Tamoxifen induces eryptosis through calcium accumulation and oxidative stress.

Chemotherapy-related anemia is a major obstacle in anticancer therapy. Tamoxifen (TAM) is an antiestrogen prescribed for breast cancer patients with hemolytic potential and apoptotic properties in nucleated cells. However, the eryptotic activity of TAM has hitherto escaped the efforts of investigators. RBCs from apparently healthy volunteers were treated with 1-50 µM of TAM for 24 h at 37 °C. Hemoglobin leakage and LDH, AST, and AChE activities were photometrically determined while K+, Na+, and Mg2+ were detected by ion-selective electrode. Flow cytometry was used to identify eryptotic cells by annexin-V-FITC, intracellular Ca2+ by Fluo4/AM, sell size and morphology by FSC and SSC signals, respectively, and oxidative stress by H2DCFDA. Whole blood was also exposed to 30 µM of TAM for 24 h at 37 °C to examine the toxicity of TAM to WBCs and platelets. TAM caused Ca2+-independent, dose-responsive hemolysis accompanied by K+, LDH, and AST leakage without improving the mechanical stability of RBCs in hypotonic environments. TAM treatment also increased the proportion of cells positive for annexin-V-FITC, Fluo4, and DCF, along with diminished FSC and SSC signals and AChE activity. Notably, TAM toxicity was aggravated by sucrose but abrogated by vitamin C, PEG 8000, and urea. Moreover, TAM exhibited distinct cytotoxic profiles against leukocytes and platelets. TAM-induced eryptosis is characterized by breakdown of membrane asymmetry, inhibition of AChE activity, Ca2+ accumulation, cell shrinkage, and oxidative stress. Vitamin C, PEG 8000, and urea may hold promise to subvert the undesirable toxic effects of TAM on RBCs.

Cytotoxicity of poly-guanidine in medulloblastoma cell lines.

Medulloblastoma (MB) is the most common pediatric brain tumor. The therapy frequently causes serious side effects, and new selective therapies are needed. MB expresses hyper sialylation, a possible target for selective therapy. The cytotoxic efficacy of a poly guanidine conjugate (GuaDex) incubated with medulloblastoma cell cultures (DAOY and MB-LU-181) was investigated. The cells were incubated with 0.05-8 µM GuaDex from 15 min to 72 h. A fluorometric cytotoxicity assay (FMCA) measured the cytotoxicity. Labeled GuaDex was used to study tumor cell interaction. FITC-label Sambucus nigra confirmed high expression of sialic acid (Sia). Immunofluorescence microscopy was used to visualize the cell F-actin and microtubules. The cell interactions were studied by confocal and fluorescence microscopy. Annexin-V assay was used to detect apoptosis. Cell cycle analysis was done by DNA content determination. A wound-healing migration assay determined the effects on the migratory ability of DAOY cells after GuaDex treatment. IC50 for GuaDex was 223.4 -281.1 nM. FMCA showed potent growth inhibition on DAOY and MB-LU-181 cells at 5 uM GuaDex after 4 h of incubation. GuaDex treatment induced G2/M phase cell cycle arrest. S. nigra FITC-label lectin confirmed high expression of Sia on DAOY medulloblastoma cells. The GuaDex treatment polymerized the cytoskeleton (actin filaments and microtubules) and bound to DNA, inducing condensation. The Annexin V assay results were negative. Cell migration was inhibited at 0.5 µM GuaDex concentration after 24 h of incubation. GuaDex showed potent cytotoxicity and invasion-inhibitory effects on medulloblastoma cells at low micromolar concentrations. GuaDex efficacy was significant and warrants further studies.

Schistosoma japonicum-derived peptide SJMHE1 ameliorates allergic symptoms and responses in mice with allergic rhinitis.

Helminth derived excretory/secretory molecules have shown efficacy in the treatment of allergic asthma in mice, but their roles in allergic rhinitis (AR) are little known. In this study, we aimed to determine the intervention effect of SJMHE1, a Schistosoma japonicum derived small molecular peptide, on ovalbumin (OVA)-induced AR mice and investigate its possible mechanism. AR was induced in BALB/c mice, following which the mice were treated with phosphate-buffered saline (PBS), OVA323-339 and SJMHE1 respectively. SJMHE1 treatment improved clinical symptoms (rubbing and sneezing), suppressed infiltrates of inflammatory cells and eosinophils in nasal mucosa, modulated the production of type-2 (IL-4 and IL-13) and anti-inflammatory (IL-10) cytokines in the nasal lavage fluids (NLF), spleen, and serum. To investigate the underlying mechanism, fluorescein isothiocyanate (FITC)-labeled SJMHE1 was subcutaneously injected into AR mice, and we found that the FITC-SJMHE1 could accumulate in spleen, but not in nasal mucosa. FITC-SJMHE1 mainly bound to CD19 positive cells (B cells), and the SJMHE1 treatment significantly increased the proportion of regulatory B cells (Bregs) and B10 cells, along with the enhancement of PR domain containing protein 1 (Prdm1) protein levels. SJMHE1 may alleviate AR by upregulating Bregs, and has great potential as a new avenue for the AR treatment.

Gemini curcumin inhibits 4T1 cancer cell proliferation and modulates the expression of apoptotic and metastatic genes in Balb/c mice model.

BACKGROUND: Despite the attractive anti-cancer effects, poor solubility and low bioavailability have restricted the clinical application of Curcumin. Recent findings show that Gemini nano-curcumin (Gemini-Cur) significantly improves the cellular uptake of Curcumin and its anti-cancer effect in tumor cells. Here, we aimed to assess the suppressive effect of Gemini-Cur on 4T1 breast cancer cells in vitro and, subsequently, in BALB/c mouse models. MATERIALS AND METHODS: Fluorescence microscopy was employed to visualize cellular uptake and morphological changes of 4T1 cells during treatment with Gemini-Cur and void curcumin. MTT and annexin V/FITC assays were performed to study the toxic effect of Gemini-Cur on mouse cancer cells. For in vivo studies, BALB/c tumor-bearing mice were used to evaluate the inhibitory effect of Gemini-Cur in comparison with mice receiving free Curcumin and nanoparticles. RESULTS: Our data showed that Gemini-Cur enters the cells and inhibits proliferation in a time- and dose-dependent manner. Annexin V/FITC confirmed apoptotic effect on 4T1 cells. In vivo studies also illustrated that tumor growth is suppressed in Gemini-Cur treated mice rather than controls. Expression studies demonstrated the modulation of apoptotic and metastatic genes, including Bax, Bcl-2, MMP-9, VEGF, and COX-2 in treated mice. CONCLUSION: In conclusion, these data demonstrate the promising anti-cancer properties of Gemini-Cur on mice models. However, further studies at molecular and cellular levels are required to conclude this therapeutic advantage.

Neuroprotective effect of a novel brain-derived peptide, HIBDAP, against oxygen-glucose deprivation through inhibition of apoptosis in PC12 cells.

BACKGROUND: The effect of a novel brain-derived peptide, hypoxic-ischemic brain damage associated peptide (HIBDAP), on apoptosis after oxygen-glucose deprivation (OGD) in PC12 cells was investigated. METHODS: The HIBDAP sequence (HSQFIGYPITLFVEKER) was coupled with the carrier peptide of the transactivator of transcription (TAT) sequence (YGRKKRRQRRR). FITC-labelled TAT-HIBDAP was observed by fluorescence microscopy. After TAT-HIBDAP treatment and OGD treatment, the PC12 cell apoptosis rate was analysed using lactate dehydrogenase (LDH) leakage and Annexin V-fluorescein isothiocyanate (FITC) assays. Mitochondrial membrane potential (ΔΨm) was examined by fluorescence microscopy. Protein expression of apoptotic factors was examined by Western blotting. RESULTS: FITC-labelled TAT-HIBDAP entered the PC12 cell nucleus. Compared with the OGD group, TAT-HIBDAP at low concentrations (1 µM, 5 µM, 10 µM) significantly reduced the apoptosis rate of PC12 cells (except at 20 µM); 5 µM TAT-HIBDAP had the most obvious effect. There were remarkable increases in ΔΨm at different concentrations (1 µM, 5 µM, 10 µM, 20 µM) of TAT-HIBDAP pretreatment, and 5 µM TAT-HIBDAP also had the most obvious effect. TAT-HIBDAP reversed the increased ratio of Bax/Bcl-2 and activation of Caspase-3 induced by OGD. CONCLUSION: TAT-HIBDAP is resistant to OGD-induced PC12 cell apoptosis by regulating the Bax/Bcl-2/Caspase-3 pathway, which may provide a novel therapeutic strategy for neonatal HIBD.

The effect of tetrastarch on the endothelial glycocalyx layer in early hemorrhagic shock using fluorescence intravital microscopy: a mouse model.

PURPOSE: To investigate vascular endothelial dysfunction based on glycocalyx impairment in massive hemorrhage and to evaluate fluid therapy. METHODS: In this randomized controlled animal study, we withdrew 1.5 mL blood and administered 1.5 mL resuscitation fluid. Mice were divided into six groups according to the infusion type and administration timing: NS-NS (normal saline), NS-HES ([hydroxyethyl starch]130), HES-NS, NS-ALB (albumin), ALB-NS, and C (control) groups. RESULTS: The glycocalyx index (GCXI) of a 40-µm artery was significantly larger in group C than in other groups (P < 0.01). Similarly, the GCXI for a 60-µm artery was significantly higher in group C than in NS-NS (P ≤ 0.05), NS-HES (P ≤ 0.01), and NS-ALB groups (P ≤ 0.05). The plasma syndecan-1 concentration, at 7.70 ± 5.71 ng/mL, was significantly lower in group C than in group NS-NS (P ≤ 0.01). The tetramethylrhodamine-labeled dextran (TMR-DEX40) fluorescence intensity in ALB-NS and HES-NS groups and the fluorescein isothiocyanate-labeled hydroxyethyl starch (FITC-HES130) fluorescence intensity in NS-HES and HES-NS groups were not significantly different from those of group C at any time point. FITC-HES130 was localized on the inner vessel wall in groups without HES130 infusion but uniformly distributed in HES130-treated groups in intravital microscopy. FITC-FITC-HES130 was localized remarkably in the inner vessel walls in group HES-NS in electron microscopy. CONCLUSIONS: In an acute massive hemorrhage mouse model, initial fluid resuscitation therapy with saline administration impaired glycocalyx and increased vascular permeability. Prior colloid-fluid administration prevented the progression of glycocalyx damage and improve prognosis. Prior HES130 administration may protect endothelial cell function.

Evaluation of Innate Lymphoid Cells (ILCs) Population in the Mouse Model of Colorectal Cancer.

BACKGROUND: Innate Lymphoid Cells (ILCs) promote tissue homeostasis, contribute to the immune defense mechanisms, and play important roles in the initiation of immune responses and chronic inflammation. OBJECTIVE: To understand the roles of innate lymphoid cells in the pathophysiology of colorectal cancer (CRC) in the mouse model. METHODS: CRC was induced using azoxymethane (AOM) and dextran sulfate sodium (DSS) in Balb/c mice (the chemically induced group=18 mice), or orthotopic injection of CT-26 cell line into the colon of another set of Balb/c mice (the orthotopic group=14mice). Normal saline was injected into 18 mice, as the sham group. After 80 days, the chemically induced group was divided into two subgroups, dysplasia (8 mice) and reparative change (10 mice), based on pathological examinations. The frequencies of ILC1, 2, and 3 were then measured in colon tissues using flow cytometry by four markers including an anti-mouse lineage cocktail (FITC anti-mCD3/FITC anti-mGr-1/FITC anti-mCD11b/ FITC anti-mCD45R (B220)/FITC anti-mTer-119), PE/Cy7 anti-mouse CD45, PE anti-mouse CD117 (c-kit), and APC anti-mouse IL-33 Rα (ST2). RESULTS: The total ILC population was significantly higher in the chemically induced reparative change compared with the sham group. ILC1 percentage in the chemically induced reparative change was significantly higher compared to those in the other three groups (Sham, chemically induced dysplasia and orthotopic dysplasia). The orthotopic dysplasia group showed more ILC3 percentage than the other groups. CONCLUSION: ILC1 and ILC3 subgroups increased significantly in reparative and dysplastic experimental CRC respectively. Thus ILC1 may have an inhibitory effect on tumor growth whereas ILC3 promotes tumor progression.

Inhibitory and Injury-Protection Effects of O-Glycan on Gastric Epithelial Cells Infected with Helicobacter pylori.

Helicobacter pylori (H. pylori) is an important pathogen that can cause gastric cancer. Multiple adhesion molecules mediated H. pylori adherence to cells is the initial step in the infection of host cells. H. pylori cholesterol-α-glucosyltransferase (CGT) recognizes and extracts cholesterol from cell membranes to destroy lipid raft structure, further promotes H. pylori adhesion to gastric epithelial cells. O-Glycan, a substance secreted by the deep gastric mucosa, can competitively inhibit CGT activity and may serve as an important factor to prevent H. pylori colonization in the deep gastric mucosa. However, the inhibitory and injury-protection effects of O-Glycan against H. pylori infection has not been well investigated. In this study, we found that O-Glycan significantly inhibited the relative urease content in the coinfection system. In the presence of O-glycan, the injury of GES-1 cells in H. pylori persistent infection model was attenuated and the cell viability was increased. We use fluorescein isothiocyanate-conjugated cholera toxin subunit B (FITC-CTX-B) to detect lipid rafts on gastric epithelial cells and observed that O-glycan can protect H. pylori from damaging lipid raft structures on cell membranes. In addition, transcriptome data showed that O-glycan treatment significantly reduced the activation of inflammatory cancer transformation pathway caused by H. pylori infection. Our results suggest that O-Glycan is able to inhibit H. pylori persistent infection of gastric epithelial cells, reduce the damage caused by H. pylori, and could serve as a potential medicine to treat patients infected with H. pylori.

ß-1,3-Glucan/chitin unmasking in the Saccharomyces cerevisiae mutant, Δmnn9, promotes immune response and resistance of the Pacific oyster (Crassostrea gigas) to Vibrio coralliilyticus infection.

Yeast cells can play a crucial role in immune activation in fish and shellfish predominantly due to the cell wall component ß-1,3-glucan, providing protection against bacterial or viral infections. However, the immunostimulatory capacity of dietary yeast cells remains poorly studied in bivalves. To understand the role of yeast cell wall components (mannan, ß-glucan and chitin) as immune activators, this study characterized the surface carbohydrate exposure of the wild-type baker's yeast Saccharomyces cerevisiae (WT) and its Δmnn9 mutant, which presents a defective mannan structure, and compared these profiles with that of ß-glucan particles, using fluorescein isothiocyanate (FITC)-labeled lectin binding analysis. Then, a first trial evaluated the immunological response in Crassostrea gigas juveniles after being fed for 24 h with an algae-based diet (100A) and its 50% substituted version (based on dry weight) with WT (50A50WT) and Δmnn9 (50A50Y), and the posterior resistance of the juveniles against Vibrio coralliilyticus infection (trial 1). The mRNA expression was measured for ß-glucan-binding protein (CgßGBP), Toll-like receptor 4 (CgTLR4), C-type lectin receptor 3 (CgCLec-3), myeloid differentiation factor 88 (CgMyD88), nuclear factor-kappa B (CgNFκB), lysozyme (CgLys), interleukin 17-5 (CgIL17-5), and superoxide dismutase (CgSOD), in oysters, before and 24 h after the bacterial inoculation. A second trial tested the effect of incorporating Δmnn9 into the 100A diet for 24 h at different substitution levels: 0, 5, 10, 25, and 50% (100A, 95A5Y, 90A10Y, 75A25Y, and 50A50Y), followed by the bacterial challenge with V. coralliilyticus (trial 2). Our findings showed that the outer cell wall surface of WT is largely composed of mannan, while Δmnn9 presents high exposure of ß-glucan and chitin, exhibiting similar FITC-lectin binding profiles (fluorescence intensity) to ß-glucan particles. A significantly higher survival after the bacterial challenge was observed in oysters fed on 50A50Y compared to those fed 50A50WT and 100A in trial 1. This better performance of 50A50Y was supported by significantly higher gene expressions of CgLys, CgSOD, CgMyD88, and CgßGBP compared to 100A, and CgSOD and CgNFκB in relation to those fed on 50A50WT, prior to the bacterial inoculation. Furthermore, improved survival was observed in oysters fed 50A50Y compared to those offered lower Δmnn9 levels and 100A in trial 2. The superior performance of Δmnn9-fed oysters is mostly associated with the elevated presence of unmasked ß-glucans on Δmnn9 cell wall surface, facilitating their interactions with oyster hemocytes. Further studies are needed to evaluate administration dose and frequency of Δmnn9 to develop strategies for long-term feeding.

Activating transcription factor 6 protects against endothelial barrier dysfunction.

BACKGROUND: Endothelial hyperpermeability is associated with sepsis and acute respiratory distress syndrome (ARDS). The identification of molecular pathways involved in barrier dysfunction; may reveal promising therapeutic targets to combat ARDS. Unfolded protein response (UPR) is a highly conserved molecular pathway, which ameliorates endoplasmic reticulum stress. The present work focuses on the effects of ATF6, which is a UPR sensor, in lipopolysaccharides (LPS)-induced endothelial hyperpermeability. METHODS: The in vitro effects of AA147 and Ceapin-A7 in LPS-induced endothelial barrier dysfunction were investigated in bovine pulmonary artery endothelial cells (BPAEC). Small interfering (si) RNA was utilized to "silence" ATF6, and electric cell-substrate impedance sensing (ECIS) measured transendothelial resistance. Fluorescein isothiocyanate (FITC)-dextran assay was utilized to assess paracellular permeability. Protein expression levels were evaluated with Western blotting, and cell viability with MTT assay. RESULTS: We demonstrated that AA147 prevents LPS-induced barrier disruption by counteracting Cofilin and myosin light chain 2 (MLC2) activation, as well as VE-Cadherin phosphorylation. Moreover, this ATF6 inducer opposed LPS-triggered decrease in transendothelial resistance (TEER), as well as LPS-induced paracellular hyperpermeability. On the other hand, ATF6 suppression due to Ceapin-A7 or small interfering RNA exerted the opposite effects, and potentiated LPS-induced endothelial barrier disruption. Moderate concentrations of both ATF6 modulators did not affect cell viability. CONCLUSIONS: ATF6 activation protects against endothelial barrier function, suggesting that this UPR sensor may serve as a therapeutic target for sepsis and ARDS.

Bromuconazole fungicide induces cell cycle arrest and apoptotic cell death in cultured human colon carcinoma cells (HCT116) via oxidative stress process.

BACKGROUND: Bromuconazole, a fungicide belonging to the triazole family, is a plant protection product used to control, repel or destroy fungi that may develop on crops. We investigated the pro-apoptotic effect of bromuconazole and the role of oxidative stress in the death mechanism induced by this fungicide in this study. METHODS: The human colon HCT116 cell line was treated with Bromuconazole (IC50/4, IC50/2, and IC50) for 24 h. Cells were collected and analysed for biomarkers of apoptotic cell death and oxidative stress as well as for the assessment of genotoxic damage. RESULTS: Our study showed that bromuconazole caused a concentration-dependent increase in cell mortality with an IC50 of 180 µM. Bromuconazole induced cell cycle arrest in the G0/G1 phase and DNA synthesis inhibition. The Comet assay showed that bromuconazole caused DNA damage in a concentration-dependent manner. Bromuconazole-induced apoptosis was observed by, Annexin-V/FITC-PI and BET/AO staining, by mitochondrial membrane depolarisation, and by increased caspase-3 activity. In addition, bromuconazole induced a significant increase in ROS and lipid peroxidation levels and a disruption in SOD and CAT activities. N-acetylcysteine (NAC) strongly prevents cytotoxic and genotoxic damage caused by bromuconazole. CONCLUSION: Bromuconazole toxicity was through the oxidative stress process, which causes DNA damage and mitochondrial dysfunction, leading to cell cycle arrest and apoptotic death of HCT116 cells.

Bromuconazole exposure induced cell cycle arrest in the G0/G1 in HCT116 cells.Bromuconazole caused DNA synthesis inhibition and degradation.Bromuconazole-induced Annexin-V/FITC-PI and BET/AO positive staining, increased caspase-3 activity and MMP.Bromuconazole enhances ROS, MDA levels and disruption of CAT and SOD activities.

Anlotinib inhibits the proliferation and induces apoptosis by inactivating the AKT pathway in androgen receptor-negative prostate cancer cells.

Prostate cancer is one of the most frequently diagnosed cancers in men. The medical treatment of metastatic prostate cancer relies heavily on androgen deprivation. The present study aimed to explore the inhibitory effect of anlotinib on androgen receptor (AR)-negative prostate cancer cell lines in vitro and investigate its mechanism of action. Two prostate cancer cell lines, DU145 and PC-3, were treated with different concentrations (0-80 µM) of anlotinib. Cell proliferation was accessed by CCK-8 assay and EdU staining. Cell nuclear morphology was observed after DAPI staining, cell apoptosis level was evaluated by Annexin-V-FITC/PI staining, and western blot was used to detect the proliferation- and apoptosis-related proteins. The potential interaction between anlotinib and AKT was revealed by molecular docking. After treatment with anlotinib, the cell proliferation rate was significantly inhibited in a dose-dependent manner. The DAPI staining showed that anlotinib could induce apoptosis. Further, Annexin V/PI double staining confirmed the occurrence of apoptosis, accompanied by the increase of cleaved caspase-3 and activated PARP. Moreover, anlotinib significantly decreased the phosphorylation of protein kinase B (AKT) and its downstream pathway proteins in prostate cells (p<0.05). Experiments further confirmed that the activation of the AKT pathway reversed the inhibitory effect of anlotinib on DU145 and PC-3 cell proliferation. In addition, molecular docking analysis revealed potential interactions between anlotinib and AKT1 at multiple sites. Overall, the present study suggested that anlotinib could inhibit the proliferation and induce apoptosis in the AR-negative prostate cancer cell lines, possibly via the inactivation of the AKT pathway.

SARS-CoV-2 spike protein inhibits megalin-mediated albumin endocytosis in proximal tubule epithelial cells.

Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.

TMBIM6 promotes diabetic tubular epithelial cell survival and albumin endocytosis by inhibiting the endoplasmic reticulum stress sensor, IRE1α.

AIM: Reduced albumin reabsorption in proximal tubular epithelial cells (PTECs), resulting from decreased megalin plasma membrane (PM) localization due to prolonged endoplasmic reticulum (ER) stress, potentially contributes to albuminuria in early diabetic kidney disease (DKD). To examine this possibility, we investigated the cytoprotective effect of TMBIM6 in promoting diabetic PTEC survival and albumin endocytosis by attenuating ER stress with an IRE1α inhibitor, KIRA6. METHODS AND RESULTS: Renal TMBIM6 distribution and expression were determined by immunohistochemistry, western blotting, and qPCR, whereas tubular injury was evaluated in db/db mice. High-glucose (HG)-treated HK-2 cells were either treated with KIRA6 or transduced with a lentiviral vector for TMBIM6 overexpression. ER stress was measured by western blotting and ER-Tracker Red staining, whereas apoptosis was determined by performing TUNEL assays. Megalin expression was measured by immunofluorescence, and albumin endocytosis was evaluated after incubating cells with FITC-labeled albumin. Tubular injury and TMBIM6 downregulation occurred in db/db mouse renal cortical tissues. Both KIRA6 treatment and TMBIM6 overexpression inhibited ER stress by decreasing the levels of phosphorylated IRE1α, XBP1s, GRP78, and CHOP, and stabilizing ER expansion in HG-treated HK-2 cells. TUNEL assays performed with KIRA6-treated or TMBIM6-overexpressing cells showed a significant decrease in apoptosis, consistent with the significant downregulation of BAX and upregulation of BCL-2, as measured by immunoblotting. Both KIRA6 and TMBIM6 overexpression promoted megalin PM localization and restored albumin endocytosis in HG-treated HK-2 cells. CONCLUSION: TMBIM6 promoted diabetic PTEC survival and albumin endocytosis by negatively regulating the IRE1α branch of ER stress.

Effects of membrane androgen receptor binding on synaptic plasticity in primary hippocampal neurons.

Androgens play an important role in the regulation of hippocampal synaptic plasticity. While the classical molecular mechanism of androgen's genomic activity is their binding to intracellular androgen receptors (iARs), they can also induce rapid non-genomic effects through specific membrane androgen receptors (mARs). In this study, we aimed to localize and characterize these mARs in primary rat hippocampal neurons. Specific punctate fluorescent signals on the cell surface, observed by testosterone-fetal bovine serum albumin conjugated fluorescein isothiocyanate (T-BSA-FITC), indicated the presence of mARs in hippocampal neurons. T-BSA-FITC binding to the cell membrane was incompletely blocked by the iAR-antagonist flutamide, and mAR binding site was competitively bound by free testosterone (T). Most neurons expressing androgen membrane binding sites are glutamatergic (excitatory), although several are γ-aminobutyric acid (GABA)ergic (inhibitory). Confocal microscopy and live-cell imaging techniques were used to observe the real-time rapid effects of androgens on hippocampal dendritic spine morphology. Immunofluorescence cell staining was used to observe their effects on the postsynaptic density protein 95 (PSD95) and synapsin (SYN) synaptic markers. While androgens did not cause a short-term increase in dendritic spine density of rat primary hippocampal neurons, they promoted the transformation of dendritic spines from thin to mushroom, promoted dendritic spine maturation, increased dendritic spine surface area, and rapidly increased PSD95 and SYN expression in the primary hippocampal neurons. Hippocampal synaptosomes were prepared using the Optiprep and Percoll density gradient two-step centrifuge methods, and the gene expression profiles of the synaptosomes and hippocampus were compared using a gene chip; PSD95 mRNA expression was detected by reverse transcription-polymerase chain reaction. Several mRNAs were detected at the synaptic site, including PSD95. Finally, the Venus-PSD95 plasmid was constructed and transfected into HT22 cells, which is a mouse hippocampal neuronal cell line. The real-time effect of androgen on synaptic protein PSD95 was observed by fluorescence recovery after photobleaching experiments, which involved the translation process of PSD95 mRNA. In conclusion, our findings increased our understanding of how androgens exert the neuroprotective mechanisms on synaptic plasticity.

Intestinal Alkaline Phosphatase Prevents Sulfate Reducing Bacteria-Induced Increased Tight Junction Permeability by Inhibiting Snail Pathway.

Tight junctions (TJs) are essential components of intestinal barrier integrity and protect the epithelium against passive paracellular flux and microbial translocation. Dysfunctional TJ leads to leaky gut, a condition associated with diseases including inflammatory bowel disease (IBD). Sulfate-Reducing Bacteria (SRB) are minor residents of the gut. An increased number of Desulfovibrio, the most predominant SRB, is observed in IBD and other diseases associated with leaky gut. However, it is not known whether Desulfovibrio contributes to leaky gut. We tested the hypothesis that Desulfovibrio vulgaris (DSV) may induce intestinal permeability in vitro. Snail, a transcription factor, disrupts barrier function by affecting TJ proteins such as occludin. Intestinal alkaline phosphatase (IAP), a host defense protein, protects epithelial barrier integrity. We tested whether DSV induced permeability in polarized Caco-2 cells via snail and if this effect was inhibited by IAP. Barrier integrity was assessed by measuring transepithelial electric resistance (TEER) and by 4kDa FITC-Dextran flux to determine paracellular permeability. We found that DSV reduced TEER, increased FITC-flux, upregulated snail protein expression, caused nuclear translocation of snail, and disrupted occludin staining at the junctions. DSV-induced permeability effects were inhibited in cells knocked down for snail. Pre-treatment of cells with IAP inhibited DSV-induced FITC flux and snail expression and DSV-mediated disruption of occludin staining. These data show that DSV, a resident commensal bacterium, can contribute to leaky gut and that snail may serve as a novel therapeutic target to mitigate DSV-induced effects. Taken together, our study suggests a novel underlying mechanism of association of Desulfovibrio bloom with diseases with increased intestinal permeability. Our study also underscores IAP as a novel therapeutic intervention for correcting SRB-induced leaky gut via inhibition of snail.

Inhibition of Src but not Syk causes weak reversal of GPVI-mediated platelet aggregation measured by light transmission aggregometry.

Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton's tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear.

C-terminal mini-PEGylation of a marine peptide N6 had potent antibacterial and anti-inflammatory properties against Escherichia coli and Salmonella strains in vitro and in vivo.

BACKGROUND: Enteropathogenic Escherichia coli and Salmonella pullorum are two important groups of zoonotic pathogens. At present, the treatment of intestinal pathogenic bacteria infection mainly relies on antibiotics, which directly inhibit or kill the pathogenic bacteria. However, due to long-term irrational, excessive use or abuse, bacteria have developed different degrees of drug resistance. N6, an arenicin-3 derivative isolated from the lugworm, has potent antibacterial activity and is poorly resistant to enzymatic hydrolysis and distribution in vivo. Polyethylene glycol (PEG) is an extensively studied polymer and commonly used in protein or peptide drugs to improve their therapeutic potential. Here, we modified the N-/C-terminal or Cys residue of N6 with liner PEGn of different lengths (n = 2, 6,12, and 24), and the effects of PEGylation of N6 on the stability, toxicity, bactericidal mechanism, distribution and efficacy were investigated in vitro and in vivo. RESULTS: The antimicrobial activity of the peptide showed that PEGylated N6 at the C-terminus (n = 2, N6-COOH-miniPEG) had potent activity against Gram-negative bacteria; PEGylated N6 at the N-terminus and Cys residues showed low or no activity with increasing lengths of PEG. N6-COOH-miniPEG has higher stability in trypsin than the parent peptide-N6. N6-COOH-miniPEG significantly regulated cytokine expression in lipopolysaccharides (LPS)-induced RAW 264.7 cells, and the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1ß were reduced by 31.21%, 65.62% and 44.12%, respectively, lower than those of N6 (-0.06%, -12.36% and -12.73%); N6-COOH-miniPEG increased the level of IL-10 (37.83%), higher than N6 (-10.21%). The data indicated that N6-COOH-miniPEG has more potent anti-inflammatory and immune-regulatory effect than N6 in LPS-stimulated RAW 264.7 cells. N6-COOH-miniPEG exhibited a much wider biodistribution in mice and prolonged in vivo half-time. FITC-labeled N6-COOH-miniPEG was distributed throughout the body of mice in the range of 0.75 - 2 h after injection, while FITC-labeled N6 only concentrated in the abdominal cavity of mice after injection, and the distribution range was narrow. N6-COOH-miniPEG improved the survival rates of mice challenged with E. coli or S. pullorum, downregulated the levels of TNF-α, IL-6, IL-1ß and IL-10 in the serum of LPS-infected mice, and alleviated multiple-organ injuries (the liver, spleen, kidney, and lung), superior to antibiotics, but slightly inferior to N6. CONCLUSIONS: The antibacterial activity, bactericidal mechanism and cytotoxicity of N6-COOH-miniPEG and N6 were similar. N6-COOH-miniPEG has a higher resistance to trysin than N6. The distribution of N6-COOH-miniPEG in mice was superior to that of N6. In exploring the modulatory effects of antimicrobial peptides on cytokines, N6-COOH-miniPEG had stronger anti-inflammatory and immunomodulatory effects than N6. The results suggested that C-terminal PEGylated N6 may provide an opportunity for the development of effective anti-inflammatory and antibacterial peptides.

Mitochondrial-targeting antioxidant MitoQ modulates angiogenesis and promotes functional recovery after spinal cord injury.

BACKGROUND: In traumatic spinal cord injury (SCI), secondary injuries, including cellular death, mitochondrial dysfunction, and vascular injury, have been considered as important causes of impaired functional recovery after SCI. Postinjury angiogenesis has been considered to be a potential strategy for SCI treatment. New-born vessels may play a key role in nerve regeneration, which indicates the importance of angiogenesis in nerve regeneration. Recent studies have revealed the crosstalk between reactive oxygen species (ROS) and angiogenesis. As the main source of cellular ROS, mitochondria have been proven to be essential to the angiogenesis process. METHODS: SCI was established in a T10 clip-compression animal model. Then, the animals received an intraperitoneal injection of MitoQ (5 mg/kg/d) on Days 0, 1, and 2 after surgery. The Basso Mouse Scale (BMS) score and footprint analysis (CatWalk analysis) were performed to evaluate functional recovery after SCI. Immunofluorescence and fluorescence assays (LEL-FITC/CD31/Iba-1/Neurofilament) were performed to evaluate angiogenesis, microglia activation and neural regeneration. RT-qPCR (VEGFR-1, VEGFR-2 and VEGFA) was performed to evaluate angiogenesis-related factor in injured spinal cord. ATP production assay and western-blotting assay (Mfn-1 and Drp-1) were performed to evaluate mitochondrial function in the injured spinal cord. BV2 cells were used as in vitro cell model. After receiving TBHP or TBHP-MitoQ treatment, ELISA and immunofluorescence assays were used to evaluate the level of VEGFA secretion from BV2 cells. A coculture system of HUVECs and BV2 cells was established. Tube formation assays and immunofluorescence assays (CD31) were performed on HUVECs in a coculture system to evaluate angiogenesis promotion. ATP production assays were performed to evaluate mitochondrial function in BV2 cells. MitoSOX Red and DCFH-DA staining were performed to evaluate mitochondrial and cellular ROS. RESULTS: In vitro MitoQ promoted the secretion of VEGFA from BV2 cells, which was verified through ELISA and immunofluorescence assays. The angiogenic promotion of MitoQ-treated BV2 cells was evaluated by tube formation and immunofluorescence assays (CD31) in a coculture system of BV2 cells and HUVECs. MitoQ inhibited cellular and mitochondrial-derived ROS in TBHP-treated BV2 cells. ATP production was increased in MitoQ-treated BV2 cells. To verify MitoQ's effect in vivo, a T10 clip-compression animal model was established successfully. MitoQ significantly promoted functional recovery, as shown by the BMS assay and gait analysis. The promotion of neural regeneration was identified through immunofluorescence assay of neurofilament. Immunofluorescence and fluorescence assays (LEL-FITC/CD31/Iba-1) and RT-qPCR (VEGFR-1, VEGFR-2 and VEGFA) indicated that MitoQ could promote angiogenesis and inhibit macrophage/microglia activation in lesion-site after SCI. Enhanced ATP production and increased Mfn-1 with decreased Drp-1 protein expression showed MitoQ could promote mitochondrial function in SCI. CONCLUSION: The mitochondrial-specific antioxidant MitoQ promotes functional recovery and tissue preservation through the enhancement of angiogenesis with the modification of mitochondrial function after SCI.

Mesenchymal Stem-Cell Remodeling of Adsorbed Type-I Collagen-The Effect of Collagen Oxidation.

This study describes the effect of collagen type I (Col I) oxidation on its physiological remodeling by adipose tissue-derived mesenchymal stem cells (ADMSCs), both mechanical and proteolytic, as an in vitro model for the acute oxidative stress that may occur in vivo upon distinct environmental changes. Morphologically, remodeling was interpreted as the mechanical rearrangement of adsorbed FITC-labelled Col I into a fibril-like pattern. This process was strongly abrogated in cells cultured on oxidized Col I albeit without visible changes in cell morphology. Proteolytic activity was quantified utilizing fluorescence de-quenching (FRET effect). The presence of ADMSCs caused a significant increase in native FITC-Col I fluorescence, which was almost absent in the oxidized samples. Parallel studies in a cell-free system confirmed the enzymatic de-quenching of native FITC-Col I by Clostridial collagenase with statistically significant inhibition occurring in the oxidized samples. Structural changes to the oxidized Col I were further studied by differential scanning calorimetry. In the oxidized samples, an additional endotherm with sustained enthalpy (∆H) was observed at 33.6 °C along with Col I's typical one at 40.5 °C. Collectively, these data support that the remodeling of Col I by ADMSCs is altered upon oxidation due to intrinsic changes to the protein's structure, which represents a novel mechanism for the control of stem cell behavior.