Acta Pharmacologica Sinica最新文献

Myocardial infarction (MI) initiates robust inflammatory responses. While moderate inflammation facilitates the clearance of necrotic debris, sustained inflammation promotes fibrosis and exacerbates MI progression. Fungal polysaccharides have shown significant anti-inflammatory activity, but role of Morchella polysaccharide (MCP) in modulating MI-associated inflammation remains unclear. In this study we investigated whether MCP ameliorated myocardial infarction injury and elucidated the underlying mechanisms focusing on the gut microbiota and associated metabolites. MI mice were established in mice by permanent ligation of the left anterior descending (LAD) coronary artery. MCP (200, 600 mg·kg^-1·d^-1. i.g.) was administered daily from D7 prior-MI induction and continued for 3 or 7 days post-MI. We showed that MCP administration significantly alleviated cardiac inflammation in post-MI mice. Metabolite screening identified 12-hydroxy-eicosapentaenoic acid (12-HEPE) as a critical mediator of MCP's anti-inflammatory effects. Intestinal metabolomic screening revealed that MCP markedly upregulated the abundance of the beneficial genus Lactobacillus. Eliminating the intestinal flora using a broad-spectrum antibiotic cocktail for 2 weeks abolished MCP-induced 12-HEPE elevation and anti-inflammation in post-MI mice. On the other hand, direct supplementation of 12-HEPE (200 µg·kg^-1·d^-1, i.p.) beginning 7 days prior to MI induction attenuated the inflammation. In conclusion, this study reveals that MCP attenuates post-MI inflammation by enriching beneficial gut bacteria such as Lactobacillus and increasing their metabolite 12-HEPE. MCP improves post-myocardial infarction (post-MI) inflammation and fibrotic repair by modulating gut microbiota and the intestinal lipid metabolite 12-HEPE. MCP treatment increases the abundance of the beneficial gut bacterium Lactobacillus. Concurrently, MCP enhances the activity of metabolic enzymes responsible for 12-HEPE synthesis within the improved intestinal microenvironment. This elevates intestinal 12-HEPE production and enhances its systemic circulation. Ultimately, increased serum 12-HPE levels attenuate cardiac inflammation and improve injury repair.

The global obesity epidemic, affecting over 650 million adults, demands innovative therapeutics. GPR75 has emerged as a promising anti-obesity target, with genetic evidence linking loss-of-function variants to protection against obesity and type 2 diabetes. However, structural insights have remained elusive due to GPR75's inherent expression and stabilization challenges. Here we present the cryo-EM structures of human GPR75 in apo and Gq-coupled states, achieved through advanced stabilization techniques including NanoBiT and molecular glue approaches. Our structures reveal unique architectural features: a completely collapsed extracellular domain eliminates the traditional orthosteric binding pocket, raising critical questions about previously reported small molecule ligands. GPR75 assumes active-like conformation in both apo and G protein complexed structures through unique molecular switches-the canonical DRY motif is replaced by HRL, abolishing the ionic lock, while a distinctive Lys134-Asp210 salt bridge stabilizes the active conformation without ligand binding. This dramatic structural divergence from conventional GPCRs necessitates alternative therapeutic strategies targeting allosteric sites or protein-protein interactions rather than orthosteric pockets. Our findings establish a crucial structural framework for developing next-generation anti-obesity therapeutics.

Current treatments for depression have focused on improving the dysregulated monoamine neurotransmitter systems in the brain. However, the conventional antidepressants based on the monoamine hypothesis usually exert side effects and unsatisfactory responses. MicroRNAs (miRNAs) are smaller noncoding RNA which are highly expressed in the brain and play important roles in the development of neurological disorders. In this study we investigated the role of miRNAs in the occurrence of depression. A rat depression model was established by exposure to chronic mild stress (CMS) over 4 weeks. In the next week, the sucrose preference test (SPT), the forced swimming test (FST), and the open field test (OFT) were used to evaluate the depression-like behaviors. Then the rats were euthanized and total RNA was isolated from rat mPFC. We showed that the level of microRNA-129-5p (miR-129-5p) was significantly increased in the mPFC of CMS rats. Overexpression of miR-129-5p in the mPFC by bilateral microinjection of lenti-miR-129-5p virus (OE-miR-129-5p) induced the depression-like behaviors in control rats, accompanied with the impairment in neuronal structures and a decrease in synaptic plasticity. In contrast, knockdown of miR-129-5p in the mPFC by bilateral microinjection of lenti-miR-129-5p sponge virus (KD-miR-129-5p) ameliorated the depression-like behaviors in CMS rats, along with the improvement in neuronal structures and an increase in synaptic plasticity. Furthermore, we demonstrated that miR-129-5p targeted to the brain-derived neurotrophic factor (BDNF) in the mPFC to contribute to the development of depression. This study suggests that miR-129-5p in the mPFC impairs the neuronal structures and reduces the synaptic plasticity after the exposure to CMS, which underlies the development of CMS-induced depression-like behaviors in rats.

Metformin, the most prescribed drug for the treatment of type 2 diabetes mellitus, increases the circulating levels of the metabolic regulator growth differentiation factor 15 (GDF15) via transcriptional regulation, with the kidneys being responsible for this increase. Since peroxisome proliferator-activated receptor (PPAR)β/δ agonists mimic many of the effects of metformin, including the rise in circulating GDF15 levels, we herein investigated whether the metformin-mediated antidiabetic effects and GDF15 upregulation were dependent on this nuclear receptor. Male Ppard^-/- and wild-type (WT) mice received a western-type high-fat diet (HFD) for 12 weeks and were treated with metformin (200 mg ·kg^-1 ·d^-1, i.g.) in the last 3 weeks. At the end of the treatment, the mice were sacrificed, and the skeletal muscle, kidney, and liver samples were collected for analyses. We showed that metformin treatment ameliorated glucose intolerance and increased hepatic and circulating GDF15 levels in WT mice, but not in Ppard^-/- mice fed a HFD. In the kidneys, metformin treatment increased the expression levels of phosphorylated AMPK and GDF15 in the WT mice, which was abolished in the Ppard^-/- mice. Both β-arrestin 1 and proprotein convertase subtilisin/kexin type 6 (PCSK6) are involved in the posttranslational maturation of GDF15. Likewise, metformin treatment increased the levels of β-arrestin 1 and PCSK6 in the kidneys of WT mice, but not Ppard^-/- mice. Furthermore, treatment of mice with a PPARβ/δ activator, GW501516 (3 mg· kg^-1 ·d^-1, i.g., for 7 days), increased the levels of these proteins in the kidneys and liver. In contrast, a PPARβ/δ antagonist GSK0660 (50 µM) prevented the increase in GDF15, β-arrestin 1, and PCSK6 levels caused by metformin in cultured podocytes. Collectively, these data uncover a regulatory axis wherein metformin, via PPARβ/δ, orchestrates glucose tolerance, AMPK activity, and GDF15 maturation.

The high relapse rate of drugs is currently a therapeutic dilemma in the treatment of substance use disorder (SUD). Emerging evidence from preclinical animal models demonstrates that pretreatment with selective dopamine D₂ receptor (D₂R) antagonists prevents reinstatement of drug-seeking. However, the role of D₂R downstream signaling in regulating relapse behavior remains unclear. In this study, we investigated the roles of G_αi-protein- and β-arrestin-dependent D₂R signaling pathways in cocaine-primed reinstatement using cocaine self-administration (SA) mouse model treated with biased ligands. We found that treatment of D₂R G_αi-protein antagonists, but not β-arrestin antagonists, significantly attenuated cocaine-primed reinstatement of drug seeking without affecting locomotor activity or anxiety levels. Administration of D₂R G_αi-protein antagonists, but not β-arrestin antagonists, increased cyclic adenosine monophosphate (cAMP) levels in the nucleus accumbens (NAc). Furthermore, treatment of D₂R G_αi-protein antagonists, but not β-arrestin antagonists, suppressed cocaine-induced neuronal activation in the NAc. Our results demonstrate that G_αi-protein-dependent D₂R signaling plays a crucial role in cocaine-primed reinstatement and suggest that D₂R G_αi-protein-biased ligands may be promising pharmacotherapeutic targets for SUD treatment.

Cerebral edema is a severe complication following ischemic stroke. Recent studies have highlighted the crucial role of the glymphatic system (GS) in the clearance of water and macromolecules. GS dysfunction involving the disorders of AQP4 polarization may be crucial in the pathophysiology of cerebral edema. β-Hydroxybutyrate (BHB), the main component of the ketone body, has been shown to alleviate neurological deficits by restoring GS function in subarachnoid hemorrhage models and to reduce Aβ deposition in Alzheimer's disease models. In this study we investigated the effects of BHB on cerebral edema following ischemic stroke and its mechanisms. The mice were fed a ketogenic diet (KD) or a normal diet for 4 weeks before transient middle cerebral artery occlusion (MCAO). Alternatively, the mice received BHB (5 g·kg^-1·d^-1) or vehicle post-MCAO. By using brain section analysis, transcranial macroimaging, two-photon in vivo imaging and MRI, we demonstrated that both KD and BHB treatment significantly enhanced GS function under normal and MCAO conditions. BHB reduced cerebral edema and infarct volume post-MCAO. Notably, delayed BHB treatment initiated 10 h post-MCAO still improved GS function, but did not influence infarct volume. Furthermore, we revealed that BHB increased α1-syntrophin expression and H3K27ac levels in α1-syntrophin (Snta1) enhancer, restoring AQP4 polarization. In addition, BHB also reduced HDAC3 expression and elevated p300 expression. These results suggest that a KD and BHB treatment enhance GS function in mice and that BHB also mitigates brain edema after MCAO. The potentiation of GS function by BHB is likely mediated by the inhibition of HDAC3 activity and the increase in p300 activity, which upregulate α1-syntrophin expression and restore AQP4 polarization.

Targeted covalent inhibitors (TCIs) are emerging as a new modality in drug discovery because of their strong binding affinity and prolonged target engagement. However, the rational design of TCIs remains a significant challenge and is hindered by the lack of methods that accurately predict the structures of covalent protein-ligand complexes. Recent advances in co-folding approaches have made substantial strides in modeling complex biomolecular structures. Despite significant progress, their performance profiles for predicting the structures of covalent protein-ligand complexes remain largely unexplored because of the absence of rigorous benchmarks. Here, we introduce CoFD-Bench, a comprehensive benchmark dataset comprising 218 recently resolved covalent complexes designed to systematically evaluate both classical docking methods (AutoDock-GPU, CovDock, and GNINA) and deep learning co-folding models (AlphaFold3 (AF3), Chai-1, and Boltz-1x). Our results demonstrate that co-folding methods achieve superior ligand RMSD accuracy and protein-ligand interaction recovery. However, their performance markedly declines for novel pocket-ligand pairs. In contrast, classical docking methods exhibit stable but modest performance, which is primarily limited by target conformations. Furthermore, computational efficiency evaluations show that co-folding methods are slower than classical approaches, posing challenges for large-scale predictions. We also reveal that AF3 has the potential to identify native covalent residues through noncovalent co-folding, with a ligand RMSD comparable to that of covalent co-folding. These findings offer a possible route to explore covalent binding without prior specification of reactive residues, which are often unknown in real-world scenarios. Our study provides crucial insights and new opportunities for future co-folding-based TCI design, informing future model applications and improvements. CoFD-Bench offers rigorous evaluation criteria, diverse docking scenarios, and various methodological baselines, positioning it as an important benchmark for future model development and assessment.

Mitochondrial DNA (mtDNA) mutations are the most common cause in aberrant mitochondrion-leading cancer, exploration of direct targeting mutated mtDNA still remains incomplete. Secoemestrin C (Sec C) is epitetrathiodioxopiperazine derived from the endophytic fungus, which exhibited a rapid and prominent anti-breast cancer effect in triple-negative breast cancer (TNBC). In this study we investigated the anticancer mechanism of Sec C, especially its effect on TNBC cells. We showed that Sec C potently inhibited the viability of both TNBC (MDA-MB-231, HS578T, BT-549) and non-TNBC (MCF-7, T47D, SK-BR-3) cells in vitro with IC₅₀ values of 1-2 μM. In MDA-MB-231 cells, treatment with Sec C (2 μM) induced DNA breakage and subsequent apoptosis. Furthermore, treatment with Sec C (2 μM) caused mtDNA damage, mitochondrial ubiquitination and subsequent mitophagy in MDA-MB-231 and MCF-7 cells. RNA-seq analysis revealed that Sec C mitigated YAP level in time and dose-dependent manner either in MDA-MB-231 and MCF-7 cells. By re-analyzing the Sec C-responsive gene network proteins, we identified SLX4 as an oncogene promoting breast cancer development, potentially by stabilizing mtDNA to suppress pathologic mitochondrion mitophagy. Specifically, Sec C initiated MDA-MB-231 cells to yield ROS that induced SLX4 ubiquitination and degradation, leading to mtDNA damage and exacerbated mitophagy and promoted YAP degradation bypassing YAP-driven DNA repair pathways. This study not only demonstrates that Sec C is a rapid and prominent anti-breast cancer drug for TNBC, but also reveals SLX4 as a novel mtDNA stabilizer supporting breast cancer progression, positioning it as both a prognostic biomarker and therapeutic target.

The adult human heart is incapable of regeneration after myocardial infarction (MI) injury. One potential therapeutic strategy is to enhance the proliferation of resident cardiomyocytes (CMs). In this study, we developed a high-content screening assay based on DNA synthesis in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) to identify small molecules that could promote CM proliferation. In the primary screening, we found that L-type calcium channel (LTCC) blockers induced DNA synthesis of hPSC-CMs. Among the 6 clinically approved calcium channel blockers tested in secondary screening and confirmatory experiments, nimodipine (NM) consistently enhanced CM proliferation both in vitro and in vivo. RNA-Seq analysis revealed that NM activated the canonical Wnt signaling pathway, while inhibiting Wnt signaling blunted the proliferative effect of NM. Lrp5, a co-receptor for Wnt ligands known to interact with LTCC, was found to mediate the effect of NM to promote nuclear localization of β-catenin and CM proliferation. In the MI mouse model established by ligating the left anterior descending coronary artery, administration of NM (10 mg/kg, i.p.) for 7 consecutive days significantly improved cardiac contractile function and enhanced resident CM proliferation, which was attenuated by co-treatment with Wnt inhibitor Wnt-C59 (10 mg/kg, i.p.). Our data suggest that L-type calcium channel blockers that induce CM proliferation may be potentially used in the treatment of MI and heart failure to promote cardiac regeneration.

Metabolic dysfunction-associated steatohepatitis (MASH), an inflammatory subtype of metabolic dysfunction-associated fatty liver disease (MAFLD), drives hepatic dysfunction and poses a significant health burden. Lipophagy dysfunction disrupts lipid droplet degradation and induces lysosomal damage, which is closely linked to MASH progression; thus, targeting lipophagy-lysosomal activation has emerged as a promising therapeutic strategy for the therapy of MASH. β-Sitosterol (β-SIT) derived from Polygonum hydropiper L. is structurally similar to cholesterol, and exhibits neuroprotective, antidiabetic and anti-obesity bioactivities. In this study, we explored the therapeutic potential of β-SIT for MASH. The mouse models of MASH were established by feeding a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD) for 10 weeks, or high-fat diet (HFD) for 12 weeks. For in vitro experiments, AML-12 cells were treated with FFA mixture (OA:PA molar ratio = 2:1) to mimic lipid overload condition. MASH mice were administered β-SIT (10 or 20 mg·kg^-1 d^-1, i.g.) for 10 weeks. We showed that β-SIT treatment dose-dependently alleviated MASH by enhancing the lipophagy-lysosomal pathway in vivo and in vitro. In FFA-stimulated AML-12 cells, we demonstrated that β-SIT (20 μM) activated autophagic flux, promoted lysosomal biogenesis, and enhanced lysosome-lipid droplet interactions, as revealed by transmission electron microscopy, multi-SIM real-time fluorescence monitoring, and lipophagy-related marker detection. By integrated approaches including bioinformatics, molecular dynamics, CETSA and functional assays, we found that β-SIT inhibited mTOR pathway activation by directly targeting Ras-related C3 botulinum toxin substrate 1 (RAC1) in MASH mice. By conducting imaging/3D reconstruction, co-immunoprecipitation, immunofluorescence colocalization, lysosomal fractionation, and biochemical analyses in FFA-stimulated AML-12 cells, we confirmed that β-SIT modulated RAC1/mTOR interactions on lysosomes to restore lipophagy function. Critically, β-SIT promoted transcription factor EB (TFEB) nuclear translocation by modulating the RAC1-mTOR axis, thereby repairing lipophagy-lysosomal defects and attenuating MASH progression. Our results suggest that targeting the RAC1-mTOR-TFEB axis is a novel mechanism of β-SIT-driven lipophagy-lysosomal regulation, and highlight β-SIT as a potential candidate for the treatment of MASH.