Methylthiazolyldiphenyl-tetrazolium Bromide Research Articles

Ailanthone disturbs cross-talk between cancer cells and tumor-associated macrophages via HIF1-α/LINC01956/FUS/β-catenin signaling pathway in glioblastoma

BackgroundAn increasing number of studies have focused on ailanthone (aila) due to its antitumor activity. However, the role of ailanthone in glioblastoma(GBM) has not been investigated before. This study aims to explore the biological function and the underlying mechanism of ailanthone in GBM.MethodsThe microarray analysis was used to screen out down-stream long non-coding RNAs (lncRNAs) targeted by ailanthone. Real-time PCR(RT-PCR) assay was used to examine LINC01956 expression levels. Colony-formation, Methylthiazolyldiphenyl-tetrazolium bromide(MTT), cell-cycle, organoids culture and in-vivo tumorigenesis assays were used to examine cell growth in vitro and in vivo. Boyden assay was used to examine cell invasion ability in vitro. RNA immunoprecipitation and RNA-protein pull-down assays were used to examine the interaction between LINC01956 and FUS protein. Chromatin Immunoprecipitation(ChIP) assay was used to examine HIF1-α-binding sites in the LINC01956 promoter.ResultsAilanthone decreased GBM cell growth in vitro and in vivo via inducing ferroptosis. Ailanthone treatment exhibited blood‒brain barrier(BBB) permeability and specifically targeted the tumor area. LINC01956 was identified as a down-stream target of Ailanthone. LINC01956 exerted as an onco-lncRNA in GBM. M2 polarization of macrophages induced by exosomes derived from glioma cells overexpressing LINC01956 accelerated GBM progression. Mechanistically, we found that LINC01956 bound to FUS and reduced its ubiquitination. LINC01956 evoked nuclear translocation of phosphorylated (p)-β-catenin by recruiting FUS. Furthermore, under hypoxic conditions, LINC01956 was regulated by HIF-1α. Ailanthone decreased the expression of LINC01956 via suppressing HIF-1α.ConclusionTaken together, our data revealed for the first time that ailanthone regulated HIF-1α/LINC01956/FUS/β-catenin signaling pathway and thereby inhibited GBM progression.

Co-culture system of breast cancer and normal cells to investigate inflammation: using doxorubicin encapsulated in adipose-derived exosomes.

Doxorubicin (DOX) chemotherapy for breast cancer is an effective treatment option, but it also has disadvantages. Exosomes (EXOs) have safely and successfully transported DOX and reduced its adverse effects; however, its use is still being explored. In this study, a co-culture system of malignant and non-malignant breast cells was used to generate an in vitro model reflecting the in vivo cellular microenvironment, and the effects of this treatment were investigated by examining inflammatory genes. Extracellular matrices (EXOs) were extracted from mesenchymal stem cells derived from human adipose tissue by ultracentrifugation. Later, Western blotting, dynamic light scattering (DLS) and transmission electron microscopy methods were used to examine the properties of the EXO. DOX was encapsulated in the EXOs by sonication and the loading rate was measured by spectrophotometry. In the current study, a co-culture system was used to investigate the cytotoxic effects of free DOX and DOX encapsulated in EXOs (EXO-DOX) on various breast cell lines, including MCF-7, MCF-10A, MDA-MB-231, and A-MSC. Additionally, the expression levels of inflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α) were examined. Methylthiazolyldiphenyl-tetrazolium bromide assay demonstrated that free DOX showed the highest cytotoxicity against MCF-10A cells, followed by MCF-7 cells. Conversely, EXO-DOX indicated a greater effect on MCF-7 cells and had a lower IC50 compared to MDA-MB-231 cells. Free DOX significantly downregulated the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α), particularly in MCF-7 and MCF-10A cells, while concurrently upregulating IL-10 expression. EXO-DOX induced a more significant alteration in cytokine expression than the control and free DOX treatment groups. The co-culture system revealed a synergistic effect of free DOX on cancer cells while simultaneously mitigating the toxic effects of DOX on normal cells. This study suggests that EXO-DOX has promising potential as a targeted drug delivery system that could potentially improve therapeutic efficacy and minimize off-target toxicity.

Carboplatin-loaded zeolitic imidazolate framework-8: Induction of antiproliferative activity and apoptosis in breast cancer cell.

The challenge with breast cancer is its ongoing high prevalence and difficulties in early detection and access to effective care. A solution lies in creating tailored metal-organic frameworks to encapsulate anticancer drugs, enabling precise and targeted treatment with less adverse effects and improved effectiveness. Zeolitic imidazolate framework-8 (ZIF-8) and carboplatin (CP)-loaded ZIF-8 were synthesized and characterized using various analytical techniques. High Resolution-transmission electron microscopy of ZIF-8 and CP@ZIF-8 indicates that the particles had a spherical shape and were nanosized. The drug release rate of CP is 98% under an acidic medium (pH 5.5) because of the dissolution of ZIF-8 into its coordinating ions, whereas 35% in a physiological medium (pH 7.4) with the addition of CP, the high porosity, and pore diameter of ZIF-8 decrease from 1243 to 1041m2/g. Breast cancer MCF-7 cells were shown greater IC50 in CP@ZIF-8 (15.01±3.03µg/mL) than free CP (34.98±4.25µg/mL) in an in vitro cytotoxicity assessment. The cytotoxicity of the CP@ZIF-8 against MCF-7 cells was studied using the methylthiazolyldiphenyl-tetrazolium bromide method. The morphological changes were examined using fluorescent staining (acridine orange-ethidium bromide and Hoechst 33258) methods. The comet assay assessed the DNA fragmentation (single-cell gel electrophoresis). The results from the study revealed that CP@ZIF-8 can be used in the treatment of breast cancer.

Irisin suppresses PDGF-BB-induced proliferation of vascular smooth muscle cells in vitro by activating AMPK/mTOR-mediated autophagy.

Restenosis is a pivotal factor that restricts the efficacy of coronary artery bypass grafting. Inhibition of vascular smooth muscle cells (VSMCs) proliferation can improve intimal hyperplasia and lumen stenosis. Irisin, a polypeptide secreted by muscle cells, has been demonstrated to have a protective role in various cardiovascular diseases. However, the effect and mechanism of irisin on VSMCs proliferation and phenotype switching remain unclear. Cell proliferation ability was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Cell cycle analysis was performed using flow cytometry, while expression levels of contractile and synthesis-related proteins were determined through RT-qPCR and Western blot. The VSMCs were infected with an adenovirus carrying GFP-LC3, and the proportion of cells showing positive expression was assessed. Additionally, the formation of autophagic lysosomes in cells was observed through transmission electron microscopy. In this study, we have demonstrated the inhibitory effects of irisin on the proliferation and phenotypic transition of platelet-derived growth factor-BB (PDGF-BB)-induced VSMCs. More importantly, we have discovered that irisin can activate the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway to mediate autophagy in PDGF-BB-induced VSMCs. The inhibitory effect of irisin on PDGF-BB-induced VSMCs proliferation was significantly attenuated by the AMPK inhibitor, Compound C. Conversely the mTOR inhibitor, rapamycin further enhanced the inhibitory effect of irisin on PDGF-BB induced VSMCs proliferation. In conclusion, our findings suggest that irisin effectively suppresses the aberrant proliferation of VSMCs following PDGF-BB stimulation by modulating autophagy levels through the AMPK/mTOR signaling pathway.

Bioactive secondary metabolites from an endophytic fungus Aspergillus sp. CCH-1E from Catharanthus roseus

This study focused on the bioactive secondary metabolites of an endophytic fungus Aspergillus sp. CCH-1E from Catharanthus roseus. The secondary metabolites from Aspergillus sp. CCH-1E were isolated by using various chromatographic methods [such as normal-phase and reversed-phase chromatography and high-performance liquid chromatography(HPLC)], and their structures were identified by various spectroscopic methods [e.g., ultraviolet(UV) spectroscopy, infrared(IR) spectroscopy, nuclear magnetic resonance(NMR) spectroscopy, and high-resolution electrospray ionization mass spectrometry(HR-ESI-MS)]. Twelve compounds were yielded and identified from Aspergillus sp. CCH-1E, which are chermesinone H(1), chermesinone I(2), chermesinone B(3), 8,11-didehydrochermesinone B(4), chermesinone C(5), chermesinone A(6), chevalone B(7), barbacenic acid(8), 3,6,8-trihydroxy-3,5,7-trimethyl-3,4-dihydroisocoumarin(9), 5-hydroxy-2-methoxy-7-methyl-1,4-naphthoquinone(10), 1-hydroxy-6,8-dimethoxy-3-methylanthracene-9,10-dione(11), and 7-drimen-9α,11,12-triol(12). Among them, compounds 1 and 2 are new compounds. The growth inhibition effects of all compounds were evaluated against non-small cell lung cancer cell lines A549 and NCI-H1650, as well as human cervical cancer cell line HeLa by using methylthiazolyldiphenyl-tetrazolium bromide(MTT). Compound 7 significantly inhibited the growth of three tumor cells with the IC_(50) values of 1.22-2.43 μmol·L~(-1), respectively. Compounds 1-6 showed moderate cell growth inhibition with the IC_(50) values of 16.24-35.28 μmol·L~(-1).

Preparation of double-loaded bitter ginseng derivative B21–DOX liposomes co-modified with SP94 and BR2 ligand and its in vitro anti-hepatocarcinogenic effect

Aim To construct a novel liposomal drug delivery system co-modified with SP94 and BR2 ligands, encapsulating both the bitter ginseng derivative B21 and doxorubicin (DOX), to achieve superior anti-tumour efficacy and reduced toxic side effects. Methods Liposomes were prepared using an organic phase reaction method, with B21 encapsulated in the lipid phase and DOX in the aqueous phase. The liposomes were further modified with SP94 and BR2 peptides. The characterisations, cytotoxicity, and in vitro targeting effects were assessed through various methods including ultraviolet spectrophotometry, high-performance liquid chromatography, nano-size analysis, ultrafiltration centrifugation, dialysis, transmission electron microscopy, flow cytometry, Methylthiazolyldiphenyl-tetrazolium bromide assay, confocal laser scanning microscopy, transwell assay, and tumorsphere assay. Results SP94/BR2-B21/DOX-LP liposomes were spherical with an average diameter of 120.87 ± 1.00 nm, a polydispersity index (PDI) of 0.223 ± 0.006, and a surface charge of −23.1 ± 1.27 mV. The encapsulation efficiencies for B21 and DOX were greater than 85% and 97% (mg/mg), respectively. The results indicated that SP94/BR2-B21/DOX-LP exhibited enhanced targeting and cytotoxicity compared to single-ligand modified and unmodified liposomes, with the combined encapsulation of B21 and DOX showing synergistic anti-hepatocarcinogenic effects. Conclusion SP94/BR2-B21/DOX-LP liposomes represent a promising targeted drug delivery system for hepatocellular carcinoma, offering improved membrane penetration, enhanced therapeutic efficacy, and reduced systemic toxicity.

Interventional Effect of Donkey Bone Collagen Peptide Iron Chelate on Cyclophosphamide Induced Immunosuppressive Mice.

Immunodeficiency can disrupt normal physiological activity and function. In this study, donkey bone collagen peptide (DP) and its iron chelate (DPI) were evaluated their potential as immunomodulators in cyclophosphamide (Cytoxan®, CTX)-induced Balb/c mice. The femoral tissue, lymphocytes, and serum from groups of mice were subjected to hematoxylin and eosin (H&E) staining, methylthiazolyldiphenyl-tetrazolium bromide (MTT) cell proliferation assays, and enzyme-linked immunosorbent assay (ELISA), respectively. Furthermore, a non-targeted metabolomics analysis based on UPLC-MS/MS and a reverse transcription polymerase chain reaction (RT-qPCR) technology were used to explore the specific metabolic pathways of DPI regulating immunocompromise. The results showed that CTX was able to significantly reduce the proliferative activity of mouse splenic lymphocytes and led to abnormal cytokine expression. After DP and DPI interventions, bone marrow tissue damage was significantly improved. In particular, DPI showed the ability to regulate the levels of immune factors more effectively than Fe2+ and DP. Furthermore, metabolomic analysis in both positive and negative ion modes showed that DPI and DP jointly regulated the levels of 20 plasma differential metabolites, while DPI and Fe2+ jointly regulated 14, and all 3 jointly regulated 10. Fe2+ and DP regulated energy metabolism and pyrimidine metabolism pathways, respectively. In contrast, DPI mainly modulated the purine salvage pathway and the JAK/STAT signaling pathway, which are the key to immune function. Therefore, DPI shows more effective immune regulation than Fe2+ and DP alone, and has good application potential in improving immunosuppression.

Optimization of lyoprotectant for recombinant human acidic fibroblast growth factor by response surface methodology

Recombinant human acidic fibroblast growth factor (rh-aFGF) is a widely used biological product, but it is unstable and its biological activity is easy to decrease. In order to maintain the long-term stability and biological activity of rh-aFGF, based on the response surface method, the freeze-drying characterization and cell proliferation rate of rh-aFGF freeze-dried powder were evaluated by scoring and Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay in this study. The optimal concentrations of trehalose, glycine and BSA were optimized, and the optimal formulation was verified by regression experiment. The results showed that trehalose, glycine and BSA had significant effects on the characterization of lyophilized rh-aFGF and cell proliferation. The optimal formulation of 5.7% trehalose, 2.04% glycine and 1.98%BSA combined with rh-aFGF could achieve the optimal freeze-dried characterization and biological activity. Using the best formulation to verify, the freeze-dried formability index of the freeze-dried powder was 23.35, and the rate of cell proliferation was 43.59%, which was close to the expected 23 and 41.69%. This study determined a freeze-dried formulation of rh-aFGF that meets the requirements of freeze-dried formalization integrity and maintains biological activity, providing reliable support for the subsequent development of related drugs.

Ginsenoside Rg1 inhibits multiple myeloma and overcomes bortezomib resistance through AMPK-mTOR pathway

BackgroundThe resistance of multiple myeloma (MM) to bortezomib (BTZ) has brought multiple challenges to its clinical use. Numerous ginsenosides have potential anti-tumor effects, however, the research on the role of Rg1 in MM has not been reported. ObjectiveTo examine the inhibitory impact of Rg1 on the growth of MM and reduce the drug resistance of MM to BTZ through in vivo and in vitro experiments, and to explore their potential mechanism. MethodsBTZ drug-resistant cell line RPMI8226R was constructed. Mouse tumor-bearing model was developed by abdominal subcutaneous injection of MM cells. MM cells were treated with AMPK inhibitor Compound C or autophagy inhibitor Chloroquine together with Rg1. RPMI8226R cells were treated with BTZ and Rg1. Cell multiplication was detected using Methylthiazolyldiphenyl-tetrazolium bromide assay. Apoptosis was assessed using flow cytometry. Immunofluorescence assay was employed to assess the autophagy markers LC3. Western blot was utilized to assess the protein expression. Immunohistochemistry was used to detect cell proliferation and apoptosis in tumor tissues. ResultsIn vitro experiments demonstrated that Rg1 could hinder the proliferation of MM cells, promote apoptosis and enhance autophagy. Rg1 could also increase the sensitivity of RPMI8226R to BTZ. In vivo experiments illustrated that Rg1 could hinder the development of MM cells in mice, weaken the proliferation of tumor cells and enhance their apoptosis. Further study found that the anti-MM impact of Rg1 was linked to AMPK-mTOR pathway, the autophagy degree of RPMI8226R was higher than that of RPMI8226, and that Rg1 could inhibit MM and overcome drug resistance through autophagy induced by AMPK-mTOR pathway. ConclusionRg1 has significant anti-MM effect and can overcome BTZ resistance, and its potential mechanism is related to the regulation of autophagy induced by AMPK-mTOR pathway. Rg1 is a promising adjuvant drug for the treatment of MM.

Baicalein Inhibits Metastasis of Oral Squamous Cell Carcinoma Cells by Regulating ERK/ELK-1/Snail Signaling.

Oral squamous cell carcinoma (OSCC) is associated with high recurrence and poor prognosis. Baicalin has multiple pharmacological effects, including anti-inflammatory and anti-proliferative activities. Here, we examine the effect of baicalein on OSCC metastasis and its potential mechanism of action. SCC-4 and CAL-27 cells were treated with different concentrations of baicalein. The proliferation of OSCC cells was evaluated by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. As for migration and metastasis, baicalein-treated OSCC cells were used for wound healing assay and Transwell assay. The levels of epithelial-mesenchymal transition-related proteins (E-cadherin, N-cadherin, vimentin) and extracellular regulated protein kinases (ERK)/ETS Transcription Factor ELK1 (ELK-1)/Snail signaling pathway-related proteins in baicalein-treated OSCC cells were evaluated by western blotting. The rates of cell proliferation and migration, along with the metastatic potential, of baicalein-treated cells were significantly lower than those of the control (p < 0.05), and the effects were concentration-dependent. Furthermore, compared to the control, baicalein significantly decreased the levels of N-cadherin and vimentin in SCC-4 and CAL-27 cells, and increased the E-cadherin level (p < 0.05). Mechanistically, baicalein downregulated the levels of p-ERK1/2, phospho-ETS Transcription Factor ELK1 (p-ELK-1), and Snail (p < 0.05). Finally, the ERK/ELK-1/Snail pathway inhibitor (U0126) promoted the effect of baicalein in inhibiting the migration and invasion of OSCC cells (p < 0.05). Baicalein abates the migration, invasion, and metastasis of OSCC cells through the ERK/ELK-1/Snail signaling pathway. This study provides a basis for the development of baicalein as a compound for the treatment of OSCC.

Targeted blocking of EGFR and GLUT1 by compound H reveals a new strategy for treatment of triple-negative breast cancer and nasopharyngeal carcinoma

BackgroundCytoplasmic epidermal growth factor receptor (EGFR) is overexpressed in both nasopharyngeal carcinoma (NPC) and triple-negative breast cancer (TNBC), while clinical outcome and prognosis vary greatly among patients treated with gefitinib, and all patients eventually develop resistance to this agent. Therefore, we propose a new concept for synthesizing multitarget compounds and reveal new therapeutic strategies for NPC and TNBC expressing EGFR. MethodsCompound H was synthesized in our previous study. Molecular docking, and cell thermal shift assays (CETSAs) and drug affinity responsive target stability(DARTS) were used to confirm the binding of compound H to EGFR and GLUT1. Methylthiazolyldiphenyl-tetrazolium bromide(MTT), annexin V-PE assays, mitochondrial membrane potential (MMP) assays, and animal models were used to evaluate the inhibitory effect of compound H on TNBC cell lines. Energy metabolism tests, Western blotting, and immunofluorescence staining were performed to evaluate the synergistic effects on EGFR- and glucose transporter type 1(GLUT1)-mediated energy metabolism. ResultsCompound H can simultaneously act on the EGFR tyrosine kinase ATP-binding site and inhibit GLUT1-mediated energy metabolism, resulting in reductions in ATP, MMP, intra-cellular lactic acid, and EGFR nuclear transfer. The anti-tumor activity of compound H is significantly superior to the combination of GLUT1 inhibitor BAY876 and EGFR inhibitor gefitinib. Compound H has remarkable anti-proliferative effects on TNBC MDA-MB231 cells, and importantly, no obvious toxicity effects of compound H were found in vivo. ConclusionsSynergistic effects of inhibition of EGFR- and GLUT1-mediated energy metabolism by compound H may present a new strategy for the treatment of TNBC and NPC.

Gefitinib Induces Apoptosis in NSCLC Cells by Promoting Glutaminolysis and Inhibiting the MEK/ERK Signaling Pathway.

Over 80% of lung cancer cases constitute non-small cell lung cancer (NSCLC), making it the most prevalent type of lung cancer globally and the leading cause of cancer-related deaths. The treatment of NSCLC patients with gefitinib has demonstrated promising initial efficacy. However, the underlying mechanism remains unclear. This study aims to investigate how gefitinib affects the mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway-mediated growth and death of NSCLC cells. In this study, the NSCLC cell line A549 was cultured in vitro and divided into a control group and a gefitinib group. The viability of the A549 cells was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect apoptosis in A549 cells, and the expression of glutamate dehydrogenase (GDH1) mRNA in these cells was determined using real-time quantitative PCR (RT-PCR). Western blotting was utilized to evaluate the protein expression levels of key components in the MEK/ERK signaling pathway, including phospho-MEK1/2, MEK1/2, phospho-ERK1/2, and ERK1/2. Additionally, intracellular glutamine content in A549 cells was measured using a colorimetric method. In contrast to the control group, the proliferation of A549 cells, the transcription level of glutamate dehydrogenase (GDH1), the intracellular glutamine content, and the protein expression levels of phospho-MEK1/2 and phospho-ERK1/2 were significantly lower in the gefitinib group. Moreover, apoptosis markedly increased. Gefitinib expedites apoptosis and diminishes proliferation in the NSCLC cell line A549 by downregulating the epidermal growth factor receptor (EGFR)/MEK/ERK signaling pathway. This effect is accomplished by fostering the expression of GDH1 to augment glutaminolysis in A549 cells.

Effect of Extrusion on Mechanical Property, Corrosion Behavior, and In Vitro Biocompatibility of the As-Cast Mg-Zn-Y-Sr Alloy.

The effect of extrusion on the microstructure, mechanical property, corrosion behavior, and in vitro biocompatibility of as-cast Mg-1.5Zn-1.2Y-0.1Sr (wt.%) alloy was investigated via tensile tests, electrochemical methods, immersion tests, methylthiazolyl diphenyltetrazolium bromide (MTT), and analytical techniques. Results showed that the as-cast and as-extruded Mg-1.5Zn-1.2Y-0.1Sr alloys comprised an α-Mg matrix and Mg3Y2Zn3 phase (W-phase). In the as-cast alloy, the W-phase was mainly distributed at the grain boundaries, with a small amount of W-phase in the grains. After hot extrusion, the W-phase was broken down into small particles that were dispersed in the alloy, and the grains were refined considerably. The as-extruded alloy exhibited appropriate mechanical properties that were attributed to refinement strengthening, dispersion strengthening, dislocation strengthening, and precipitation strengthening. The as-cast and as-extruded alloys exhibited galvanic corrosion between the W-phase and α-Mg matrix as the main corrosion mechanism. The coarse W-phase directly caused the poor corrosion resistance of the as-cast alloy. The as-extruded alloy obtained via hydrogen evolution and mass loss had corrosion rates of less than 0.5 mm/year. MTT, high-content screening (HCS) analysis, and cell adhesion tests revealed that the as-extruded alloy can improve L929 cell viability and has great potential in the field of biomedical biodegradable implant materials.

Type I collagen extracellular matrix facilitates nerve regeneration via the construction of a favourable microenvironment.

The extracellular matrix (ECM) provides essential physical support and biochemical cues for diverse biological activities, including tissue remodelling and regeneration, and thus is commonly applied in the construction of artificial peripheral nerve grafts. Nevertheless, the specific functions of essential peripheral nerve ECM components have not been fully determined. Our research aimed to differentially represent the neural activities of main components of ECM on peripheral nerve regeneration. Schwann cells from sciatic nerves and neurons from dorsal root ganglia were isolated and cultured in vitro. The cells were seeded onto noncoated dishes, Matrigel-coated dishes, and dishes coated with the four major ECM components fibronectin, laminin, collagen I, and collagen IV. The effects of these ECM components on Schwann cell proliferation were determined via methylthiazolyldiphenyl-tetrazolium bromide (MTT), Cell Counting Kit-8, and 5-ethynyl-2'-deoxyuridine (EdU) assays, whereas their effects on cell migration were determined via wound healing and live-cell imaging. Neurite growth in neurons cultured on different ECM components was observed. Furthermore, the two types of collagen were incorporated into chitosan artificial nerves and used to repair sciatic nerve defects in rats. Immunofluorescence analysis and a behavioural assessment, including gait, electrophysiology, and target muscle analysis, were conducted. ECM components, especially collagen I, stimulated the DNA synthesis and movement of Schwann cells. Direct measurement of the neurite lengths of neurons cultured on ECM components further revealed the beneficial effects of ECM components on neurite outgrowth. Injection of collagen I into chitosan and poly(lactic-co-glycolic acid) artificial nerves demonstrated that collagen I facilitated axon regeneration and functional recovery after nerve defect repair by stimulating the migration of Schwann cells and the formation of new blood vessels. In contrast, collagen IV recruited excess fibroblasts and inflammatory macrophages and thus had disadvantageous effects on nerve regeneration. These findings reveal the modulatory effects of specific ECM components on cell populations of peripheral nerves, reveal the contributing roles of collagen I in microenvironment construction and axon regeneration, and highlight the use of collagen I for the healing of injured peripheral nerves.

Formononetin Restrains Tumorigenesis of Breast Tumor by Restraining STING-NF-κB and Interfering with the Activation of PD-L1.

Breast cancer (BC), a common tumor in women, has high morbidity and mortality. Formononetin, an active ingredient in red clover and Astragalus membranaceus, has a wide range of pharmacological applications, including as an anticancer agent. Since immunotherapy is a hot topic in the treatment strategy of BC, it was dedicated to appraising the specific mechanism of formononetin in BC immunotherapy in this research. Different formononetin concentrations (0, 20, 40, 60, 80, 100 μM) were used to treat BC cells transfected with pcDNA3.1-Programmed death ligand 1 (PD-L1) or Short-hairpin RNA (sh)-PD-L1. Cells were separated into four subgroups: CTRL, pcDNA3.1-PD-L1, sh-CTRL, and sh-PD-L1. Cell viability and cell cycle were assessed through Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and flow cytometry. Programmed death ligand 1 (PD-L1) mRNA concentration was validated via quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Cell metastasis was evaluated via cloning assay and transwell assay. The p-STING/stimulator of interferon genes (STING), p-p65/p65, and PD-L1 concentrations were determined by western blot. Formononetin restrained the proliferation of MCF-7 and MDA-MB-468 cells, and reduced PD-L1 mRNA, p-STING/STING, and p-p65/p65 protein concentrations. Whereas PD-L1 inhibition restrained the viability of BC cells, pcDNA3.1-PD-L1 intervention had the opposite result. STING pathway inhibitor C-176 combined with formononetin treatment further restrained cell proliferation, colony formation, and cell invasion, in contrast to cells treated with formononetin alone. Formononetin can restrain the proliferation of BC cells, which may be mediated through the interference of PD-L1 and suppression of the activation of the STING-NF-κB pathway.

Ginsenoside Rd Induces Differentiation of Myeloid Leukemia Cells via Regulating ERK/GSK-3β Signaling Pathway.

To investigate the role of ginsenoside Rd (GRd) in acute myeloid leukemia (AML) cell differentiation. AML cells were treated with GRd (25, 50, 100 and 200 µg/mL), retinoic acid (RA, 0.1g/L) and PD98059 (20 mg/mL) for 72 h, cell survival was detected by methylthiazolyldiphenyl-tetrazolium bromide and colony formation assays, and cell cycle was detected by flow cytometry. Cell morphology and differentiation were observed by Wright-Giemsa staining, peroxidase chemical staining and cellular immunochemistry assay, respectively. The protein expression levels of GATA binding protein 1 (GATA-1), purine rich Box-1 (PU.1), phosphorylated-extracellular signal-related kinase (p-ERK), ERK, phosphorylated-glycogen synthase kinase-3β (p-GSK3β), GSK3β and signal transducer and activator of transcription 1 (STAT1) were detected by Western blot. Thirty-six mice were randomly divided into 3 groups using a random number table: model control group (non-treated), GRd group [treated with 200 mg/(kg·d) GRd] and homoharringtonine (HTT) group [treated with 1 mg/(kg·d) HTT]. A tumor-bearing nude mouse model was established, and tumor weight and volume were recorded. Changes of subcutaneous tumor tissue were observed after hematoxylin and eosin staining. WT1 and GATA-1 expressions were detected by immunohistochemical staining. The cell survival was inhibited by GRd in a dose-dependent manner and GRd caused G0/G1 cell arrest (p<0.05). GRd treatment induced leukemia cell differentiation, showing increased expressions of peroxidase and specific proteins concerning erythrogenic or granulocytic differentiation (p<0.05). GRd treatment elicited upregulation of p-ERK, p-GSK-3β and STAT1 expressions in cells, and reversed the effects of PD98059 on inhibiting the expressions of peroxidase, GATA-1 and PU.1 (P<0.05). After GRd treatment, tumor weight and volume of mice were decreased, and tumor cells underwent massive apoptosis and necrosis (P<0.05). WT1 level was decreased, and GATA-1 level was significantly increased in subcutaneous tumor tissues (P<0.05 or P<0.01). GRd might induce the differentiation of AML cells via regulating the ERK/GSK-3β signaling pathway.

Discovery of indole analogues from Periplaneta americana extract and their activities on cell proliferation and recovery of ulcerative colitis in mice

Background: As an important medicinal insect, Periplaneta americana (PA) has been applied for the treatment of wounds, burns, and ulcers with fewer side effects and a reduced recurrence rate, which provides great potential for developing new drugs based on its active constituents.Materials and methods: The main chromatographic peaks determined by high performance liquid chromatography (HPLC) in the PA concentrated ethanol-extract liquid (PACEL) were separated, purified, and identified by semi-preparative LC, mass spectrum, and 1H NMR spectroscopic analysis. The biological activities of the identified compounds were investigated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method based on in vitro human skin fibroblasts (HSF) and in vivo experiments based on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) mouse model. Furthermore, RT-qPCR of six genes related to inflammation or intestinal epithelial cell proliferation was employed to investigate the molecular mechanism of the indole analogues recovering UC in mice.Results: Five indole analogues were purified and identified from PACEL, including tryptophan (Trp), tryptamine (pa01), 1,2,3,4-tetrahydrogen-β-carboline-3-carboxylic acid (pa02), (1S, 3S)-1-methyl-1,2,3,4-tetrahydrogen-β-carboline-3-carboxylic acid (pa03), and (1R, 3S)-1-methyl-1,2,3,4-tetrahydrogen-β-carboline-3-carboxylic acid (pa04), among which the pa02 and pa04 were reported in PA for the first time. In vitro and in vivo experiments showed that PACEL, Trp, and pa02 had promoting HSF proliferation activity and intragastric administration of them could alleviate symptoms of weight loss and colon length shortening in the UC mice. Although recovery activity of the compound pa01 on the colon length was not as obvious as other compounds, it showed anti-inflammatory activity in histological analysis. In addition, The RT-qPCR results indicated that the three indole analogues could alleviate DSS-induced intestinal inflammation in mice by inhibiting pro-inflammatory cytokines (MMP7, IL1α) and down-regulating BMP8B expression.Conclusion: This study reported the isolation, purification, structure identification, and biological activity of the active indole analogues in PACEL. It was found for the first time that the PA extract contained many indole analogues and Trp, which exhibited good proliferation activity on HSF fibroblasts as well as anti-UC activity in mice. These indole analogues probably are important components related to the pharmacological activity in PA.

Polycaprolactone/Testicular Extracellular Matrix/Graphene Oxide-Based Electrospun Tubular Scaffolds for Reproductive Medicine: Biomimetic Architecture of Seminiferous Tubules.

Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively replicating the spatial characteristics of native tissue, poses a challenge in maintaining the requisite cellular arrangement essential for spermatogenesis. In order to mimic the structural properties of seminiferous tubules, the objective is to fabricate a biocompatible tubular scaffold. Following the decellularization process of the testicular tissue, validation of cellular remnants' elimination from the specimens is conducted using 4',6-diamidino-2-phenylindole staining, hematoxylin and eosin staining, and DNA content analysis. The presence of extracellular matrix (ECM) components is confirmed through Alcian blue, Orcein, and Masson's trichrome staining techniques. The electrospinning technique is employed to synthesize the scaffolds using polycaprolactone (PCL), extracted ECM, and varying concentrations of graphene oxide (GO) (0.5%, 1%, and 2%). Subsequently, comprehensive evaluations are performed to assess the properties of the synthetic scaffolds. These evaluations encompass Fourier-transform infrared spectroscopy, scanning electron microscopy imaging, scaffold degradation testing, mechanical behavior analysis, methylthiazolyldiphenyl-tetrazolium bromide assay, and in vivo biocompatibility assessment. The PCL/decellularized extracellular matrix with 0.5% GO formulation exhibits superior fiber morphology and enhanced mechanical properties, and outperforms other groups in terms of in vitro biocompatibility. Consequently, these scaffolds present a viable option for implementation in "in vitro spermatogenesis" procedures, holding promise for future sperm production from spermatogonial cells.

The Effects of Pifithrin-µ on Spermatogonial Stem Cell Viability and Pluripotency

Introduction: Spermatogonial stem cells (SSCs) offer remarkable competencies for animal reproduction and overcoming human disease as a result of their differentiation capability. We evaluated the effect of small molecule pifithrin-mu (PFT-µ), a well-known inhibitor of P53 on SSC biological processes such as viability, apoptosis, and gene expression pattern. Methods: The SSCs were isolated from the testes of adult NMRI mice and then cultured in DMEM/F12 medium containing 10% FBS. Then, they were characterized by the immunocytochemistry technique by high PLZF and low c-Kit expressions. SSC colony formation assay was carried out and their viability was estimated by methylthiazolyldiphenyl-tetrazolium bromide (MTT, or 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay upon exposure to PFT-µ (0, 0.6, 1.2, 2.5, and 5 µm). The apoptosis percentages were also measured using FACS analysis, and finally, Oct4 and Stra8 expression at mRNA levels was assessed using real-time quantitative PCR. Results: The 0.6 and 1.2 µm PFT-µ improved the viability of SSC based on MTT assay results; however, 2.5 and 5 µm PFT-µ reduced SSC viability compared with the control group. Moreover, PFT-µ at lower concentrations enhanced the colony size of SSCs and diminished their apoptosis. As well, exposure to PFT-µ upregulated Oct4 expression while downregulating the meiotic entry marker, Stra8. Conclusion: Based on findings, optimized concentrations of PFT-µ can decrease SSC apoptosis, and conversely potentiate their pluripotency and self-renewal capacities in vitro.

Abstract P2185: Hydrogen Sulfide Donor Inhibits Cisplatin-induced Foam Cell Death In An In Vitro Model Of Atherosclerosis

Introduction: Cisplatin (CPDD) is used to treat various cancers, including testicular, lung, head and neck, and ovarian. CPDD use is associated with long-term cardiovascular morbidity and mortality risk, including acute coronary syndrome (ACS). Pro-inflammatory activation and death of macrophage foam cells (MFC) within the atheroma contribute to plaque destabilization and coronary artery thrombosis. The mechanisms underlying CPDD-induced ACS remain largely obscure. Hydrogen sulfide (H 2 S) has been shown to regulate cell survival and reduce the residual inflammatory risk in the setting of myocardial infarction. Hypothesis: We hypothesize that H 2 S protects MFC following CPDD administration. Methods: Human monocyte THP-1 cells were differentiated into MFC with 150 nM of phorbol 12-myristate 13 acetate (PMA) for 48 hours, followed by 100 μg/mL of OxLDL in serum-free media for 24 hours. Lipid content was assessed by Oil Red O staining (Fig A). A Methyl thiazolyl diphenyl-tetrazolium bromide (MTT) assay was used to assess the cytotoxic effects on MFC subjected to 24-hour treatment with 25 μM CPDD following 30-minute pre-treatment with 40 μM sodium sulfide (Na 2 S, a H 2 S donor) (Fig B). Results: Differentiated cells exposed to OxLDL showed a 25% increase in lipid accumulation compared to untreated cells (Fig A, **p&lt;0.01 vs THP-1). Na 2 S alone showed no effect on cell death. Treatment with CPDD induced ~20% increase in cell death while pre-treatment with Na 2 S reduced MFC death by ~50% (Fig B, **p&lt;0.01 vs OxLDL, *p&lt;0.05 vs OxLDL+CPDD). Conclusion: Our results indicate that CPDD may contribute to plaque destabilization by inducing MFC death, which is attenuated with H 2 S treatment.