Cholestatic liver injury (CLI) is a rapid progressive liver disorder characterized by the accumulation of bile acids (BA). Although pregnane X receptor (PXR) is a critical regulator of BA metabolism, the synergistic mechanisms of natural compounds targeting these pathways remain unclear. In this study, we demonstrated a positive correlation between BA accumulation and disease severity in clinical samples. Further, we identified Schisandrin B (Sin B), a lignan from Schisandra chinensis, as a potent hepatoprotective agent in α-naphthyl isothiocyanate (ANIT)- induced CLI. We demonstrated that Sin B treatment reduced BA levels and inflammation in ANIT-induced WRL68 cells, liver lobule chips, and mice. Notably, Sin B activated PXR, increased the levels of UDP-glucuronosyltransferase 1A1 (UGT1A1), CYP3A4 (in humans) / CYP3A11 (in mice) and MRPs, and enhanced TFEB transcriptional activity and autophagic flux in vivo and in vitro. Knockout of hepatic Pxr or Tfeb blocked these effects of Sin B. Mechanistic investigation revealed that Sin B is directly binds to PXR at residues S106, G144, and W299, inducing conformational changes in the ligand-binding domain (LBD) was verified through target fishing, molecular dynamics (MD) simulations, drug affinity responsive target stability assay, isothermal titration calorimetry and surface plasmon resonance. Our findings provide structural and functional insights into the dual-pathway mechanism of Sin B and support its therapeutic potential for CLI.

ETHNOPHARMACOLOGICAL RELEVANCE:In traditional Chinese medicine (TCM), colorectal cancer (CRC) is commonly associated with patterns such as damp-heat accumulation, heat toxin, and yin deficiency. Sanwu Huangqin Decoction (SWHQD) is a classical traditional Chinese medicine (TCM) formula composed of three medicinal herbs-Huangqin (Scutellaria baicalensis Georgi), Kushen (Sophora flavescens Ait.), and Dihuang (Rehmannia glutinosa Libosch.). It has been traditionally prescribed for clearing heat, eliminating dampness, and nourishing yin, particularly in the treatment of gastrointestinal disorders. Clinically, SWHQD has been used as an adjunctive intervention for CRC management. However, the precise pharmacological mechanisms underlying its anti-CRC activity remain incompletely characterized. AIM OF THE STUDY:This study sought to evaluate the anti-tumorigenic efficacy of SWHQD against CRC and to determine whether ferritinophagy-mediated ferroptosis contributes to its mechanism of action. MATERIALS AND METHODS:Human CRC cell lines (HCT116 and SW480) and a xenograft tumor model in BALB/c nude mice were used to evaluate the anti-tumor effects of SWHQD in vitro and in vivo. Ferroptosis-related indicators, including intracellular Fe2+, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH), were measured. The role of ferritinophagy was examined through analysis of the NCOA4/FTH1 pathway and NCOA4 gene silencing. RESULTS:SWHQD significantly suppressed CRC cell proliferation in vitro and inhibited tumor growth in vivo. Treatment induced hallmark features of ferroptosis, including elevated intracellular Fe2+, increased lipid ROS and MDA levels, and depletion of GSH, all of which were significantly abrogated by the ferroptosis inhibitors. Mechanistically, SWHQD upregulated nuclear receptor coactivator 4 (NCOA4), strengthened the interaction between NCOA4 and ferritin heavy chain 1 (FTH1), promoted autophagic degradation of FTH1, and consequently triggered iron release, thereby activating ferritinophagy-dependent ferroptosis. Silencing of NCOA4 markedly attenuated these biochemical and functional responses. CONCLUSIONS:SWHQD suppresses CRC through a mechanism involving the NCOA4/FTH1 pathway to induce ferritinophagy-dependent ferroptosis. These findings provide mechanistic evidence supporting the traditional application of SWHQD and highlight its potential as a complementary therapeutic strategy for CRC.

Heart failure with preserved ejection fraction (HFpEF) is a complex cardiovascular disorder characterized by diastolic dysfunction, metabolic dysregulation and limited therapeutic options. Post-translational modifications (PTMs) are key regulators of cardiac metabolism, but the role of butyrylation in HFpEF pathogenesis remains unclear. This study explored the mechanistic role of butyrylation in myocardial energy metabolism of HFpEF and evaluate the therapeutic potential of ginsenoside Rb3 (G-Rb3). A "two-hit" (high-fat diet + L-NAME) mouse model and a phenylephrine (PE)-induced hypertrophic and metabolically stressed cellular model were established. Myocardial PTM screening identified butyrylation as the target for proteomic analysis. G-Rb3 efficacy was evaluated in vivo and in vitro, with mechanistic studies involving Sirtuin 4 (SIRT4) inhibitor and overexpression experiments to confirm its regulatory role. Male mice model displayed earlier and more severe HFpEF phenotypes than females, thus justifying their use in mechanistic studies. Butyrylome analysis revealed hyperbutyrylation of succinate-CoA ligase subunit alpha (SUCLG1) at K90, which impaired its enzymatic function in tricarboxylic acid (TCA) cycle, resulting in reduced succinate and ATP production. SIRT4 was identified as a key regulator of SUCLG1 debutylation, with downregulated SIRT4 expression leading to SUCLG1 hyperbutyrylation. G-Rb3 directly bound SIRT4, reversing SUCLG1 hyperbutyrylation, restoring TCA cycle flux and ATP levels, improving diastolic dysfunction and metabolic abnormalities. These effects were nullified by SIRT4 inhibition, confirming SIRT4 as G-Rb3’s critical target. Our study reveals SUCLG1 butyrylation as a novel metabolic regulator in the pathogenesis of HFpEF, orchestrated by SIRT4. G-Rb3, a SIRT4-interacting regulator, rescues this axis, offering a mechanism-based therapy for HFpEF.

The ornamental plant Ficus microcarpa produces diverse bioactive triterpenoids. We functionally characterized eight oxidosqualene cyclases (FmOSC1-8), which together generate eight distinct triterpenol skeletons, underpinning the species chemical diversity. FmOSC1 was identified as a rare multifunctional ψ-taraxasterol synthase, the first of its kind in Moraceae. Key residues differentiating FmOSC1 from the α-amyrin-predominant FmOSC2 were pinpointed. Furthermore, a chromosomally linked gene pair (FmOSC3/FmCYP716A520) was shown to sequentially produce lupane-type derivatives. This cytochrome P450 (FmCYP716A520) exhibited broad substrate flexibility, processing intermediates from FmOSC1, FmOSC2, and FmOSC6 to form ψ-taraxastane-, ursane-, and oleanane-type products, respectively. Remarkably, CYP716A homologues from five other plant species could also utilize the FmOSC1 product, yielding oxidized derivatives and revealing conserved catalytic versatility within this enzyme subfamily. Our findings provide fundamental genetic insights into triterpenoid biosynthesis in F. microcarpa and offer valuable biocatalytic tools for synthetic biology applications.

10-Hydroxyl mesaconitine (10-OH MA) is a new potential toxicity marker of Radix Aconiti Lateralis Preparata (Fuzi), which is widely used for the treatment of cardiovascular diseases, rheumatoid arthritis, and other illnesses. Recently, we found that 10-OH MA concentrations in the plasma and urine of outpatients are comparable to those of aconitine, one of the most important toxicity markers of Fuzi. Published studies have shown that CYP3A plays important roles in the disposition and toxicity of Fuzi. This study aims to elucidate the role of cytochrome P450 enzymes in the metabolism and toxicity of 10-OH MA. 10-OH MA was transformed to 14 and 12 metabolites when incubated with human and mouse liver microsomes, respectively. The major metabolic pathways included hydroxylation, dehydrogenation, and demethylation. Human recombinant enzyme metabolic assays revealed that CYP3A5 was mainly involved in 10-OH MA metabolism, followed by CYP3A4, CYP3A7, CYP1A2, and CYP2C8. CYP3A inhibitor ketoconazole significantly inhibited the metabolism of 10-OH MA in vitro. The dose-normalized area under the plasma concentration-time curve from time 0 to t (AUC0-t/Dose) and tissue 10-OH MA concentrations were significantly increased in Cyp3a-inhibited C57BL/6J mice administered with 10-OH MA, which was accompanied by a sharp decrease in the LD50 value and enhanced cardiotoxicity and neurotoxicity. Moreover, diltiazem markedly increased the AUC0-t/Dose of 10-OH MA by 17.6-fold. Additionally, 10-OH MA competitively inhibited CYP3A5 and CYP3A4 in vitro, with apparent Ki values of 5.0 and 13.6 μmol/L, respectively. In conclusion, CYP3A5 and CYP3A4 are the pivotal enzymes responsible for the metabolism and toxicity of 10-OH MA. Coexposure to 10-OH-MA and CYP3A inhibitors likely significantly increased 10-OH MA toxicity. SIGNIFICANCE STATEMENT: 10-Hydroxyl mesaconitine (10-OH MA) is a potential toxicity marker of Radix Aconiti Lateralis Preparata (Fuzi), the metabolism of which is predominantly mediated by CYP3A5 and CYP3A4. CYP3A inhibition markedly increases systemic exposure and cardiotoxicity/neurotoxicity of 10-OH MA. In addition, 10-OH MA competitively inhibits CYP3A, indicating the risks of coadministration with CYP3A inhibitors.

Ganglioside GM3 is a well-established cutaneous melanoma-associated carbohydrate antigen. Efforts toward its clinical translation as a cancer vaccine have been hampered by poor immunogenicity. This study describes the design and synthesis of a fully synthetic self-adjuvanting GM3-based conjugate vaccine (1) using lipid A mimetics as a carrier. Immunological evaluations demonstrated that conjugate 1 effectively elicited GM3-specific immune responses. The antisera induced by this conjugate recognized, bound to, and facilitated the destruction of GM3-positive cancer cells. Combination with the adjuvants QS-21 and EcMPLA dramatically enhanced the immunogenicity and antitumor efficacy of conjugate 1 at a dose of 2 μg of GM3 per mouse. The IgG titers of 1/EcMPLA/QS-21 were more than twice those of GM3-KLH/Al. Furthermore, 1/EcMPLA/QS-21 conferred strong protection in tumor-challenge models, significantly delaying tumor growth and extending survival. These findings demonstrate the potential of conjugate 1 combined with EcMPLA and QS-21 as a candidate vaccine for melanoma immunotherapy.

Cinnamaldehyde, a bioactive constituent derived from Cinnamomum cassia Presl, exhibits diverse pharmacological effects, including vasodilatory and antihypertensive properties. However, its pharmacological impact and underlying mechanism concerning ischemic heart failure (IHF) remain poorly understood. This study aimed to investigate the cardioprotective effects of cinnamaldehyde on IHF and thereby elucidate its potential mechanism both in vivo and in vitro. To do so, a hypoxic injury model was established using AC16 cells, and male C57BL/6J mice underwent left anterior descending (LAD) artery ligation for eight weeks before receiving varying doses of cinnamaldehyde from the fourth week onward. Cardiac function and morphology were assessed via M-mode echocardiography, H&E staining, and Masson staining. Western blotting, co-immunoprecipitation (IP) assays, cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) analysis, and siRNA transfection were employed to evaluate the mechanism. Cinnamaldehyde significantly improved cardiac function, ameliorated cardiac fibrosis, and reduced myocardial inflammation in LAD-induced IHF mice. Concurrently, it protected cardiomyocytes and inhibited the inflammatory response in oxygen-glucose-deprived (OGD)-treated AC16 cells. Mechanistically, cinnamaldehyde was directly bound to USP18 and thus upregulated its expression. Further investigation revealed that cinnamaldehyde inhibited [Formula: see text]-adrenergic receptor ([Formula: see text]-AR) ubiquitination, thereby increasing its protein level. It also suppressed the TAK1/NF-κB pathway. Crucially, silencing USP18 eliminated both the cardioprotective and anti-inflammatory effects of cinnamaldehyde while also halting the inhibition of both [Formula: see text]-AR ubiquitination and the TAK1/NF-κB pathway. Cinnamaldehyde thus collectively enhances cardiac function against IHF by upregulating USP18 to thereby subsequently suppress both [Formula: see text]-AR ubiquitination and the activation of the TAK1/NF-κB pathway.

Background Colorectal cancer (CRC) is a leading cause of cancer death worldwide, mainly due to cancer cell proliferation and migration. Although chondroitin sulfate (CS) is involved in cancer progression, its regulatory mechanisms remain unclear. Purpose To investigate the role and mechanism of chondroitin polymerizing factor 2 (CHPF2) and CS in CRC progression, as well as to evaluate the therapeutic potential of ponicidin. Methods The correlation between CHPF2 and prognosis was analyzed in clinical samples. Mechanistically, ponicidin was found to target CHPF2, suppress CS synthesis, and consequently block the Wnt/β-catenin pathway. Its anti-tumor efficacy was validated in cellular, organoid, and animal models. Results We identified CHPF2, a key enzyme in CS synthesis, as a critical driver of CRC. CHPF2 is significantly overexpressed in CRC tissues, and its high expression correlates with advanced disease stage and poor patient prognosis. Functionally, CHPF2 drives tumor cell proliferation, migration, and survival by enhancing CS production. Mechanistically, CS promotes the activation of the Wnt/β-catenin signaling pathway and epithelial-mesenchymal transition (EMT). This effect is associated with CS-dependent modifications of Wnt1; however, further investigation is required to determine whether Wnt1 is directly modified by CS chains or indirectly affected via CS-modified proteoglycans. Furthermore, the natural diterpenoid ponicidin derived from Rabdosia rubescens directly targets CHPF2 and inhibits its enzymatic activity, thereby reducing CS biosynthesis and subsequently blocking the Wnt/β-catenin signaling pathway. The anti-tumor efficacy of ponicidin was validated in cellular models, patient-derived organoids, and primary CRC models, demonstrating its potent inhibitory effects on tumor growth and metastasis. Conclusion Our study reveals the oncogenic role of the CHPF2/CS axis in CRC, establishes CHPF2 as a novel therapeutic target, and provides compelling preclinical evidence supporting ponicidin as a promising CHPF2-targeted agent for CRC treatment.

Six new sesquiterpenoids glasesquiterpenoids A-F (1-6), together with two known ones, 7 and 8, were coisolated from the root of Lindera glauca. Their structures were elucidated by x-ray diffraction, quantum chemical calculations, and spectroscopic methods. Glasesquiterpenoid A (1) with an unprecedented 4/7 bicyclic carbon skeleton was the first example reported in the class of sesquiterpenoids. In the bioassays, compounds 6‒8 exhibited good inhibitory activity of NO production with IC50 values of 2.4, 5.5, and 4.0 µM, respectively, comparable to the positive control indomethacin (IC50 = 24.1 µM).

BACKGROUND:Irinotecan (CPT-11) is widely used for colorectal cancer treatment, with delayed-onset diarrhea as its primary side effect. Xiao-Chai-Hu-Tang (XCHT) has been clinically observed to alleviate chemotherapy-induced diarrhea, but its underlying mechanisms remain unclear. PURPOSE:This study aimed to elucidate the regulatory mechanisms through which XCHT alleviated CPT-11-induced diarrhea using an integrated multi-omics approach. METHODS:The chemical components of XCHT were detected using UHPLC-QE-Orbitrap-MS, and 12 active components were quantified via UHPLC-QQQ-MS/MS to ensure quality stability. CPT-11-induced diarrheal mice were established to evaluate the therapeutic effects of XCHT. Differential metabolites in liver and intestinal tissues among control, CPT-11, and XCHT mice were analyzed by untargeted metabolomics, followed by molecular network analysis using IPA. Bile acids were quantified using targeted UHPLC-QQQ-MS/MS method. Proteomics of colon tissues identified differentially expressed proteins, with functional enrichment conducted via GO and KEGG. Western blot and flow cytometry were used to validate the potential biopathway. RESULTS:Total 1108 chemical components were identified in XCHT, mainly including flavonoids, terpenoids and phenylpropanoids. XCHT significantly improved diarrhea symptoms and reduced intestinal inflammation. Metabolomics revealed 117 and 53 differential metabolites in the liver and intestine, respectively, with both tissues showing alterations in bile acid metabolism. Targeted bile acid analysis showed that CPT-11 inhibited the synthesis and uptake of bile acids in the liver and enhanced bile excretion, resulting in an increase in bile acids in the gallbladder. Meanwhile, CPT-11 impaired the reabsorption of bile acids in the intestine, leading to a decrease in total bile acid levels, reduction of circulating bile acids, and accumulation of conjugated bile acids in the colon. XCHT reversed these abnormal phenomena, restoring the intestinal bile acid homeostasis. Proteomics identified 86 differentially expressed proteins in colon tissues, with significant enrichment in 'Focal adhesion' pathway. Further verification indicated that CPT-11 abnormally activated the focal adhesion kinase (FAK) and its downstream RhoA/ROCK pathways, then reduced tight junction proteins ZO-1 and Occludin, damaging the intestinal barrier. XCHT inhibited this pathway, restored the expression of tight junction proteins, balanced Th1/Th2 differentiation, and alleviated intestinal inflammation. Cholestyramine, a bile acid chelator, also alleviated CPT-11-induced diarrhea by reducing the accumulation of bile acids in the intestine, confirming the key role of bile acids in the treatment of CPT-11-induced diarrhea. CONCLUSION:This study revealed that the combined administration of XCHT could restore bile acid reabsorption, reduced the accumulation of conjugated bile acids in the colon, inhibited the abnormally activated FAK-RhoA/ROCK pathway, improved intestinal epithelial barrier function and alleviate diarrhea. These findings provide a new entry point for the treatment of chemotherapy-induced diarrhea (CID) and offer data support for the combined medication regimen of XCHT and CPT-11.

ETHNOPHARMACOLOGICAL RELEVANCE:Xiao-Chai-Hu-Tang (XCHT) is a classical multi-herb formula widely used for gastrointestinal symptoms and peptic ulcers. Our previous preclinical studies have shown that XCHT attenuated irinotecan-induced severe delayed-onset diarrhea (SDOD). However, the clinical safety of XCHT combined with irinotecan remains unclear. AIM OF THE STUDY:To evaluate the clinical safety of concomitant XCHT administration with an irinotecan-based regimen (FOLFIRI) in patients with advanced colorectal cancer. MATERIALS AND METHODS:Six postmenopausal women with advanced colorectal cancer who had not previously been treated with irinotecan were enrolled. Patients received XCHT once daily for 5 consecutive days (9 g, p.o.). On day 4, irinotecan (180 mg/m2, i.v.) and XCHT were administered simultaneously. The diarrhea severity was evaluated following standard criteria in National Cancer Institute Common Terminology Criteria for Adverse Events version 5.0 (NCI-CTC AE V5.0). Routine safety parameters (e.g. blood, hepatic and renal function tests) were performed before the next cycle of chemotherapy. The blood samples were collected on day 4. Then XCHT ingredients and its metabolites in plasma were identified by ultra-high performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF/MS) and the plasma contents of XCHT ingredients, irinotecan and its major metabolites, were determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). RESULTS:During Cycle 1 safety monitoring, grade 1 diarrhea was observed in 5/6 patients and grade 2 diarrhea in 1/6 patient; no grade 3-4 diarrhea was observed. Pharmacokinetic (PK) results revealed that the systemic exposure of irinotecan, 7-ethyl-10-hydroxycamptothecin (SN-38), and SN-38 glucuronide (SN-38G) were similar to those in historical controls. Forty compounds were identified, mainly including flavonoids, saponins, phenols, alkaloids, gingerols and fatty acid related compounds. Twelve ingredients of XCHT, including baicalin, wogonoside, baicalein, wogonin, glycyrrhizic acid, glycyrrhetinic acid, formononetin, liquiritin, zingerone, ginsenoside Rb1, ginsenoside Rd and ginsenoside Rg3 have been detected in plasma. CONCLUSION:In this preliminary pilot study, co-administration of XCHT with FOLFIRI was well tolerated under the assessed conditions. The findings provide preliminary short-term safety and systemic pharmacokinetic information to inform the design of larger randomized controlled trials (RCTs).

Four undescribed phenolic glycosides, pubescenosides V-Y (1-4), and nine known compounds (5-13) were isolated from the roots of Ilex pubescens. Structural elucidation was performed using various techniques, including infrared spectroscopy, ultraviolet spectroscopy, high-resolution electrospray ionization mass spectrometry, electronic circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy. The cardioprotective effects of the compounds were evaluated using an oxygen-glucose deprivation-induced injury model in H9c2 cells. At 10 µM, all compounds were nontoxic; however, compounds 5 and 12 significantly increased the cell viability, positioning them as promising lead compounds for cardioprotective drug development.

Phenolic hydroxyl groups significantly influence the efficiency of low-rank coal direct liquefaction, yet their specific role in thermal reactivity is unclear. This study deciphers the fundamental mechanism by which phenolic hydroxyl groups govern the thermal reactivity of Naomaohu long flame coal (NL), a low-rank coal, identifying them as a key regulator of its chemical and physical structure. Through hydrothermal treatment (240-320 degrees C) of demineralized NL, the phenolic hydroxyl concentration in it achieved a selective reduction from 2.94 to 0.08 mmol/g, while the carboxyl group remained stable at 0.21-0.25 mmol/g. A decrease in these groups was found to promote aromatic condensation and a rise in C-C/C-H bonds, which in turn triggered pore structure collapse, surface smoothing, and specific surface area decreasing. This structural coarsening was directly linked to a raised energy barrier for thermal reaction, with the activation energy being increased from 28.26 to 43.11 kJ/mol. At the electronic level, it was demonstrated that the reaction energy barrier is lowered by phenolic hydroxyls through a homogenization of the molecular electrostatic potential and a weakening of the critical C-O bonds. Thus, the role of phenolic hydroxyls is conclusively resolved, and their concentration is established as a primary factor controlling coal reactivity, which provides immediate implications for the strategic upgrading of low-rank coal.

Traditional Chinese Medicine (TCM) offers valuable therapeutic strategies for chronic and infectious diseases, yet the inherent complexity of its multi-component, multi-target formulations and synergistic effects presents substantial challenges to pharmacological mechanistic understanding. Structural pharmacology of Chinese Medicine has emerged as a transformative discipline, integrating structural biology, computational chemistry, and pharmacology to elucidate the precise mechanisms underlying TCM efficacy. This review synthesizes technological advancements that enable the characterization of synergistic mechanisms and dynamic molecular interactions in TCM. Key advancements include high-resolution structural techniques such as X-ray crystallography, cryo-electron microscopy, sophisticated computational approaches such as AI-driven predictive modeling, and advanced analytical platforms. We critically examine persistent technical hurdles, such as capturing transient binding events and modeling complex multi-component system dynamics. Finally, we outline future research trajectories to establish a predictive and adaptable scientific foundation for TCM modernization, facilitating its evidence-based global integration and application in precision medicine.

Huangqin decoction (HQD), a traditional Chinese prescription composed of Scutellariae Radix (Huang-Qin in Chinese), Paeonia Lactiflora (Shao-Yao), Glycyrrhizae Radix et Rhizoma (Gan-Cao), and Jujubae Fructus (Da-Zao), has been used for heat-induced diarrhea (Re-Xie) for over 1800 years. Its modern modified pharmaceutical formulation, YIV906, was currently the first traditional Chinese prescriptions botanical medicine to enter the Food and Drug Administration clinical trial phase for cancer treatment. Despite its long history, the modern scientific explorations of its phytochemistry composition and Pharmacological activities still need to be systematically summarized. By analyzing and summarizing the current research of HQD, this review aimed to clarify existing research gaps and propose recommendations for future researches. The search terms “Huangqin decoction” and “Huangqin tang” were utilized to obtain the relevant literature from online databases including Web of Science, PubMed, Google Scholar, SciFinder, and China National Knowledge Infrastructure. Furthermore, traditional medical books in China were also collected. In total, 192 chemical constituents were identified in HQD using analytical methods. The quality evaluation of HQD primarily focused on its chemical fingerprint. In addition, pharmacokinetic studies on HQD centered on the active components present in its constituent herbs. Pharmacological studies showed HQD had anti-ulcerative colitis, anti-cancer, anti-bacterial properties, and regulated metabolism. Its main mechanisms involved signaling pathways like PI3K/AKT/mTOR, miR-185-3p/MLCK/pMLC, and IFN-γ/JAK/ETS, which collectively maintained immune balance, intestinal microbiota, and epithelial barrier integrity. In clinical applications, HQD was commonly used for the treatment of digestive system diseases, cancer, and metabolic syndrome. However, there remained some limitations in the investigation of HQD. Future studies focusing on the elucidation of chemical constituents, the improvement of quality control, and the clarification of functional mechanisms are worthy of exploration.

Background Early-life stress (ELS) is a key risk factor for adolescent depression. Si-Ni-San (SNS), a classic traditional Chinese medicine formula, has shown antidepressant potential, yet its effects on the dorsal raphe nucleus (DRN)-nucleus accumbens (NAc) serotonergic circuit remain unclear. Purpose This study aimed to investigate whether SNS alleviates adolescent depression by restoring DRN-NAc serotonergic circuit function and to identify the serotonin receptor mediating its synaptic effects in the NAc. Study design Firstly, the antidepressant efficacy of SNS was evaluated in a mouse model of ELS. Subsequently, its underlying mechanism was explored through integrated neurophysiological, molecular, and pharmacological analyses. Methods Depressive- and anxiety-like behaviors were assessed using behavioral tests (sucrose preference, tail suspension, forced swim, open field, and elevated plus maze). In vivo electrophysiolog was employed to monitor DRN neuronal activity. Chemogenetic manipulation was employed to regulate the DRN-NAc serotonergic circuit, while 5-HT4R function was assessed through pharmacological intervention and viral knockdown. Synaptic and molecular mechanisms were examined using Western blotting, qPCR, ELISA, and immunofluorescence. Results SNS alleviated depressive-like behaviors, enhanced neural activity and low-frequency oscillations in the DRN, and restored 5-hydroxytryptamine (5-HT) levels in the NAc. Mechanistically, SNS upregulated tryptophan hydroxylase 2 (TPH2) while downregulating indoleamine 2,3-dioxygenase 1 (IDO1), thus promoting 5-HT synthesis. Critically, the antidepressant effects of SNS were blocked by either chemogenetic inhibition of the DRN-NAc serotonergic circuit or pharmacological blockade of 5-HT4R in the NAc. Meanwhile, the knockdown of 5-HT4R abolished the ameliorative effects of SNS on depressive-like behaviors and associated synaptic remodeling, including the upregulation of brain-derived neurotrophic factor, postsynaptic density protein 95, and mushroom spine density. Conclusion These results demonstrate that SNS alleviates depressive-like behaviors in adolescent male mice by restoring DRN-NAc serotonergic circuit function, enhancing 5-HT bioavailability, and promoting 5-HT4R-dependent synaptic plasticity in the NAc, revealing a circuit- and receptor-specific therapeutic mechanism.

Colorectal cancer (CRC) is one of the most common and deadly types of cancer globally. Ferroptosis, a type of regulated cell death that relies on iron, has become a promising target for treating CRC. Alkannin, a natural compound from Lithospermum erythrorhizon, exhibits anti-tumor activity, yet its mechanism in CRC is unclear. This study investigated alkannin's role in regulating ferroptosis via the Keap1/Nrf2/GPX4 axis. Using network pharmacology and experimental validation in HCT116 and SW480 cells and a xenograft mouse model, we found that alkannin markedly inhibited the viability, proliferation, and migratory capacity of CRC cells, demonstrating significant anti-tumor activity. Network pharmacology revealed a primary association between alkannin's therapeutic effects and the induction of ferroptosis, along with the regulation of oxidative stress pathways, with a notable focus on the Keap1/Nrf2 axis. Experimental evidence confirmed that alkannin induced ferroptosis, as reflected by increased intracellular Fe2+ levels and lipid peroxidation, along with reduced glutathione (GSH) content. These effects were reversed by ferroptosis inhibitors, which also attenuated alkannin-induced cytotoxicity. Mechanistically, alkannin enhanced Keap1 protein stability by suppressing its ubiquitination. It promoted the interaction between Keap1 and Nrf2, leading to decreased Nrf2 expression and inhibition of its nuclear translocation, thereby downregulating the expression of glutathione peroxidase 4 (GPX4), a key suppressor of ferroptosis. Genetic silencing of Keap1 significantly diminished alkannin-induced ferroptotic cell death. In vivo, alkannin effectively inhibited tumor growth in xenografted nude mice. Furthermore, it induced ferroptosis in tumor tissues, as evidenced by similar biochemical changes, which were counteracted by co-administration of a ferroptosis inhibitor. Consistently, alkannin upregulated Keap1 expression while reducing the protein levels of Nrf2 and GPX4 in tumor tissues. In conclusion, alkannin induces ferroptosis in CRC by stabilizing Keap1 to inhibit the Nrf2/GPX4 pathway, supporting its potential as a CRC therapeutic agent.

Brain metastasis (BrM) is the most common complication with the highest mortality in clinical tumor patients. Its underlying pathogenesis remains unclear, and effective therapeutic compounds are currently unavailable. Herein, we first revealed that S100A14 is a critical molecular mediator of tumor-derived exosomes (EVs)-driven BrM. DIA-based proteomics demonstrated consistently high S100A14 expression in BrM-EVs from both cells (53.2-fold in A549 BM3 cells and 2.4-fold in 4T1 BM2 cells) and clinical patient samples (5.1-fold). Intracardiac injection mice model confirmed that S100A14-overexpression EVs promoted BrM in breast (4T1: 88.89% vs. 44.45%; MDA-MB-231: 66.67% vs. 33.33%) and lung cancer (LLC: 50% vs. 33.33%; A549: 83.33% vs. 33.33%). Mechanistically, S100A14 directly targeted PIAS3 to reprogram astrocytes (AS) by activating STAT3 signaling and triggering secretion of pro-inflammatory chemokines (CCL2/CCL5/CXCL5), which recruit immunosuppressive MDSCs. Crucially, we identified the natural compound, germacrone, as a promising therapeutic agent that effectively inhibited BrM progression in both lung and breast cancer without significant toxicity. Germacrone directly binds to S100A14 in AS to disrupt the S100A14-PIAS3 interaction, inhibit STAT3 activation, and MDSCs recruitment. Our study elucidated a novel mechanism by which tumor-derived S100A14 EVs promote tumor BrM. Moreover, germacrone emerged as a prospective therapeutic agent for preventing BrM by specifically targeting S100A14.

Background: Tumor-associated macrophages (TAMs), especially SPP1+TAMs are associated with poor prognosis of colorectal cancer (CRC). However, the underlying mechanism remains unclear, and the therapeutic targets have yet to be identified. Methods: Single-cell RNA sequencing (scRNA-seq) data were used to explore the interactions between SPP1+TAMs and CRC cells. TAM co-culture model, liver metastasis models and clinical tissue microarray (n=42) were used to validate the key secreted cellular factor and its associated receptor that mediated the interactions between SPP1+TAMs and CRC cells. Results: We found that migration inhibitory factor (MIF) was the most important signaling molecule involved in the interaction between SPP1+TAMs and CRC cells, as revealed by cellular interaction analysis of integrated scRNA-seq datasets. Interestingly, SPP1 was co-expressed with MIF receptor CD44 on SPP1+TAMs. When SPP1+TAMs was activated, CD44 was crucial for MIF-mediated angiogenesis. Our data showed that CRC cells activated SPP1+TAMs, which was abolished by blocking the MIF signaling both in vitro and in vivo. Furthermore, the pathological role of MIF is suggested by the elevated expression of MIF and activation of SPP1+TAMs in CRC patients, as demonstrated in clinical tissue microarray. Further mechanistic studies revealed that POU2F2 was a crucial transcription factor mediating MIF-driven activation of SPP1+TAMs, and that BCL9L was a direct downstream target of POU2F2. Conclusions: Our findings suggest that the MIF/CD44/POU2F2/BCL9L signaling axis is involved in the proangiogenic capacity of activated SPP1+TAMs, thereby enhances CRC metastasis. Targeting this novel signaling axis can effectively suppress the SPP1+TAM activation, which represents a promising and pivotal strategy for managing CRC metastasis.