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.

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.

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.

Meat quality and lean meat percentage are important to pig industry. Muscle and adipose tissues are regarded as secretory organs that release myokines and adipokines to mediate muscle-adipose tissue crosstalk, which regulates lipid deposition and skeletal muscle development. The Taoyuan Black (TB) pig, a Chinese indigenous breed, exhibits abundant lipid deposition and is known for excellent meat quality. The aim of this study was to explore the dynamics of muscle development and lipid deposition in TB pigs at different ages and to identify myokines and adipokines that contribute to lipid accumulation. Forty TB and 40 Duroc pigs were fed the same diet and slaughtered for analysis at age of 60, 120, 180, and 210 d. The “window phase” of lipid deposition in TB pigs was defined from 120 to 180 d of age according to carcass traits, intramuscular fat (IMF), and adipocyte area. Comparative transcriptome of the longissimus dorsi (LD) muscle and subcutaneous adipose revealed upregulated energy and lipid metabolism pathways in TB pigs at 180 d compared to 120 d ( P < 0.05). Many myokines and adipokines associated with IMF and peripheral fat deposition have been identified via database comparison. Myokine IGF2 in TB pigs was downregulated at 180 d compared to 120 d ( P < 0.05) and had negative correlation with backfat thickness, perirenal fat percentage, and fat percentage ( P < 0.05), adipokines SFRP5 and AGT were upregulated in TB pigs at 180 d compared to 120 d and different between TB and Duroc pigs ( P < 0.05). This study provides new insights into the interaction between muscle and adipose tissues, as well as potential targets for nutritional regulation of IMF, lean meat percentage, and meat quality.

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.

Introduction Dietary fiber (DF) regulates immune response and barrier function by interacting with epithelial cells and immune cells. However, the differences in the regulation of intestinal health of different pig breeds by DF remain obscure. Methods A total of 60 healthy pigs (20 pigs/breed) from Taoyuan black (TB), Xiangcun black (XB), and Duroc (DR) pigs (body weight = 11.00 ± 1.00 kg) were fed two different levels (low and high) of DF for 28 days to evaluate the differences in the modulation of intestinal immunity and barrier function by DF in different pig breeds. Results TB and XB pigs had higher plasma Eos level, Eos%, and Lym% but lower Neu level compared with the DR pigs when fed low DF (LDF). The TB and XB pigs had higher plasma Eos, MCV, and MCH levels and Eos% while lower Neu% compared with the DR pigs when fed high DF (HDF). HDF decreased IgA, IgG, IgM, and sIgA concentrations in the ileum of TB and XB pigs compared with the DR pigs, while the plasma IgG and IgM concentrations of TB pigs were higher than those of the DR pigs. Moreover, compared with the DR pigs, HDF decreased the levels of IL-1β, IL-17, and TGF-β in the plasma, and IL-1β, IL-2, IL-6, IL-10, IL-17, IFN-γ, TGF-β, and TNF-α in the ileum of TB and XB pigs. However, HDF did not affect the mRNA expression of cytokines in the ileum of TB, XB, and DR pigs, while HDF increased the TRAF6 expression of TB pigs compared with the DR pigs. In addition, HDF increased the Claudin abundance of TB and DR pigs compared with the pigs feeding with LDF. Moreover, in the LDF and HDF groups, the XB pigs had higher protein abundances of Claudin and ZO-1 compared with the TB and DR pigs. Conclusions DF regulated the TB and DR pigs' plasma immune cells, the XB pigs showed enhanced barrier function, and the DR pigs had increased ileal inflammation, which indicates that Chinese indigenous pigs are more DF tolerant than the DR pigs.

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.

Longevity individuals have lower susceptibility to chronic hypoxia, inflammation, oxidative stress, and aging-related diseases. It has long been speculated that “rejuvenation molecules” exist in their blood to promote extended lifespan. We unexpectedly discovered that longevity individuals exhibit erythrocyte oxygen release function similar to young individuals, whereas most elderly show reduced oxygen release capacity. Untargeted erythrocyte metabolomics profiling revealed that longevity individuals are characterized by youth-like metabolic reprogramming and these metabolites effectively differentiate the longevity from the elderly. Quantification analyses led us to identify multiple novel longevity-related metabolites within erythrocytes including adenosine, sphingosine-1-phosphate (S1P), and glutathione (GSH) related amino acids. Mechanistically, we revealed that increased bisphosphoglycerate mutase (BPGM) and reduced MFSD2B protein levels in the erythrocytes of longevity individuals collaboratively work together to induce elevation of intracellular S1P, promote the release of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from membrane to the cytosol, and thereby orchestrate glucose metabolic reprogramming toward Rapoport–Luebering Shunt to induce the 2,3-BPG production and trigger oxygen delivery. Furthermore, increased glutamine and glutamate transporter expression coupled with the enhanced intracellular metabolism underlie the elevated GSH production and the higher anti-oxidative stress capacity in the erythrocytes of longevity individuals. As such, longevity individuals displayed less systemic hypoxia-related metabolites and more antioxidative and anti-inflammatory metabolites in the plasma, thereby healthier clinical outcomes including lower inflammation parameters as well as better glucose–lipid metabolism, and liver and kidney function. Overall, we identified that youthful erythrocyte function and metabolism enable longevity individuals to better counteract peripheral tissue hypoxia, inflammation, and oxidative stress, thus maintaining healthspan.