Jun Chang

Voltage-gated sodium (Nav) channel Nav1.2, encoded by SCN2A, is a major determinant of neuronal excitability and an important contributor to epilepsy-associated hyperexcitability. However, small-molecule inhibitors with well-defined Nav1.2 pharmacology remain limited. Here, using an automated electrophysiological screening platform, we identified Lvguidingan (LV), an antiepileptic candidate that has entered phase II clinical evaluation in China, as a hit inhibitor of Nav1.2 and systematically characterized its pharmacological properties. Manual patch-clamp recordings showed that LV inhibited Nav1.2 and displayed differential inhibitory effects across other tested sodium channel subtypes. LV exhibited clear state- and use-dependent inhibition of Nav1.2 and shifted channel gating toward less available states. Molecular modeling and mutational analysis further identified key binding determinants underlying LV-mediated Nav1.2 inhibition. At the neuronal level, LV suppressed the firing of hippocampal neurons and reduced action potential amplitude. Importantly, LV also inhibited two epilepsy-associated SCN2A gain-of-function variants. Together, these results identify LV as a Nav1.2 inhibitor with defined biophysical and structural features and inhibitory activity against disease-associated SCN2A variants, providing new insight into the Nav1.2-related mechanism underlying the antiepileptic activity of LV.

Cardiac fibrosis remains a critical determinant of adverse outcomes in heart disease, yet effective anti-fibrotic therapies are lacking. While multiple semaphorin family members participate in cardiovascular pathophysiology, the role of semaphorin 4f (Sema4f) in cardiac fibrosis remains unexplored. This study investigates the role and mechanisms of Sema4f in fibrotic remodeling post-myocardial infarction (MI). We employed flow cytometry to characterize cell type-specific Sema4f expression patterns in post-MI hearts. Lineage-specific knockout mice (fibroblast vs. myeloid) were subjected to left anterior descending ligation to assess functional consequences. Proteomic analysis of Sema4f-deficient cardiac fibroblasts was conducted to identify downstream effectors. Key pathways were subsequently validated using pharmacological inhibitors. We found that Sema4f expression was markedly upregulated during the fibrotic phase post-MI, primarily due to fibroblast activation. Fibroblast-, but not myeloid-, specific Sema4f deletion significantly reduced fibrosis and improved cardiac function. Proteomic profiling revealed that Sema4f deficiency led to downregulation of pro-fibrotic gene expression, which was associated with impaired actin cytoskeletal remodeling and decreased nuclear translocation of YAP/TAZ. Pharmacological inhibition of either actin remodeling or YAP/TAZ activity attenuated fibrosis, whereas YAP/TAZ activation abolished the anti-fibrotic effects of Sema4f knockout. Our study provides the first evidence demonstrating the functional role of Sema4f in cardiac fibroblast activation and fibrosis progression. We have identified a fibroblast-specific mechanism mediated by the Sema4f-actin cytoskeleton-YAP/TAZ axis, offering novel mechanistic insights into fibrosis regulation and revealing a promising therapeutic target for cardiac fibrosis with potential clinical applications. Graphical abstract

Obesity, a major global health challenge associated with metabolic and cardiovascular disorders, has drawn increasing attention to the therapeutic potential of white adipose tissue (WAT) browning. Although the lysine methyltransferase SETD7 has been implicated in various cardiovascular and metabolic diseases, its role in adipose thermogenesis remains unclear. Here, we reported that SETD7 was upregulated in inguinal WAT (iWAT) of obese mice and was primarily localized to mature adipocytes. Setd7 knockdown ( Setd7 ^⁺/⁻ ) mice exhibited enhanced thermogenic gene expression and iWAT browning upon cold exposure or β3-adrenergic stimulation, whereas thermogenic activity in brown adipose tissue (BAT) remained largely unaffected. In vitro, SETD7 knockdown did not alter adipogenesis but potently augmented thermogenic capacity in beige adipocytes, while SETD7 overexpression exerted the opposite effect. Mechanistically, RNA-Seq analysis revealed that SETD7 deficiency upregulated Adcy7 transcription, leading to increased Sirt1 levels and enhanced Creb1 phosphorylation, thereby activating the thermogenic program. Notably, Setd7 ^{⁺/ –} mice resisted high-fat diet (HFD)-induced obesity, exhibiting reduced weight gain, elevated energy expenditure, and improved metabolic health. Together, these findings identify SETD7 as a negative regulator of iWAT thermogenesis and suggest that targeting SETD7 may represent a promising strategy for combating obesity.

Ligstroside (LIG) is a phenolic secoiridoid glycoside naturally occurring in olives and structurally related to the widely studied olive constituent oleuropein. Although LIG has been reported to exhibit diverse biological activities, it has received comparatively limited attention, and the molecular targets underlying its bioactivity remain largely unknown, thereby limiting exploration of its pharmacological potential. To facilitate target identification and mechanistic investigation, we designed and synthesized a LIG-derived photoaffinity probe (PAL-LIG) through minimal modification of the LIG scaffold. The probe incorporates a photoactivatable diazirine group that enables UVinduced covalent crosslinking with proximal proteins together with an alkyne handle that allows subsequent bioorthogonal conjugation via copper-catalyzed azide–alkyne cycloaddition (CuAAC). Following photo-crosslinking, probe-labeled proteins can be conjugated with azide-functionalized reporter tags, including biotin for streptavidinbased enrichment and pulldown or TAMRA for fluorescence detection and cellular imaging. Using this strategy, PAL-LIG enables enrichment of potential LIG-binding proteins as well as fluorescence-based visualization of probe labeling in cells. Importantly, PAL-LIG retains biological activity comparable to LIG, as demonstrated by its similar ability to promote ATP production in mouse C2C12 myoblasts, indicating that installation of the photoaffinity and alkyne handles minimally perturbs the intrinsic bioactivity of LIG. Collectively, PAL-LIG provides a chemical tool for investigating the direct protein targets and molecular mechanisms of LIG.

Introduction/Objectives: Procyanidins are abundant dietary flavonoids with diverse pharmacological activities; however, their Absorption, Distribution, Metabolism, and Excretion (ADME) remain incompletely characterized. MT-8, a procyanidin B3 derivative with an ethyl group at the C8 position of the A-ring, is a promising preclinical candidate for cerebral ischemia treatment and is currently in the Investigational New Drug (IND) application stage. This study aimed to systematically characterize the ADME profile of MT-8 in male Sprague-Dawley (SD) rats following intravenous administration to provide a reference for ADME studies of procyanidin-related compounds. Methods: A single intravenous dose of 24 mg/kg (100 μCi/kg) [¹⁴C]MT-8 was administered to male SD rats. Given the observed instability of MT-8 in plasma due to catechol oxidation, 10 mM tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was employed as a stabilizer in all sample preparations. Pharmacokinetics, tissue distribution, mass balance, metabolite profiling, and metabolic enzyme phenotyping were comprehensively evaluated. Results: Pharmacokinetic analysis revealed rapid elimination of [¹⁴C]MT-8-related substances, with a terminal half-life of 1.77 hours. The blood-to-plasma radioactivity ratio (1.68) indicated preferential distribution into blood cells. Tissue distribution revealed the highest radioactivity concentrations in the small intestine, kidneys, and liver. Mass balance analysis showed total radioactive recovery of 95.49% within 168 hours, with clearance of >90% of drug-related substances within 24 hours, predominantly via feces (85.57%) and bile (62.78% in bile duct-cannulated rats), with only 9.91% in urine. Metabolite profiling identified 32 metabolites in plasma, urine, feces, and bile, with methylation and glucuronidation as the major metabolic pathways. In vitro phenotyping referred to catechol-O-methyltransferase (COMT) involvement in methylation and UDP-glucuronosyltransferase 1A1 (UGT1A1) and 1A9 (UGT1A9) in glucuronidation. Discussion: After verifying the mechanism of the instability of MT-8 in plasma, we tracked the in vivo disposition of MT-8 via radioisotope labeling technique and acquired credible pharmacokinetic, mass balance, tissue distribution, and metabolite identification results. Phenotypes of two enzymes were determined to further the investigation of the methylation and glucuronidation process of in vivo clearance of MT-8. Conclusion: Collectively, MT-8 undergoes rapid clearance, broad distribution, extensive metabolism, and predominant biliary/fecal excretion. These findings provide critical support for MT-8’s IND application and subsequent clinical development.

Epigenetic regulation plays a pivotal role in adipocyte development and thermogenesis. Ash2l, a key component of the COMPASS (Complex of Proteins Associated with Set1) histone methyltransferase, regulates gene expression through epigenetic mechanisms. This study explored the role of Ash2l in adipose tissue thermogenesis and obesity-related metabolic dysfunction. Ash2l was initially identified through transcriptomic analysis, and its expression was further validated in mouse models of high-fat diet (HFD), cold exposure, and CL316,243 stimulation. In vitro gain- and loss-of-function experiments were conducted to assess the role of Ash2l in adipogenesis and thermogenesis. To knockdown Ash2l in vivo, adeno-associated viruses carrying short hairpin RNA targeting Ash2l (AAV-shAsh2l) were injected into either the brown adipose tissue (BAT) or the inguinal white adipose tissue (iWAT). The functional consequences of Ash2l deficiency were evaluated in mice under room temperature, cold exposure, and HFD conditions. Finally, chromatin immunoprecipitation sequencing (ChIP-seq) was employed as an exploratory analysis to identify genomic regions associated with Ash2l during adipocyte development. Our findings demonstrate that Ash2l modulates the expression of both adipogenic and thermogenic genes in adipocytes. Mice with BAT- or iWAT-knockdown of Ash2l displayed defective cold-induced thermogenesis, aggravated diet-induced obesity, and systemic metabolic dysregulation. Moreover, Ash2l knockdown in BAT under cold exposure or HFD conditions also attenuated thermogenic activity in iWAT, an effect that may be mediated by reduced secretion of FABP4. These findings establish Ash2l as a critical regulator of adipogenesis and thermogenesis. This study provides important insights into the epigenetic role of Ash2l in maintaining metabolic homeostasis under conditions of nutritional excess.

Beige adipocytes play a key role in non-shivering thermogenesis. SEMA3E, a member of the class 3 semaphorin family, is involved in various pathological processes, but its role in adipocyte differentiation and thermogenesis remains unclear. Here, we found SEMA3E expression increased in inguinal white adipose tissue (iWAT) following cold exposure or β-adrenergic agonist CL316,243 stimulation. In vitro, loss- and gain-of-function experiments revealed that SEMA3E promoted beige adipocyte differentiation and enhanced thermogenic genes expression. In vivo, fat transplantation experiments indicated that SEMA3E promoted adipogenesis. Furthermore, adeno-associated virus (AAV)-mediated SEMA3E knockdown in iWAT impaired thermogenesis in mice exposed to cold or CL316,243. RNA-Seq analysis linked SEMA3E to mitochondrial oxidative phosphorylation, and its knockdown reduced mitochondrial respiration by downregulating respiratory chain components expression and lowering mitochondrial oxygen consumption rate. Mechanistically, gene set enrichment analysis suggested SEMA3E regulated beige adipocyte differentiation via the Wnt/β-catenin pathway. SEMA3E knockdown delayed β-catenin degradation, while inhibiting this pathway with IWR-1 rescued the suppressed differentiation and thermogenic genes expression. In conclusion, these findings highlight the crucial role of SEMA3E in beige adipocyte differentiation and thermogenesis.

Background: Skeletal muscle atrophy is a debilitating condition associated with diverse diseases and clinical interventions. Although triggers such as glucocorticoid excess or direct muscle injury disrupt the balance between protein homeostasis and tissue repair, the upstream molecular signals that actively preserve muscle integrity remain largely unknown. Semaphorin 4A (Sema4A) is a Class IV transmembrane semaphorin traditionally recognized for its roles in neural development and immune regulation; however, its function in skeletal muscle maintenance has not been explored. Methods: We first examined Sema4A expression in established models of muscle catabolism. Subsequently, adeno‐associated virus (AAV)‐mediated Sema4A restoration was employed to evaluate its therapeutic potential in both dexamethasone‐induced atrophy and acute injury mouse models. Mechanistic studies were performed in C2C12 myotubes using gain‐ and loss‐of‐function approaches. Results: Sema4A expression was significantly downregulated under atrophic conditions ( p < 0.05). Restoration of Sema4A effectively attenuated dexamethasone‐induced muscle loss (4.766 vs. 5.075 mg/g B.W., p = 0.0414) and accelerated functional recovery (6.219 × 10 ⁻² vs. 7.124 × 10 ⁻² N/g B.W., p = 0.0422). We identified Plexin B2 (Plxnb2) as the functional receptor mediating these effects. At the molecular level, Sema4A signalling suppressed FoxO3a nuclear translocation (FoxO3a positive cells: vector‐Dex 27.99% vs. OE‐Sema4A‐Dex 12.49%, p < 0.05), thereby inhibiting the expression of key atrogenes, while simultaneously reactivating the PI3K‐AKT‐mTOR anabolic pathway. This reprogramming of intracellular metabolic signalling was further associated with the establishment of a reparative immune microenvironment, a process potentially modulated by muscle‐derived factors such as Gdf15. Conclusions: Our study identifies Sema4A as a novel protective regulator that mitigates muscle atrophy and enhances regeneration through a dual mechanism: restoring intracellular anabolic signalling and fostering a proregenerative immune niche. These findings highlight Sema4A as a promising therapeutic target for the treatment of muscle‐wasting disorders.

Ischemic stroke (IS) is a leading cause of mortality and disability worldwide, but effective therapeutic options are limited. In this study, a chemical proteomic strategy is employed using the active compound procyanidin B3 (PB3) as a chemical probe to identify the therapeutic targets for IS. It is discovered that the Ras GTPase‐activating protein SH3 domain‐binding protein 1 (G3BP1) is a key target of PB3, which exerts a neuroprotective effect by inhibiting the degradation of stress granules and reducing apoptosis. Based on this finding, 14 PB3 derivatives are designed and synthesized, among which compound 6c exhibited potent neuroprotective activity and favorable blood‐brain barrier permeability. This study not only establishes G3BP1 as a promising therapeutic target for IS but also highlights the potential of PB3 and its derivatives for the development of IS therapeutic agents.