Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP

Target identification in natural products plays a critical role in the development of innovative drugs. Bufalin, a compound derived from traditional medicines, has shown promising anti-cancer activity; however, its precise molecular mechanism of action remains unclear. Here, we employ artificial intelligence, molecular docking, and molecular dynamics simulations to elucidate the molecular mechanism of Bufalin. Using an integrated multi-predictive strategy, we identify CYP17A1, ESR1, mTOR, AR, and PRKCD as the potential targets of Bufalin. Subsequent validation via surface plasmon resonance, biotin pulldown, and thermal shift assays confirms Bufalin’s direct binding to ESR1, which encodes estrogen receptor alpha (ERα). Molecular docking analyses pinpoint Bufalin’s selective interaction with Arg394 on ERα. Molecular dynamic simulations further show that Bufalin acts as a molecular glue, enhancing the interaction between ERα and the E3 ligase STUB1, thereby promoting proteasomal degradation of ERα. Given the therapeutic potential of ERα degradation in overcoming endocrine resistance, we investigate the inhibitory effect of Bufalin on endocrine-resistant models and prove Bufalin reverses Tamoxifen resistance in vitro, in vivo, and in patient-derived breast cancer organoids from tamoxifen-relapsed cases. Collectively, our findings indicate that Bufalin functions as a molecular glue to degrade ERα, offering a potential therapeutic strategy for reversing Tamoxifen resistance.

Chronic kidney disease (CKD) progression is tightly associated with renal fibrosis, which is regulated by macrophage M2 polarization. The intestinal metabolite trimethylamine N-oxide (TMAO) has been reported to promote CKD, yet its underlying mechanism remains unclear. Here, we elucidated a mechanism wherein TMAO excreted through the kidneys alters the pyruvate metabolism of renal tubular epithelial cells, resulting in the production of lactic acid. Local lactic acid accumulation in the kidney promotes adjacent macrophage M2 polarization, a process speculated to be mediated by specific lactylation of macrophage genes. Through lactylation omics analysis, we identified histone H4 lysine 12 (H4K12) as the most significantly up-regulated lysine residue subjected to lactylation. Subsequent chromatin immunoprecipitation sequencing (ChIP-seq) assays revealed H4K12 lactylation on several glycometabolism gene promoters and genes. Furthermore, we found that this lactylation-mediated epigenetic regulation requires the assistance of the “porter”protein p300, as knockdown of p300 weakened the trend towards M2 polarization induced by lactic acid. Using an in vivo unilateral ureteral obstruction (UUO) mouse model, we verified the M2 polarization effect of TMAO and its detrimental role in CKD, as well as the protective effect of the TMAO inhibitor iodomethylcholine (IMC) on CKD. Clinical data validated the up-regulated TMAO’s effect on renal M2 polarization and fibrosis. Our findings suggest that CKD patients exhibit increased TMAO levels, which modulate the production of lactic acid by renal intrinsic cells. Epigenetic regulations mediated by lactic acid, particularly H4K12la on macrophage genes involved in glycometabolism, may contribute to M2 polarization. Targeting TMAO or its downstream pathways could have potential therapeutic benefits in CKD. Schematic diagram showing the whole TMAO modulation process. CKD dysfunction of microbiota leads to elevated TMA. TMA metabolized through liver into TMAO which excreted 90% through kidney. Renal tubular epithelial cells contact with TMAO and secrete lactic acid affecting adjacent macrophages more into M2 type through gene histone H4K12la under the help of p300 as a carrier. These genes include a large amount of glucose metabolism related genes which could at least partially explain this M2 polarization.

Identifying novel therapeutic targets and drugs is crucial for treating triple-negative breast cancer (TNBC). Bufalin, a key active ingredient of the traditional Chinese medicine HuaChansu , has been employed in tumor therapy. Here, SPR-LC-MS/MS is employed to characterize the targets of Bufalin and found that serine/threonine kinase 33 (STK33) possesses a strong binding affinity to Bufalin. Combining molecular docking, SPR analysis, and Biotin-pulldown analysis, it is demonstrated that STK33 can bind Bufalin. Notably, STK33 is highly expressed in TNBC and is associated with poor prognosis in TNBC patients. STK33 knockdown inhibits TNBC cell growth both in vitro and in vivo. Mechanistically, STK33 phosphorylates and stabilizes CCAR1, which promotes tumor growth and metastasis, thereby driving tumor progression. Further analyses confirmed that Methionine 245 of STK33 is required for STK33-Bufalin interaction, and Bufalin treatment promotes the degradation of STK33 protein by destroying the STK33-HSP90 complex. Through in vitro, in vivo, and in patient-derived TNBC organoids, it is observed that Bufalin inhibited the TNBC cell proliferation by targeting STK33. This study not only establishes Bufalin as a putative STK33 degrader to suppress TNBC but also identifies STK33 as a pro-cancer factor in TNBC, presenting a potential therapeutic target for TNBC.

While lipid nanoparticles (LNPs) are widely used as efficient drug delivery systems in therapeutic modalities such as messenger ribonucleic acid (mRNA) vaccines, limitations in terms of targeting specificity continue to hinder their applications in precision medicine, particularly in the targeted delivery of drugs to specific retinal cell types, such as Müller cells. To enhance the targeting capability of LNPs toward Müller cells, the aim was to develop a novel LNP delivery system based on cell membrane (CM)-coating technology. Primary and immortalized Müller CMs were used to coat LNPs, and their targeting efficiency, transfection capability, and sustained action were systematically evaluated using in vitro co-culture assays and in vivo animal models. The LNPs coated with immortalized Müller CMs achieved significantly higher transfection efficiencies and better targeting for Müller cells compared to that of those coated with primary CMs; moreover, the former LNPs exhibited sustained drug release and targeting effects for up to three days, both in vitro and in vivo . Further proteomic analysis and functional validation of Achaete-scute homolog 1-loaded LNPs (LNP-Ascl1) and Müller CM-fused LNP-Ascl1 (CM-LNP-Ascl1) revealed that the latter likely promoted efficient Ascl1 delivery through vimentin (VIM)-mediated specific cellular recognition. This synergistic mechanism activated the Wnt/Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway and its downstream effector, matrix metalloproteinase 9 (MMP-9), establishing a positive feedback loop that facilitated cell proliferation. In summary, the immortalized Müller CM-coated LNPs outperformed their primary CM-coated counterparts in terms of both their targeting ability and sustained action. The stable membrane protein profile of immortalized membranes offers a promising strategy for refining precision drug delivery systems and demonstrates the potential for advancing targeted therapies for retinal diseases.

Ferroptosis induction is particularly promising for cancer therapy when the apoptosis pathway is compromised. Current strategies in nanomedicine for inducing ferroptosis primarily focus on promoting the accumulation of reactive oxygen species (ROS). However, the presence of intracellular antioxidants, such as nuclear factor erythroid 2-related factor 2 (Nrf2), can limit the effectiveness of such therapy by activating detoxification systems and eliminating ROS. To overcome this challenge, we developed a synergistic ferroptosis-inducing agent by modifying manganese (Mn 2+ )–1,8-dihydroxy-3-hydroxymethyl-anthraquinone (aloe-emodin, AE) with polyvinyl pyrrolidone (PVP) to create nanoparticles (MAP NPs). In the tumor microenvironment, these NPs degraded and released AE and Mn(II), facilitating the generation of ROS and Mn(IV) through a Fenton-like reaction between hydrogen peroxide (H 2 O 2 ) and Mn(II). Mn(IV) subsequently interacts with glutathione (GSH) to induce a cyclic catalytic effect, and the depletion of GSH diminished the activation of glutathione-dependent peroxidase 4 (GPX4). Furthermore, AE inhibits the activity of Nrf2 and depleted GSH, thereby synergistically enhancing antitumor efficacy. Here it is demonstrated that MAP NPs effectively generate a robust ROS storm within tumor cells, suggesting that high-performance ferroptosis therapy is effective. Additionally, the inclusion of Mn(II) in the MAP NPs enables real-time monitoring of therapeutic efficacy via magnetic resonance T 1 -weighted contrast imaging.

Inflammatory macrophages (M1 macrophages) and interleukin-1β (IL-1β) serve as critical mediators of inflammatory response and antimicrobial defense in the immune system. Our preliminary investigation identified the HEAT repeat protein (maestro heat-like repeat family member 7, MROH7) as a potential regulator of IL-1β; however, its function in macrophages remains unexplored. In this study, we demonstrated that MROH7 inhibits IL-1β production in M1 macrophages. Mechanistically, MROH7 facilitates the acetylation of lipopolysaccharide-binding protein (LBP) through accumulating intracellular arachidonic acid (AA), thereby promoting its degradation and inhibiting the nuclear factor κB (NF-κB) signaling pathway. Additionally, mice with the myeloid depletion of Mroh7 exhibit an aggravated inflammatory response in lipopolysaccharide (LPS)-induced systemic inflammation. In summary, our study establishes MROH7 as a regulator in macrophage-mediated inflammation, providing critical insights into potential therapeutic targets for inflammatory disorders.

Background Functional dyspepsia (FD), characterized by complex pathophysiology and limited therapeutic options, is a prevalent gastrointestinal disorder. Wei-Dong Granules (WDGs) demonstrate significant clinical efficacy in FD treatment; however, their underlying mechanisms require elucidation. Purpose To investigate the therapeutic effects of WDGs on FD and delineate the associated molecular mechanisms. Methods An FD rat model was established using neonatal iodoacetamide-induced transient gastric injury followed by tail clamping and alternate-day fasting in adulthood. Rats received WDGs via gavage. Gastric motility (food intake, gastric emptying rate, residual food volume), behavioral symptoms, serum gastrointestinal hormones (Motilin, Gastrin, Ghrelin, CCK), and inflammatory factors were assessed. Gastric mucosal damage and repair were evaluated by H&E, Masson, TUNEL, and CD45 staining histologically. After that, UPLC-MS was carried out to identify chemical profiling of WDGs. Transcriptomics of gastric tissues and gut microbiota analysis of intestinal contents were performed, followed by multi-omics integration. ELISA, qPCR, immunohistochemistry, western blot, Masson staining, TUNEL staining, and CD45 staining were conducted to validate the proposed mechanism. Results WDG treatment significantly improved FD symptoms, evidenced by increased food intake and gastric emptying rate, decreased gastric residual volume, and alleviated behavioral abnormalities. WDGs mitigated gastric mucosal damage and promoted glandular and mucosal regeneration. It regulated gastrointestinal hormones (elevated Motilin, Gastrin, Ghrelin; decreased CCK) and reduced IL-6 levels. UPLC-MS identified 174 chemical components in WDG, with 132 confirmed by standards, predominantly flavonoids (50), organic oxides (26), and prenol lipids (23). Multi-omics analysis indicated that WDGs modulated gut microbiota dysbiosis (e.g., Bacteroides, Peptostreptococcus, Ruminococcus), promoted short-chain fatty acid (SCFA) production, activated the vagus nerve-hypothalamus Ghrelin pathway via the gut-brain axis, regulated gastrointestinal hormone secretion, enhanced antioxidant capacity, and facilitated gastric mucosal repair. Subsequent validation confirmed that WDGs alleviated FD through multi-targeted actions encompassing enhanced motility, anti-inflammation, antioxidant effects, and mucosal repair. Conclusion WDGs effectively treat FD by orchestrating gut microbiota-SCFA-gut-brain axis signaling, which enhances gastric motility, reduces inflammation and oxidative stress, and promotes mucosal repair. This integrated study elucidates the mechanism of WDGs and provides a scientific foundation for its clinical application in FD therapy.

Increasing investigations indicate that neurotransmitters shape immune cell function; however, current results about glycine (Gly) in inflammatory macrophage responses are conflicting. Here, we found that Gly transporters support interleukin-1β (IL-1β) production in inflammatory macrophages, while Gly receptors inhibit it. Inflammatory macrophages have higher expression of Gly transporter 1 (GlyT1; also known as SLC6A9). Notably, SLC6A9 inhibition leads to extracellular accumulation of Gly and limits IL-1β production in inflammatory macrophages. Mechanically, extracellular Gly suppresses phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT1)/mammalian target of rapamycin (mTOR) signaling through the Gly receptor alpha-4 (Glrα4), thereby inhibiting activation of the NOD-like receptor 3 (NLRP3) inflammasome and IL-1β production. Furthermore, Gly supplementation or myeloid-specific SLC6A9 depletion alleviates the lipopolysaccharide (LPS)-induced inflammatory response in vivo . Collectively, our findings reveal a previously uncharacterized mechanism for the Gly-ergic system in regulating inflammatory macrophage function, providing a potential alleviating target for macrophage-associated diseases.

Cancer cells rely on lipogenesis in addition to exogenous lipid uptake, and fatty acid synthase (FASN) is aberrantly overexpressed in myeloid leukemia, yet its role in leukemogenesis is unclear. We show that FASN is essential for leukemogenesis. Its genetic ablation impairs leukemic cell growth, survival, and clonogenicity in vitro , and reduces disease burden in vivo , without significantly affecting normal hematopoiesis. We further identify a platensimycin derivative compound MS-C19 as a potent FASN inhibitor. MS-C19 suppresses growth and clonogenicity in clinical acute myeloid leukemia (AML) samples. Mechanistically, FASN inhibition or deficiency activates lysosomal and inflammatory gene programs, inducing lysosomal membrane permeabilization and associated cell death but not lysosome biogenesis. We further identify that GRN, a lysosomal and neuroinflammatory gene, is potently transcribed by TFEB upon FASN inhibition. GRN depletion reverses the anti-leukemic effects of FASN loss. Our findings establish FASN as a therapeutic target and support its pharmacological inhibition by MS-C19 for leukemia treatment.

Background Ambra1 has recently been identified as a key regulatory factor in the progression of mantle cell lymphoma (MCL). The objective of this study was to investigate the biological role and molecular mechanism of Ambra1 in MCL. Methods The m6A modification level of Ambra1 was detected by MeRIP-qPCR. Wild-type and mutant Ambra1 plasmids were constructed to verify the direct regulation of Ambra1 by METTL3-mediated m6A modification. The influence of METTL3/m6A/YTHDF2/Ambra1 on the viability, proliferation, migration, apoptosis, and cell cycle of MCL cells was evaluated by standard in vitro assays. RIP and RNA pull-down assays were performed to validate Ambra1 as a downstream target of YTHDF2. Xenograft tumor models were established using BALB/c nude mice to confirm the in vivo phenotype of METTL3 and Ambra1 silencing. Results Ambra1 was downregulated in MCL cells by METTL3-mediated m6A modification. Furthermore, knocking down METTL3 in the MCL cells inhibited their proliferation, migration, and invasion through the upregulation of Ambra1, while METTL3 overexpression had the opposite effect. The m6A reader protein YTHDF2 downregulated Ambra1 expression by binding to Ambra1-m6A. YTHDF2 knockdown inhibited the growth of MCL cells through Ambra1, while YTHDF2 overexpression had the opposite effect. Mechanistically, METTL3 downregulated Ambra1 in the MCL cells in an m6A-YTHDF2-dependent manner to inhibit apoptosis. Finally, METTL3 knockdown inhibited MCL progression in vivo by inducing Ambra1 expression. Conclusion METTL3 promotes MCL progression through YTHDF2-mediated degradation of Ambra1 mRNA, suggesting that the METTL3/YTHDF2/Ambra1 may serve as a potential therapeutic target for MCL.