Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594

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.

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.

Objective. Here, we aimed to explore the main mechanism of Yaobishu (YBS) in lumbar disc herniation (LDH). Methods and Results. Eighteen compounds that might act on LDH were obtained through a combination of network pharmacology prediction and identification by high-performance liquid chromatography-mass spectrometry. The key compounds were palmitic acid and trans-4-hydroxy-3-methoxycinnamate (cinnamate). KEGG analysis demonstrated that palmitic acid target genes mainly regulate the PPAR signaling pathway, Ras signaling pathway, and fatty acid metabolism. Cinnamate target genes were primarily involved in chemical carcinogenesis-receptor activation, lipid and atherosclerosis, the HIF-1 signaling pathway, and nitrogen metabolism. The rat LDH model was constructed using autologous nucleus pulposus tissue implantation. Differential expression gene (DEGs) related to metabolism (CDKN1A and UHRF1), inflammation (S100A9 and SOCS3), autophagy (DCN and LEPR), and apoptosis (CTSW and BCL2A1) in dorsal root ganglion (DRG) tissues of the control and LDH groups was evaluated by RNA-Seq. TNF-α stimulated DRG neuronal cells were used to establish an in vitro LDH model. YBS, palmitic acid, and cinnamate reduced the expression of substance P, CGRP, S100A9, CTSW, and cleaved caspase-3, while enhancing the expression of CDKN1A, UHRF1, PCNA, Ki67, SOCS3, DCN, LEPR, and BCL2A1, as well as telomerase activity. Pearson’s correlation analysis confirmed that DCN was positively correlated with BCL2A1, indicating that autophagy might be negatively correlated with apoptosis in LDH. YBS, palmitic acid, and cinnamate reduced the Siegal neurological score and serum IL-1β and IL-18 levels, while increasing changes in the hind paw mechanical withdrawal threshold. The RNA-Seq results further showed that YBS downregulated S100A9 and CTSW expression, while upregulating SOCS3, CDKN1A, UHRF1, DCN, LEPR, and BCL2A1 expression. Conclusion. YBS and its compounds, palmitic acid, and cinnamate, attenuated LDH by regulating the inflammatory, metabolic, autophagic, and apoptotic pathways. Our results might improve the theoretical and experimental basis for clinical applications of LDH disease treatment.

Background: A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is involved in the occurrence and development of myocardial fibrosis. Here, we sought to explore the specific regulatory mechanism of ADAMTS1 in cardiac fibrosis post-myocardial infarction (CFPMI). Methods: Blood samples from patients with myocardial fibrosis were collected. A CFPMI mouse model and in vitro models involving human or mouse cardiac fibroblasts treated with TGF-β1 or Ang II were constructed. ChIP was used to confirm that SMAD2 binds to ADAMTS1, and Co-IP was used to verify the interaction between ADAMTS1 and HDAC6. Cellular models with SMAD2 knockdown, ADAMTS1 regulation, and HDAC6 inhibitor treatment were used to study their roles in fibrosis. Finally, AAV-shRNA-HDAC6 and ADAMTS1 inhibitor effects were verified in vivo. Results: ADAMTS1 levels were higher in myocardial fibrosis patients' serum. Increased ADAMTS1 and p-SMAD2 were found in fibrotic mouse hearts and human cardiac fibroblasts stimulated with fibrotic factors. ChIP validated the binding of SMAD2 to ADAMTS1. Mechanistically, SMAD2 regulated ADAMTS1 expression during TGF-β1-induced fibrosis in human and mouse cardiac fibroblasts. Overexpression of ADAMTS1 enhanced the production of collagen fiber proteins in human and mouse cardiac fibroblasts induced by TGF-β1. Moreover, HDAC6 expression was elevated in CFPMI mouse hearts and ADAMTS1 inhibited HDAC6 to regulate fibrosis. ADAMTS1 interacted with HDAC6 during fibrosis. In vivo, shRNA-HDAC6 and ADAMTS1 inhibitor treatment alleviated myocardial fibrosis and improved cardiac function after CFPMI. Conclusions: Targeting ADAMTS1/HDAC6 alleviated TGF-β1/SMAD2-associated cardiac fibrosis in CFPMI. This study may provide a novel theoretical basis for the treatment of myocardial fibrosis.

Macrophage M2 polarization plays a pivotal role in breast cancer development. The present study aimed to investigate the interplay of the Leupaxin (LPXN)/HDAC6/EGR2 axis in breast cancer and its impact on macrophage M2 polarization. Our findings indicate that LPXN overexpression in breast cancer tissues correlates with M2 macrophage polarization. To investigate LPXN's potential role, we conducted siRNA-mediated silencing in macrophages. In a breast cancer cell-macrophage co-culture system, LPXN silencing was associated with reduced cancer cell proliferation, decreased M2 polarization markers, and diminished HDAC6 expression. BIOGRD and experimental data suggest a regulatory relationship between LPXN and HDAC6. Notably, HDAC6 inhibition partially reversed the pro-M2 effects of LPXN overexpression. Further mechanistic studies revealed that HDAC6 interacts with EGR2, functioning as its deacetylase and negatively regulating EGR2 expression. EGR2 silencing partially attenuated the anti-M2 effects observed with LPXN knockdown. In murine breast cancer models, LPXN silencing was linked to increased M1 macrophage markers and reduced tumor burden. These findings suggest LPXN may influence breast cancer progression through HDAC6/EGR2-mediated regulation of macrophage polarization. In conclusion, our study demonstrated that the LPXN/HDAC6/EGR2 axis promotes breast cancer progression by augmenting macrophage M2 polarization. Graphical

Ischemic stroke is a serious health hazard that lacks effective treatment strategies. This study aims to investigate baicalin’s effect on tight junctions and immune cell infiltration after ischemic stroke injury. Rat brain microvascular endothelial cells (BMECs) were treated with OGD/R to establish an in vitro model. Caspase-3, Bax, Bcl-2, zonula occludens-1 (ZO-1), occludin, claudin-5, tumor necrosis factor (TNF)- α , interleukin (IL)-6, inducible nitric oxide synthase (iNOS), Toll-like receptor (TLR) 2, TLR4, and nuclear factor-kappa B (NF- κ B) expressions were detected using qRT-PCR and western blotting. ZO-1, TNF- α , iNOS, IL6, CD31, and ZO-1 expressions were examined using immunofluorescence. A tube formation assay was performed to measure angiogenesis. An ischemia-reperfusion model in rats was established by middle cerebral artery occlusion. The infarct volume was observed using 2,3,5-triphenyltetrazolium chloride staining. TNF- α , iNOS, and IL6 levels in the serum were tested using ELISA. Flow cytometry was performed to examine immune cell inflammatory infiltration. Baicalin had no significant effect on the proliferation of normal BMECs. Baicalin inhibited apoptosis, protected against tight junction injury, and alleviated the inflammatory response in OGD/R-induced BMECs and IR rats, with the highest dose (25 μ g/mL) exerting a superior effect. Baicalin decreased the neurological function score, infarct volume, and brain water content, relieved brain morphological changes, and inhibited immune cell infiltration in vivo . In conclusion, baicalin could reduce BMECs apoptosis, protect tight junctions, and resist immune cell infiltration, thereby alleviating ischemic stroke. Our findings potentially provide a novel treatment strategy for ischemic stroke.

Ethnopharmacological relevance Probiotic fermentation is a mild and safe biological method to boost the performance of herbs. Portulaca oleracea L. (PO), with folklore records of purgative, anti-dermatological and anti-epidemic effects, has been demonstrated to possess anti-inflammatory, immunomodulatory, and antioxidant properties. However, the potential of PO for the treatment of atopic dermatitis (AD) has not been sufficiently explored. Aim of study This study aimed to evaluate the therapeutic benefits of PO and fermented Portulaca oleracea L. (FPO) and explore their intrinsic mechanisms. Methods By utilizing 2,4-dinitrofluorobenzene-induced AD mice as a model, the histopathology of the lesions was observed using H&E and toluidine blue staining methods; the levels of immunoglobulin E (Ig E), histamine (HIS), and thymic stromal lymphopoietin (TSLP) in serum were measured using ELISA , whereas, the expression of inflammatory cytokines in skin lesion was measured using ELISA and immunohistochemistry experiments. The expression of tumor necrosis factor-α ( TNF-α ), IKKα , NF-κB mRNA was measured using qPCR; and the expression of TNF-α、p-IKKα, p-IκBα, p-NF-κB was measured using western blotting . Results Both 20 mg/mL PO and FPO alleviated mast cell infiltration and lesion pathology, reduced serum levels of Ig E, HIS and TSLP, down-regulated the expression of AD-related inflammatory cytokines, such as, TNF-α, interferon-γ, and interleukin-4, and increased filaggrin expression. Furthermore, they inhibited the expression of TNF-α, IKKα, and NF-κB genes and TNF-α, p-IKKα, p-NF-κB and p-IκBα proteins associated with the NF-κB signaling pathway. Conclusions PO and FPO has a positive therapeutic potential on AD, indicating that it may be employed as alternative therapies for AD.

Aims Myocardial hypertrophy is a key pathological basis for heart failure, closely related to disturbances in cardiac lipid metabolism and impaired mitochondrial function. Long-chain acyl-CoA synthetase 6 (ACSL6) is a pivotal enzyme in fatty acid metabolism, but its role in myocardial hypertrophy remains unclear. This study aims to investigate the role of ACSL6 in myocardial hypertrophy and its potential mechanisms. Materials and methods Mouse models of myocardial hypertrophy induced by isoproterenol (ISO) and neonatal mouse cardiomyocyte (NMCM) hypertrophy models intervened by angiotensin II (Ang II) were established. Using lentivirus-mediated ACSL6 overexpression, co-immunoprecipitation, mass spectrometry, lipidomics, transmission electron microscopy, and molecular biology techniques, the functions and mechanisms of ACSL6 were explored. Key findings ACSL6 was downregulated in ISO-induced myocardial hypertrophic mouse tissues and Ang II-treated NMCMs, with expression decreasing as Ang II intervention duration increased. ACSL6 overexpression significantly alleviated myocardial hypertrophy, improved cardiac function, and mitigated cell damage and hypertrophic marker upregulation. Additionally, ACSL6 overexpression inhibited myocardial lipid synthesis and accumulation, ameliorated lipid metabolic disorder, and enhanced mitochondrial function in ISO-induced mice. Mechanistically, KRT17 bound to ACSL6, competing with E3 ubiquitin ligase MIB1, protecting ACSL6 from ubiquitination and degradation. KRT17 knockdown reversed the protective effects of ACSL6 overexpression, exacerbating lipid accumulation and mitochondrial dysfunction. Significance ACSL6 alleviated myocardial hypertrophy by ameliorating cardiac lipid synthesis and mitochondrial function. KRT17 stabilized ACSL6 expression by inhibiting its ubiquitination and degradation, mediating ACSL6's protective effects. Targeting the KRT17-ACSL6 axis emerged as a promising strategy for treating myocardial hypertrophy.

This study aimed to elucidate the underlying mechanisms regarding microRNA-708-5p (miR-708-5p) in lung adenocarcinoma (LUAD). Here, the co-culture system of LUAD cells and macrophages, as well as a xenograft mouse model, were established. High levels of miR-708-5p were observed in LUAD. Exosomal miR-708-5p facilitated M2-like phenotype polarization, whereas miR-708-5p inhibition blocked the polarization. Exosomal miR-708-5p was identified as a pivotal signaling molecule for macrophages to mediate tumor cell proliferation, invasion, migration and IFN-γ production in T cells. In addition, miR708-5p was observed to induce PD-L1 expression, and PD-L1 silencing inhibited macrophage-induced tumor cell growth behavior and regulated CD8 T cell activity. In xenograft models, miR-708-5p inhibition and PD-L1 silencing attenuated macrophage-induced tumor growth, induced IFN-γ secretion and CD8 expression, and modulated the PTEN/AKT/mTOR pathway. In LUAD patients, there was an upregulation of both miR-708-5p and PD-L1 expression, accompanied by the activation of PTEN/AKT/mTOR. In conclusion, this study demonstrated the induction of M2 macrophage polarization and PD-L1 expression by exosomal miR-708-5p. We observed that exosomal miR-708-5p mediated the PTEN/AKT/mTOR pathway, diminished CD8 T cell activity and accelerated LUAD progression. The inhibition of specific exosomal miRNA secretion and anti-PD-L1 in the LUAD microenvironment may represent a promising avenue for LUAD immunotherapy.

Background Heparin reduces myocardial ischemia-reperfusion (I/R) injury, which is associated with pyroptosis. As a derivative of heparin, non-anticoagulant heparin (NAH) is rarely researched in this field. This study aims to explore the mechanisms of NAH in myocardial I/R injury and pyroptosis. Methods Cardiomyocytes (H9C2) were exposed to hypoxia/reoxygenation (H/R) to simulate myocardial I/R injury in vitro . Cells were treated with NAH, ov-gasdermin D (GSDMD), ov-caspase 11, H 2 O 2 , N -acetyl-L-cysteine (NAC), and recombinant HMGB1 (rHMGB1). The binding of NAH to HMGB1 was detected by molecular docking and DARTS. For in vivo validation, C57BL/6 J male mice underwent myocardial I/R surgery and received NAH and rHMGB1 treatment. Results NAH inhibited H/R-induced pyroptosis of H9C2 cells as evidenced by decreased caspase 11/GSDMD activation, decreased IL-18/IL-1β/LDH release, and increased ATP yields. These effects were attenuated by caspase 11 or GSDMD-N overexpression. Similar to NAC, NAH inhibited H/R and H 2 O 2 -induced oxidative stress. Moreover, NAH reversed the promoting effects of rHMGB1 on cell pyroptosis and oxidative stress. Mechanistically, NAH bound to HMGB1, blocking HMGB1/RAGE interaction. In mice, NAH alleviated myocardial infarction, injury, fibrosis, pyroptosis, and oxidative stress. These effects were reversed by rHMGB1. Conclusions NAH protects against myocardial I/R injury by inhibiting GSDMD-mediated pyroptosis via the HMGB1/RAGE pathway. NAH may serve as a potential drug for treating myocardial I/R injury.