic acid (FA), a key methyl donor, attenuates inflammatory damage through epigenetic regulation of AMPD2. Bisulfite pyrosequencing was used to detect the DNA methylation level of AMPD2 in patients and experimental models. The function of AMPD2 and the role of FA were investigated using a mouse model of ICH and lipopolysaccharide (LPS)-stimulated microglia. The interaction between AMPD2 and deoxycytidine kinase (DCK) and its pathway activity were assessed using gain-and-loss-of-function assays, NF-κB regulation assays, and co-immunoprecipitation (Co-IP) assays. Furthermore, the methylation inhibitor 5-azacytidine (5-AZA) was used to validate the epigenetic mechanism of FA. AMPD2 expression was significantly upregulated after ICH, a phenomenon negatively correlated with its DNA methylation levels. AMPD2 knockdown attenuated neuroinflammation and improved neurological outcomes, whereas AMPD2 overexpression exacerbated inflammatory responses. FA treatment suppressed AMPD2 expression by promoting its DNA methylation, thereby mitigating neuroinflammation and neuronal injury. Mechanistically, AMPD2 interacted with DCK and promoted its ubiquitin proteasome dependent degradation, leading to disinhibition of the NF-κB pathway and enhanced release of pro inflammatory cytokines such as IL 1β and IL 6. Inhibition of NF-κB or rescue of DCK expression reversed the pro inflammatory effects of AMPD2. This study explore that AMPD2 promotes inflammation by binding to DCK and facilitating its ubiquitination and reveals the mechanism that FA improves neurological function after ICH by altering the DNA methylation level in the promoter region of AMPD2 gene. thereby improving the recovery of neurological function through AMPD2/DCK/NF-κB signaling pathway.
Allergic contact dermatitis (ACD) is a common and debilitating disease with a lifetime prevalence of 20%. Recently, the mast cell Mas-related G protein-coupled receptor B2 (MrgprB2) has been implicated in the pathogenesis of pruritus in ACD. Huanglian Ointment (HLO) demonstrates significant clinical efficacy in promoting skin healing, treating eczema, and alleviating skin inflammation and itching. However, the mechanism underlying the antipruritic effects of HLO has not yet been elucidated. This study aims to elucidate the potential mechanism underlying the therapeutic effect of HLO against ACD via the MrgprB2-mediated neuroimmune axis. In this study, scratching behavioral assays, transgenic mice (MrgprB2-/- mice), histopathological analysis, immunofluorescence, whole-cell patch-clamp technique, HPLC analysis, UHPLC-MS/MS, molecular docking and Bio-layer Interferometry (BLI) were employed to investigate the potential antipruritic mechanisms of HLO and berberine. Here, we demonstrated that HLO ameliorated pruritus in the oxazolone-induced ACD model by inhibiting mast cell activation and the subsequent release of tryptase, thereby reducing the abnormal excitability of dorsal root ganglion (DRG) neurons. In addition, knockout of MrgprB2 receptor could significantly reduce oxazoline-induced pruritus, tryptase release, and the abnormal excitability of DRG neurons. Notably, the therapeutic effect of HLO was significantly weakened in MrgprB2-/- mice, indicating that the mechanism underlying the antipruritic effect of HLO is potentially related to the MrgprB2-mediated neuroimmune axis. Furthermore, component analysis of HLO combined with molecular docking screening suggested that berberine could serve as one of the potential key bioactive components. BLI assays implied a relatively preferable binding interaction between berberine and MrgprX2 protein. Further data also indicated that berberine might mitigate pruritus in ACD model, and such anti-pruritic activity is presumably correlated with the MrgprB2 receptor. In conclusion, our findings indicated that HLO could effectively alleviate pruritus, and this effect may be linked to the MrgprB2-related neuroimmune axis. Berberine may potentially act as an active component involved in HLO's antipruritic pharmacological effects.