Zishen Huoxue (ZSHX) Decoction can ameliorate myocardial ischaemia by regulating the mitochondrial quality control network. However, the identification of new molecular targets is necessary for ZSHX’s control of mitochondrial protein homeostasis and metabolic activities. Utilizing animal and cellular models with NDUFS4^CKO or DUSP1^CKO, along with single-cell sequencing, metabolomics, network pharmacology, and in vivo/in vitro interventions, the study found that ischemia-reperfusion (I/R) injury triggers endoplasmic reticulum stress and mitochondrial metabolic reprogramming, accompanied by downregulation of DUSP1 and NDUFS4. Network pharmacology suggested ZSHX’s role in regulating mitochondrial activity during inflammatory damage, while metabolomics confirmed that ZSHX alters metabolite composition and expression in I/R-affected tissues. Single-cell sequencing further linked I/R to disrupted mitochondrial energy metabolism and cell death, and in vitro experiments demonstrated that ZSHX preserves mitochondrial proteostasis, inhibits endoplasmic reticulum stress, restores calcium balance, upregulates DUSP1/NDUFS4 expression, and controls metabolic reprogramming to reduce myocardial inflammatory injury. Kaempferol, the primary active component of ZSHX, drives these protective effects by enhancing DUSP1/NDUFS4 expression, thereby preventing endoplasmic reticulum stress and inflammatory bursts, preserving mitochondrial function, and re-encoding mitochondrial metabolic processes post-I/R injury.

Calcific aortic valve disease (CAVD) is a serious heart valve condition with increasing global prevalence. Currently, transcatheter aortic valve implantation (TAVI) or surgical aortic valve replacement (SAVR) represents the only available treatment strategy, as no pharmaceutical therapies for CAVD are approved. The aim of this study was to identify compounds capable of inhibiting osteogenic differentiation of human aortic valve interstitial cells (hVICs), a process critically implicated in CAVD pathogenesis, and to elucidate the underlying molecular mechanism. From an in-house library of 88 compounds screened via dot-blotting, we identified chipericumin D, a natural compound extracted from Hypericum monogynum L., as a candidate exhibiting potent inhibitory activity against hVIC osteogenic differentiation. Network pharmacology analysis, molecular docking, drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) collectively demonstrated direct binding of chipericumin D to the epidermal growth factor receptor (EGFR). Furthermore, chipericumin D suppressed activation of the EGFR/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway in hVICs cultured under osteogenic medium (OM) conditions. These findings indicate that chipericumin D is a promising therapeutic candidate for CAVD, and provide preliminary evidence that EGFR constitutes a novel molecular target for CAVD intervention.

Triple-negative breast cancer (TNBC) is the most challenging breast cancer subtype to treat due to the absence of effective targeted therapies. In this study, we demonstrate that elevated expression of microtubule affinity-regulating kinase 2 (MARK2), but not other MARK family members (MARK1, MARK3, and MARK4), correlates with poor prognosis in TNBC patients. Silencing MARK2 impairs TNBC progression via inhibition of mutant p53 (mutp53) signaling. In contrast, silencing any of the other three MARKs either enhances or does not affect TNBC cell growth or migration and has no impact on mutp53 expression. Notably, direct knockdown of mutp53 recapitulates the effects of MARK2 ablation in TNBC cells, further supporting a functional linkage. Moreover, ectopic expression of either wild-type (WT) MARK2 or its kinase-dead (KD) mutant enhances mutp53 signaling and promotes TNBC progression; however, MARK2 overexpression does not alter wild-type p53 (wtp53) expression or cell growth in luminal breast cancer cells. Significant inverse correlations are also observed between the expression levels of MARK2, THBS1, or HBEGF (two direct target genes of mutp53) and both overall and disease-free survival in TNBC patients harboring mutTP53, whereas no such association exists between MARK2 and survival in breast cancer subtypes expressing wtTP53. MARK2 is predominantly localized in the nucleus of TNBC cells, where it interacts with and stabilizes mutp53 through its UBA and Spacer domains. Consistent with this, MARK2-ΔUBA or MARK2-ΔSpacer mutant proteins fail to bind mutp53 or sustain its signaling, thereby acting as dominant-negative inhibitors that suppress TNBC progression. Collectively, our findings indicate that suppressing MARK2 expression, rather than inhibiting its kinase activity, may represent an effective therapeutic strategy for TNBC with mutTP53.

Lung squamous cell carcinoma (LUSC) is a prevalent and aggressive form of lung cancer with limited therapeutic options. Sanguinarine (SAG), a prominent benzophenanthridine alkaloid derived from Zanthoxylum nitidum (Roxb.) DC, exhibits established anti-tumor activity; however, its molecular mechanisms in LUSC remain incompletely defined. In this study, the anti-cancer effects and underlying mechanisms of SAG were systematically investigated in vitro and in vivo. Cell viability and death were evaluated using methyl thiazolyl tetrazolium (MTT) assays, colony formation assays, flow cytometry, transmission electron microscopy (TEM), and Western blotting (WB). Drug affinity responsive target stability (DARTS) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS), molecular docking, cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) were employed to identify and validate molecular targets of SAG. The results demonstrated that SAG simultaneously induces apoptosis and ferroptosis in LUSC cells by directly targeting the endoplasmic reticulum (ER) chaperone binding immunoglobulin protein (BiP). Silencing of BiP markedly attenuated SAG-induced apoptosis and ferroptosis, confirming its essential role in this process. Mechanistically, SAG up-regulates BiP expression and activates the protein kinase R-like endoplasmic reticulum kinase (PERK)/eIF2α/C/EBP homologous protein (CHOP)/GADD34 signaling axis of ER stress (ERS), ultimately leading to dual induction of apoptosis and ferroptosis in vitro and in vivo.

Gouty arthritis (GA) is an inflammatory disorder characterized by the deposition of monosodium urate (MSU) crystals in joint tissues. Pulchinenoside B4 (B4) has broad-spectrum anti-inflammatory properties, but its role and potential mechanism in the pathogenesis of GA are still unclear. The purpose of this study is to comprehensively elucidate the therapeutic effect and mechanism of B4 on GA by integrating transcriptome analysis and in vitro and in vivo experiments. In the MSU-induced mouse GA model, B4 treatment significantly improved ankle edema and reduced inflammatory cell infiltration. Through the analysis of transcriptome sequencing results, we identified multiple differentially expressed long non-coding RNAs (lncRNAs), such as Nod1, Rbck1 and Pycard. In the in-depth exploration of the mechanism, we focused on the NOD-like receptor signaling pathway, NF-κB signaling cascade, and B4-regulated macrophage polarization. In vitro and in vivo models, we confirmed that B4 significantly inhibited the expression and activation of key components of NLRP3 inflammasome (such as ASC, Caspase-1 and IL-1β) by qPCR, Western blot and immunofluorescence. Flow cytometry and immunofluorescence analysis further showed that B4 could prevent MSU-induced macrophage polarization to pro-inflammatory M1 phenotype.Based on these results, this study elucidated the mechanism of B4 improving MSU-induced GA inflammatory response by inhibiting NLRP3 inflammasome activation and blocking M1 macrophage polarization. These results suggest that B4 has great potential as a candidate drug for the treatment of GA.

Nephropathy 1 Formula (N1F), a traditional Chinese medicine (TCM), has demonstrated promising clinical efficacy in diabetic nephropathy (DN). However, its underlying protective mechanisms remain insufficiently defined. In this study, a type 2 diabetes mellitus (T2DM) mouse model was established using a high-fat diet (HFD) and streptozotocin (STZ). Additionally, DN was simulated in vitro via exposure of mouse glomerular mesangial cells (MES-13) to high glucose (HG) and trimethylamine-N-oxide (TMAO). To elucidate the mechanistic basis of N1F’s renoprotective effects, an integrative approach combining metabolomics, transcriptomics, and 16S ribosomal ribonucleic acid (rRNA) gene sequencing was employed. N1F treatment reduced the urinary albumin-to-creatinine ratio (UACR), preserved renal function, and attenuated histopathological damage and renal fibrosis in diabetic mice. Mechanistically, N1F modulated systemic TMAO levels and energy metabolism, altered gut microbiota composition, and suppressed microbial production of TMAO-related metabolites. Under hyperglycemic conditions, TMAO induced excessive mitochondrial reactive oxygen species (mROS), impaired mitochondrial dynamics, and disrupted cellular energy metabolism. In contrast, N1F normalized mROS levels, restored mitochondrial structure and function, enhanced oxidative phosphorylation (OXPHOS), increased ATP production, and reduced glycolytic dependency. Furthermore, N1F downregulated the expression of key pyroptosis-related proteins—including NOD-like receptor family pyrin domain-containing 3 (NLRP3), N-terminal gasdermin D (GSDMD), cleaved-Casp1, interleukin-1β (IL-1β), and IL-18—in both in vivo and in vitro models, indicating suppression of pyroptosis via inhibition of the TMAO-mROS-NLRP3 signaling axis. Collectively, these findings demonstrate that N1F exerts protective effects against DN by targeting mitochondrial dysfunction and pyroptotic injury, supporting its potential as a therapeutic strategy for DN.

Ultraviolet (UV) exposure accelerates skin aging and increases the risk of skin-related diseases. Amentoflavone (AMF), the major compound isolated from Selaginella tamariscina, exhibits potent antioxidant and anti-inflammatory activities. This study aimed to investigate the therapeutic effects and mechanisms of S. tamariscina extract (STE) and AMF on UVB-induced skin photoaging. In vitro and in vivo photoaging models were established to evaluate the protective effects of STE and AMF. The therapeutic target of AMF was identified using network pharmacology, bioinformatic analysis, and molecular docking. In vitro, STE significantly reduced UVB-induced oxidative stress, inflammation, and apoptosis. In vivo, both STE and AMF effectively mitigated UVB-induced skin injury. Mechanistically, AMF directly interacted with AMP-activated protein kinase (AMPK), thereby promoting autophagy and protecting cells from UVB-induced damage. In conclusion, STE and its active compound AMF alleviate UVB-induced photoaging via activation of the AMPK signaling pathway, supporting their potential use in skin photoaging therapy.

Cisplatin and its metabolites can cause severe gastrointestinal mucosal damage, leading to varying degrees of intestinal injury in nearly all patients following administration. Although the anti-tumor effectiveness of R-ginsenoside Rg3 (R-Rg3, a key chemical component of Shen Yi Capsule) as the first-line drug is widely recognized in the clinic, there is less concern about the improved effects of R-Rg3 against intestinal toxicity caused by concurrent cisplatin chemotherapy. This study aimed to investigate the protective effects of R-Rg3 against cisplatin-induced intestinal toxicity and to explore its potential molecular targets and mechanisms. Rats in the R-Rg3 treatment group were treated with R-Rg3 (7, 14 mg·kg⁻¹, p.o.) for 7 days, and a single dose of cisplatin (14 mg·kg⁻¹, i.p.) was administered via intraperitoneal injection to rats in the cisplatin group and R-Rg3 treatment group on the 7^th day. Intestinal epithelial cell line 6 (IEC-6) were pretreated with R-Rg3 (1.25, 2.5, 5 μmol·L⁻¹) for 24 h followed by cisplatin treatment (3 μmol·L⁻¹, 24 h). The in vivo results showed that, R-Rg3 treatment for 7 days markedly alleviated cisplatin-induced oxidative stress and mitochondrial dysfunction in vivo, while suppressing excessive autophagy and reducing intestinal damage. In IEC-6 cells, R-Rg3 pretreatment inhibited mitophagy and promoted the restoration of lysosomal function. Autophagy inhibitors 3-methyladenine (autophagosome formation inhibitor, 1 μmol·L⁻¹) and Bafilomycin A1 (proton pump inhibitor, 8 μmol·L⁻¹) were used to verify the mechanism of R-Rg3 action. Importantly, application of 3-methyladenine and Bafilomycin A1 verified that R-Rg3 could alleviate gastrointestinal dysfunction by restoring the cisplatin-induced autophagic flux blockade. In conclusion, this study identifies a previously underappreciated protective role of R-Rg3 against cisplatin-induced intestinal injury. Furthermore, it suggests that pharmacological modulation of the mitochondria-lysosome axis by R-Rg3 may effectively mitigate oxidative stress-mediated autophagic flux impairment caused by cisplatin.

Ten previously undescribed flavonoids, seladoeflavones J-Q, C, and E (1-10), together with fifteen known biflavones (11-25), were isolated from the whole herbs of Selaginella doederleinii. The structures of the new compounds were elucidated using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). By comparing experimental spectral data with NMR calculations, the structures of compounds 1-5, 7, and 8 were assigned. The absolute stereochemistries of compounds 5-10 were confirmed by comparing their circular dichroism (CD) spectra with reported data. Notably, the originally proposed structures of seladoeflavones C and E were revised and found to be identical to those of compounds 7 and 8, respectively. All isolated compounds were evaluated for cytotoxic activity against a panel of cancer cell lines. Most notably, 2'',3''-dihydroochnaflavone (14) and involvenflavone G (19) exhibited significant anti-proliferative and pro-apoptotic effects in laryngeal cancer cells (Hep-2 and FaDu). Mechanistic studies revealed that compound 14 induced apoptosis by suppressing the protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, whereas compound 19 downregulated endoplasmic reticulum (ER) stress pathways. These findings indicate that compounds 14 and 19 possess strong potential as anti-laryngeal cancer agents, providing robust evidence for the traditional use of S. doederleinii in the treatment of laryngeal cancer.