DMEM高糖完全培养基

Background Triple-negative breast cancer (TNBC) is characterized by high invasiveness and metastasis potential. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is strongly associated with breast cancer progression, although the underlying mechanisms are largely unknown. Methods The gene expression profiles of TNBC samples were downloaded from the TCGA database, and ubiquitination enzymes related to immune regulation were screened. UCHL1 expression in the TNBC tissues and in adipose-derived mesenchymal stem cells (ADSCs) stimulated in vitro with pro-inflammatory cytokines were analyzed. Exosomes were isolated from these stimulated ADSCs and transfected with scrambled (si-NC) or UCHL1-specific (si-UCHL1) siRNA constructs. TNBC cells were treated with the ADSCs-derived exosomes (ADSCs-Exos) and then co-cultured with macrophages or T cells. Finally, the tumorigenic potential of the ADSCs-Exos was evaluated by injecting the exosomes into mice bearing TNBC xenografts. Results UCHL1 was highly expressed in TNBC tissues and the stimulated ADSCs. The exosomes derived from stimulated ADSCs increased the viability and migration capacity of TNBC cells in vitro, and significantly increased Ki-67 expression through UCHL1. Furthermore, ADSCs-Exos induced M2 polarization of THP-1 monocytes by upregulating CD206 and Arg-1, and downregulating TNF-α and iNOS, and also decreased the proportion of CD3 + CD8 + T cells. Mechanistically, UCHL1 regulated the STAT3 and PD-L1 signaling pathways through HDAC6. Exosomes derived from the control and cytokine-stimulated ADSCs also promoted tumor growth in vivo, and increased the expression of UCHL1, CD206, HDAC6, STAT3, and PD-L1. However, UCHL1 knockdown reversed the pro-tumorigenic effects of the ADSCs-derived exosomes in vivo and in vitro. Conclusion Pro-inflammatory factors (IFN-γ + TNF-α) stimulating ADSCs-Exos enhance immune evasion in triple-negative breast cancer by regulating the HDAC6/STAT3/PD-L1 pathway via UCHL1 transporter. Thus, UCHL1 inhibition may enhance the response of TNBC to immunotherapy. Graphical Abstract

Background Osteosarcoma (OS) is a bone tumor characterized by a high recurrence rate and poor prognosis. Arjunolic acid (AA), the most abundant triterpene component in Cyclocarya paliurus, is reported to have anti-tumor effects. Its specific role in OS is still unknown, which we aim to investigate in our study.Methods An OS mouse model was established to investigate the effects of AA. Subsequently, M2 macrophages and M0 macrophages pretreated with AA were co-cultured with OS cells. The impact of AA on OS cell behavior (proliferation, apoptosis, migration, and invasion) was evaluated via EdU staining, flow cytometry, and Transwell assays. Concurrently, the expression of M1- and M2-associated genes (CD86, CD163, IL-6, Arg1) was quantified to assess AA’s regulatory role in macrophages within the tumor microenvironment (TME). Knockdown or overexpression of Wnt3a in AA-treated M0 macrophages to determine whether AA modulates Wnt3a-mediated M2 polarization, which was further validated in vivo.Results In vivo, AA inhibited tumor progression in OS mice. Concurrently, AA-treated macrophages inhibited OS cell malignant behavior, and AA inhibited OS cell-mediated macrophage M2-type polarization. Mechanistically, AA inhibits the malignant behavior of OS cells and inhibits tumor progression in OS mice by suppressing Wnt3a-mediated macrophage M2 polarization. Additionally, AA-induced macrophage conversion to a pro-inflammatory phenotype in the TME of OS mice.Conclusion Our experiment demonstrated that AA from Cyclocarya paliurus inhibits Wnt3a-mediated M2 macrophage polarization to suppress the progression of osteosarcoma, providing a pharmacological foundation for developing therapies against OS.

Objective Glioblastoma (GBM) has poor clinical prognosis due to limited treatment options. In addition, the current treatment regimens for GBM may only slightly prolong patient survival. The aim of this study was to assess the role of BMAL1 in the immune microenvironment and drug resistance of GBM. Methods GBM cell lines with stable BMAL1 knockdown or LDHA overexpression were constructed, and functionally characterized by the CCK8, EdU incorporation, and transwell assays. In vivo GBM model was established in C57BL/6J mice. Flow cytometry, ELISA, immunofluorescence, and RT-qPCR were performed to detect macrophage polarization. Lactate production, pathological changes, and the expression of glycolytic proteins were analyzed by HE staining, immunohistochemistry, biochemical assays, and Western blotting. Results BMAL1 silencing inhibited the malignant characteristics, lactate production, and expression of glycolytic proteins in GBM cells, and these changes were abrogated by overexpression of LDHA or exogenous lactate supplementation. Furthermore, BMAL1 knockdown induced M1 polarization of macrophages, and inhibited M2 polarization and angiogenesis in GBM cells in conditioned media. Overexpression of LDHA or presence of exogenous lactate inhibited BMAL1-induced M1 polarization and angiogenesis. Finally, BMAL1 silencing and bevacizumab synergistically inhibited glycolysis, angiogenesis and M2 polarization, and promoted M1 polarization in vivo, thereby suppressing GBM growth. Conclusion BMAL1 silencing can sensitize GBM cells to bevacizumab by promoting M1/M2 polarization through the LDHA/lactate axis.

The dorsal root ganglion (DRG) plays a critical role in mediating neuropathic pain (NP) following peripheral nerve injury (PNI), although the underlying mechanism remains unclear. This study investigated how Schwann cell (SCs)-derived extracellular vesicles (SC-EVs) regulate neuronal ferroptosis in NP after PNI. After validating isolated SCs (S-100) and DRG neurons (NeuN), SC-EVs were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting for exosomal markers (Alix/CD9/CD63) and the absence of Calnexin. Co-localization of PKH67 with β-Tubulin-III confirmed SC-EVs uptake by DRG neurons. Biochemical assays and flow cytometry demonstrated SC-EVs suppressed ferroptosis in both LPS-stimulated DRG neurons and chronic constriction injury (CCI) rat models, while simultaneously inhibiting apoptosis, inflammation, and NP progression. Mechanistically, RT-qPCR and western blotting revealed aberrant expression of PPARγ, p53, SAT1, and ALOX15 in LPS/CCI models. Co-immunoprecipitation demonstrated that binding between PPARγ and p53 inhibits SAT1/ALOX15-mediated ferroptosis in DRG neurons. Notably, SC-EVs delivered MFG-E8 to upregulate PPARγ and suppress the activation of the p53/SAT1/ALOX15, thereby attenuating neuronal ferroptosis and ameliorating CCI-induced NP. In conclusion, MFG-E8 delivered via SC-EVs alleviates NP after PNI by modulating the PPARγ/p53/SAT1/ALOX15 signaling axis to inhibit CCI-induced ferroptosis, offering novel therapeutic insights.

Background Glioma represents the predominant malignant brain tumor. This investigation endeavors to elucidate the impact and prospective mechanisms of glycolysis-related lncARSR on glioma. Methods LncARSR level was assessed in normal glial cells and glioma cells. Cell proliferation, migration, and invasion measurements were conducted through CCK-8, wound healing, and transwell assay. Flow cytometry was utilized to measure cell apoptosis and cell cycle. Biochemical assay kits and immunoblotting were employed to measure the content of glycolysis-related indicators and protein expression, respectively. We analyzed the impact of both lncARSR knockdown and overexpression of the Signal Transducer and Activator of Transcription 3 (STAT3) on Hexokinase 2 (HK2) using dual luciferase reporter assays and Chromatin Immunoprecipitation (ChIP) experiments. Further assessment of the impact of lncARSR on glioma progression was conducted through animal experiments. Results LncARSR was expressed at elevated levels in glioma cells compared to normal glial cells. Overexpressing lncARSR enhanced proliferation, migration, invasion, and G2/M phase arrest in glioma cells and also increased glucose, lactate, ATP production, as well as the expression of HK2, PFK1, PKM2, GLUT1, and LDHA. STAT3 binding to the HK2 gene promoter was weakened following the knockdown of lncARSR. Upregulation of STAT3 reversed the suppressed functions of knocking down lncARSR on cell proliferation, migration, invasion, G2/M phase arrest, and glycolysis and counteracted its promotional effect on cell apoptosis. In vivo , knocking down lncARSR inhibits glioma growth and ki67 and PCNA expression. Conclusion LncARSR promotes the development of glioma by enhancing glycolysis through the STAT3-HK2 axis.

Epilepsy is a common neurological disorder, and the exploration of potential therapeutic drugs for its treatment is still ongoing. Vitamin D has emerged as a promising treatment due to its potential neuroprotective effects and anti-epileptic properties. This study aimed to investigate the effects of vitamin D on epilepsy and neuroinflammation in juvenile mice using network pharmacology and molecular docking, with a focus on the mammalian target of rapamycin (mTOR) signaling pathway. Experimental mouse models of epilepsy were established through intraperitoneal injection of pilocarpine, and in vitro injury models of hippocampal neurons were induced by glutamate (Glu) stimulation. The anti-epileptic effects of vitamin D were evaluated both in vivo and in vitro. Network pharmacology and molecular docking analysis were used to identify potential targets and regulatory pathways of vitamin D in epilepsy. The involvement of the mTOR signaling pathway in the regulation of mouse epilepsy by vitamin D was validated using rapamycin (RAPA). The levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were assessed by enzyme-linked immunosorbent assay (ELISA). Gene and protein expressions were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining was used to analyze the apoptosis of hippocampal neurons. In in vivo experiments, vitamin D reduced the Racine scores of epileptic mice, prolonged the latency of epilepsy, and inhibited the production of TNF-α, IL-1β, and IL-6 in the hippocampus. Furthermore, network pharmacology analysis identified RAF1 as a potential target of vitamin D in epilepsy, which was further confirmed by molecular docking analysis. Additionally, the mTOR signaling pathway was found to be involved in the regulation of mouse epilepsy by vitamin D. In in vitro experiments, Glu stimulation upregulated the expressions of RAF1 and LC3II/LC3I, inhibited mTOR phosphorylation, and induced neuronal apoptosis. Mechanistically, vitamin D activated the mTOR signaling pathway and alleviated mouse epilepsy via RAF1, while the use of the pathway inhibitor RAPA reversed this effect. Vitamin D alleviated epilepsy symptoms and neuroinflammation in juvenile mice by activating the mTOR signaling pathway via RAF1. These findings provided new insights into the molecular mechanisms underlying the anti-epileptic effects of vitamin D and further supported its use as an adjunctive therapy for existing anti-epileptic drugs.

Fibrosis drives GMC progression, with FKBP prolyl isomerase 10 (FKBP10) playing a key role. This study aimed to explore the molecular mechanism by which FKBP10 regulates GMC. Expression levels of FKBP10, HSP47, SMAD3, autophagy, and fibrosis-related indicators were analyzed for correlations. Histological staining was used to assess tissue damage and fibrosis. The GMC rat model was constructed by injecting methanol penicillin. The interaction between FKBP10 and HSP47 was also detected. Results showed that FKBP10 expression was up-regulated in the gluteal muscle of GMC patients and rats, accompanied by obvious damage and fibrosis. Elevated levels of TGFβ1, α-SMA, collagen I, collagen III, vimentin, fibronectin, p62, and LC3, along with decreased levels of MMP9 and LC3II/I, Beclin 1, p62, and ATG7, indicated weakened autophagy. FKBP10 expression correlated negatively with autophagy indicators and positively with HSP47 and fibrosis indicators. FKBP10 was found to interact with HSP47. Knockdown of FKBP10 down-regulated the levels of HSP47 and p-SMAD3/SMAD3. Further, knockdown of FKBP10, HSP47 and rapamycin (RAPA) partially reversed the TGFβ1-induced effect. Conversely, 3-methyl adenine (3-MA) and HSP47 overexpression enhanced TGFβ1-induced effects. In GMC rats, FKBP10 knockdown reduced tissue damage and fibrosis, reversed HSP47, p-SMAD3/SMAD3, fibrosis and autophagy indicators levels, and reduced autophagy and LC3 levels. In summary, silencing FKBP10 inactivated the HSP47/SMAD3 signaling pathway, inhibited fibrosis and ameliorated autophagy defects, thereby alleviating GMC.

Objective This work aims to investigate the mechanism of Jiawei Danxuan Koukang (JDK) and Quercetin in oral submucous fibrosis (OSF) carcinogenesis. Design We established an OSF model for rats by injecting Arecoline into the oral mucosa of rats to study the impact of JDK and Quercetin on the progression of OSF and OSCC. Then, the viability, proliferation, and migration ability of Arecoline-induced hOMF, CAL27 and SCC-25 cells in JDK and quercetin intervention were detected. Results The oral mucosal epithelial cells of OSF model and OSF rats were atrophy and thinning, α-SMA, CollageI, Vimentin, Snail, AR and eukaryotic translation initiation factor 5A2 (eIF5A2) expression increased apparently, and E-cadherin expression decreased. The intervention of JDK and Quercetin reversed the changes in oral mucosal epithelial cells and OSF rats. The levels of AR in CAL27 and SCC-25 cells were higher than those in hOMF cells, and Arecoline intervention increased the levels of AR in hOMF, CAL27 and SCC-25 cells. Overexpression of AR up-regulated eIF5A2 to enhance the viability, proliferation and migration of hOMF, CAL27 and SCC-25 cells, and promoted EMT. Quercetin reversed changes in cell feature, and EMT levels in oe-AR intervention. Conclusions JDK and Quercetin inhibited OSF carcinogenesis by inhibiting the AR/eIF5A2 signal-mediated EMT.

MicroRNA-146a-5p (miR-146a-5p) actively participates in the process of cerebral ischemia–reperfusion (CI/R) injury. Dysregulation of the tumor necrosis factor receptor-associated factor 6 (TRAF6)/nuclear factor kappa-B (NF-κB) p65 axis is closely associated with inflammatory response. This study aimed to investigate the potential involvement of miR-146a-5p and TRAF6/NF-κB p65 in mediating CI/R progression in vitro. HT-22 cells were challenged with oxygen–glucose deprivation/reoxygenation (OGD/R) to simulate CI/R in vitro. HT-22 cells were transfected with miR-146a-5p mimics or TRAF6 overexpression constructs. The impact of miR-146a-5p on apoptosis, inflammation, and TRAF6/NF-κB p65 activation were investigated. OGD/R inhibited HT-22 cell viability, induced apoptosis, reduced miR-146a-5p levels and activated the TRAF6/NF-κB p65 pathway. MiR-146a-5p mimics reduced pro-inflammatory factor release, limited apoptosis-related protein expression, and inactivated the TRAF6/NF-κB p65 pathway in OGD/R-challenged HT-22 cells. Mechanistically, miR-146a-5p was verified to bind to TRAF6 3’UTR. TRAF6 overexpression reversed the beneficial effects of miR-146a-5p mimics on apoptosis, inflammation, and TRAF6/NF-κB p65 activation. This work revealed that miR-146a-5p targeted TRAF6 and suppressed the TRAF6/NF-κB p65 pathway, thereby reducing OGD/R-induced inflammation and apoptosis in HT-22 cells. These findings suggest the potential of the miR-146a-5p/TRAF6/NF-κB p65 axis in the treatment of CI/R.