Mouse spermatogenesis entails the maintenance and self-renewal of spermatogonial stem cells (SSCs), which require a complex web-like signaling network transduced by various cytokines. Although brain-derived neurotrophic factor (BDNF) is expressed in Sertoli cells in the testis, and its receptor tropomyosin receptor kinase B (TrkB) is expressed in the spermatogonial population containing SSCs, potential functions of BDNF for spermatogenesis have not been uncovered. Here, we generate BDNF conditional knockout mice and find that BDNF is dispensable for in vivo spermatogenesis and fertility. However, in vitro , we reveal that BDNF -deficient germline stem cells (GSCs) exhibit growth potential not only in the absence of glial cell line-derived neurotrophic factor (GDNF), a master regulator for GSC proliferation, but also in the absence of other factors, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin. GSCs grown without these factors are prone to differentiation, yet they maintain expression of promyelocytic leukemia zinc finger ( Plzf ), an undifferentiated spermatogonial marker. Inhibition of phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), and Src pathways all interfere with the growth of BDNF-deficient GSCs. Thus, our findings suggest a role for BDNF in maintaining the undifferentiated state of spermatogonia, particularly in situations where there is a shortage of growth factors.
Animals ; Male ; Brain-Derived Neurotrophic Factor/genetics* ; Spermatogonia/cytology* ; Mice ; Spermatogenesis/genetics* ; Mice, Knockout ; Cell Differentiation ; Glial Cell Line-Derived Neurotrophic Factor/genetics* ; Promyelocytic Leukemia Zinc Finger Protein/genetics* ; Cell Survival/physiology* ; Signal Transduction/physiology* ; Cell Proliferation/physiology*

Objective: This study aimed to use an air-liquid interface (ALI) exposure system to simulate the inhalation exposure of motorcycle exhaust particulates (MEPs) and then investigate the benchmark dose (BMD) of MEPs by evaluating cell relative viability (CRV) in lung epithelial BEAS-2B cells. Methods: The MEPs dose was characterized by measuring the number concentration (NC), surface area concentration (SAC), and mass concentration (MC). BEAS-2B cells were exposed to MEPs at different concentrations Results: Our results reveal that BMD of NC and SAC were estimated by the best-fitting Hill model, while MC was estimated by Polynomial model. The BMDL for CRV following ALI exposure to MEPs were as follows: 364.2#/cm Conclusion These results indicate that MEPs exposure
Benchmarking/statistics & numerical data* ; Bronchi/physiology* ; Cell Line ; Cell Survival/drug effects* ; Epithelial Cells/physiology* ; Humans ; Motorcycles ; Particulate Matter/adverse effects* ; Vehicle Emissions/analysis*

RNA helicases, the largest family of proteins that participate in RNA metabolism, stabilize the intracellular environment through various processes, such as translation and pre-RNA splicing. These proteins are also involved in some diseases, such as cancers and viral diseases. Autophagy, a self-digestive and cytoprotective trafficking process in which superfluous organelles and cellular garbage are degraded to stabilize the internal environment or maintain basic cellular survival, is associated with human diseases. Interestingly, similar to autophagy, RNA helicases play important roles in maintaining cellular homeostasis and are related to many types of diseases. According to recent studies, RNA helicases are closely related to autophagy, participate in regulating autophagy, or serve as a bridge between autophagy and other cellular activities that widely regulate some pathophysiological processes or the development and progression of diseases. Here, we summarize the most recent studies to understand how RNA helicases function as regulatory proteins and determine their association with autophagy in various diseases.
Animals ; Antiviral Agents/pharmacology* ; Autophagy ; Beclin-1/metabolism* ; Carcinogenesis ; Cell Survival ; DEAD Box Protein 58/metabolism* ; Disease Progression ; Gene Expression Regulation ; Homeostasis ; Humans ; Immune System/physiology* ; Neoplasms/metabolism* ; RNA Helicases/metabolism* ; RNA Splicing ; Receptors, Immunologic/metabolism*

OBJECTIVE: To investigate the effects of autophagy on osteogenic differentiation of stem cells from the apical papilla (SCAPs) in the presence of tumor necrosis factor- (TNF-) stimulation . METHODS: SCAPs treated with TNF- (0, 5, and 10 ng/mL) with or without 5 mmol/L 3-MA were examined for the expression of autophagy marker LC3-Ⅱ using Western blotting. The cells were transfected with GFP-LC3 plasmid and fluorescence microscopy was used for quantitative analysis of intracellular GFP-LC3; AO staining was used to detect the acidic vesicles in the cells. The cell viability was assessed with CCK-8 assays and the cell apoptosis rate was analyzed using flow cytometry. The cells treated with TNF- or with TNF- and 3-MA were cultured in osteogenic differentiation medium for 3 to 14 days, and real- time PCR was used to detect the mRNA expressions of osteogenesis-related genes (ALP, BSP, and OCN) for evaluating the cell differentiation. RESULTS: TNF- induced activation of autophagy in cultured SCAPs. Pharmacological inhibition of TNF--induced autophagy by 3-MA significantly decreased the cell viability and increased the apoptosis rate of SCAPs ( < 0.05). Compared with the cells treated with TNF- alone, the cells treated with both TNF- and 3-MA exhibited decreased expressions of the ALP and BSP mRNA on days 3, 7 and 14 during osteogenic induction ( < 0.05) and decreased expression of OCN mRNA on days 3 and 7 during the induction ( < 0.05). CONCLUSIONS Autophagy may play an important role during the osteogenic differentiation of SCAPs in the presence of TNF- stimulation.
Autophagy ; drug effects ; physiology ; Cell Differentiation ; drug effects ; physiology ; Cell Survival ; drug effects ; Cells, Cultured ; Dental Papilla ; cytology ; Green Fluorescent Proteins ; Humans ; Osteogenesis ; physiology ; Stem Cells ; drug effects ; physiology ; Transfection ; Tumor Necrosis Factor-alpha ; administration & dosage ; antagonists & inhibitors ; pharmacology

OBJECTIVE: To study the protective effect of enhanced peroxisome proliferator activated receptor γ (PPARγ) pathway against apoptosis of long-term cultured primary nerve cells. METHODS: A natural aging model was established in primary rat nerve cells by long-term culture for 22 days. The cells were divided into control group, 0.1, 1.0, 5.0, and 10 μmol/L GW9662 intervention groups, and 0.1, 1.0, 5.0, and 10 μmol/L pioglitazone intervention groups. The cell viability was assessed using MTT assay and the cell morphological changes were observed after the treatments to determine the optimal concentrations of GW9662 and pioglitazone. Double immunofluorescence labeling and flow cytometry were used to observe the changes in the number of viable cells and cell apoptosis following the treatments; immunocytochemical staining was used to assess the changes in the anti-oxidation ability of the treated cells. RESULTS: The optimal concentrations of GW9662 and pioglitazone determined based on the cell viability and morphological changes were both 1 μmol/L. Compared with the control group, GW9662 treatment significantly lowered while pioglitazone significantly increased the total cell number and nerve cell counts ( < 0.05), and nerve cells in the cell cultures maintained a constant ratio at about 80% in all the groups ( > 0.05). GW9662 significantly enhanced while pioglitazone significantly lowered the cell apoptosis rates compared with the control group ( < 0.05). GW9662 obviously lowered SOD activity and GSH content in G group ( < 0.05) and increased MDA content in the cells ( < 0.05), and pioglitazone resulted in reverse changes in SOD, GSH and MDA contents in the cells ( < 0.05). CONCLUSIONS Activation of PPARγ pathway protects long-term cultured primary nerve cells by enhancing cellular anti-oxidant capacity and reducing cell apoptosis, suggesting a potential strategy for anti-aging treatment of the nervous system through intervention of the PPARγ pathway.
Anilides ; administration & dosage ; pharmacology ; Animals ; Apoptosis ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Cellular Senescence ; physiology ; Neurons ; cytology ; PPAR gamma ; metabolism ; Pioglitazone ; administration & dosage ; pharmacology ; Rats

BACKGROUND: Serum antinuclear antibodies (ANAs) are positive in some patients with chronic lymphocytic leukemia (CLL), but the prognostic value of ANAs remains unknown. The aim of this study was to evaluate the role of ANAs as a prognostic factor in CLL. METHODS: This study retrospectively analyzed clinical data from 216 newly diagnosed CLL subjects with ANAs test from 2007 to 2017. Multivariate Cox regression analyses were used to screen the independent prognostic factors related to time to first treatment (TTFT), progression free survival (PFS) and overall survival (OS). Receiver operator characteristic curves and area under the curve (AUC) were utilized to assess the predictive accuracy of ANAs together with other independent factors for OS. RESULTS: The incidence of ANAs abnormality at diagnosis was 13.9%. ANAs positivity and TP53 disruption were independent prognostic indicators for OS. The AUC of positive ANAs together with TP53 disruption was 0.766 (95% confidence interval [CI]: 0.697-0.826), which was significantly larger than that of either TP53 disruption (AUC: 0.706, 95% CI: 0.634-0.772, P = 0.034) or positive ANAs (AUC: 0.595, 95% CI: 0.520-0.668, P < 0.001) in OS prediction. Besides, serum positive ANAs as one additional parameter to CLL-international prognostic index (IPI) obtained superior AUCs in predicting CLL OS than CLL-IPI alone. CONCLUSION This study identified ANAs as an independent prognostic factor for CLL, and further investigations are needed to validate this finding.
ADP-ribosyl Cyclase 1 ; blood ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Antinuclear ; blood ; Autoimmunity ; physiology ; Female ; Humans ; Kaplan-Meier Estimate ; Leukemia, Lymphocytic, Chronic, B-Cell ; blood ; mortality ; Male ; Middle Aged ; Multivariate Analysis ; Mutation ; genetics ; Proportional Hazards Models ; Retrospective Studies ; Survival Analysis ; Tumor Suppressor Protein p53 ; blood ; Young Adult ; ZAP-70 Protein-Tyrosine Kinase ; blood

OBJECTIVE: To investigate the effects of Ganoderma lucidum polysaccharides (GL-PS) on human fibroblasts and skin wound healing in Kunming male mice and to explore the putative molecular mechanism. METHODS: Primary human skin fibroblasts were cultured. The viability of fibroblasts treated with 0, 10, 20, 40, 80, and 160 μg/mL of GL-PS, respectively were detected by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide (MTT). The migration ability of fibroblasts treated with 0, 10, 20, and 40 μg/mL of GL-PS were measured by transwell assay. The secretion of the C-terminal peptide of procollagen type I (CICP) and transforming growth factor-β1 (TGF-β1) in the cell supernatant was tested by enzyme-linked immunosorbent assay. The expression of β-catenin was detected by Western blot. Furthermore, the Kunming mouse model with full-layer skin resection trauma was established, and was treated with 10, 20, and 40 mg/mL of GL-PS, respectively as external use. The size of the wound was measured daily, complete healing time in each group was recorded and the percentage of wound contraction was calculated. RESULTS: Compared with the control group, 10, 20, and 40 μg/mL of GL-PS significantly increased the viability of fibroblasts, promoted the migration ability of fibroblasts, and up-regulated the expressions of CICP and TGF-β1 in fibroblasts (Plt;0.05 or Plt;0.01). The expression of β-catenin in fibroblasts treated with 20 and 40 μg/mL of GL-PS was significantly higher than that of the control group (Plt;0.01). Furthermore, after external use of 10, 20, and 40 mg/mL of GL-PS, the rates of wound healing in mice were significantly higher and the wound healing time was significantly less than the control group (Plt;0.05 or Plt;0.01). CONCLUSION A certain concentration of GL-PS may promote wound healing via activation of the Wnt/β-catenin signaling pathway and up-regulation of TGF-β1, which might serve as a promising source of skin wound healing.
Animals ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Fibroblasts ; drug effects ; Humans ; Male ; Mice ; Polysaccharides ; pharmacology ; Reishi ; chemistry ; Skin ; drug effects ; injuries ; Transforming Growth Factor beta1 ; physiology ; Wound Healing ; drug effects ; beta Catenin ; physiology

The present study was designed to analyze the metabolites of all-trans-retinal (atRal) and compare the cytotoxicity of atRal versus its derivative all-trans-retinoic acid (atRA) in human retinal pigment epithelial (RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol (atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 μmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful (91 vs. 29 pmol/mL), suggesting that atRA conversion facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species (ROS) overproduction, heme oxygenase-1 (HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1 (PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4^-/-RDH8^-/- mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4^-/-RDH8^-/- mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.
ATP-Binding Cassette Transporters/physiology* ; Alcohol Oxidoreductases/physiology* ; Animals ; Cell Survival/drug effects* ; Cells, Cultured ; Humans ; Inactivation, Metabolic ; Mice ; Retina/metabolism* ; Retinal Pigment Epithelium/metabolism* ; Swine ; Tretinoin/pharmacology*

OBJECTIVEThe current study aims to investigate the effect of Hemagglutinating virus of Japan envelope (HVJ-E) on induction of apoptosis and autophagy in human prostate cancer PC3 cells, and the underlying mechanisms.

METHODSPC3 cells were treated with HVJ-E at various multiplicity of infection (MOI), and the generated reactive oxygen species (ROS), cell viability, apoptosis, and autophagy were detected, respectively. Next, the role of ROS played in the regulation of HVJ-E-induced apoptosis and autuphagy in PC3 cells were analysed. In the end, the relationship between HVJ-E-induced apoptosis and autuophagy was investigated by using rapamycin and chloroquine.

RESULTSFlow cytometry assay revealed that HVJ-E treatment induced dose-dependent apoptosis and that the JNK and p38 MAPK signaling pathways were involved in HVJ-E-induced apoptosis in PC3 cells. In addition, HVJ-E was able to induce autophagy in PC3 cells via the class III PI3K/beclin-1 pathway. The data also implyed that HVJ-E-triggered autophagy and apoptosis were ROS dependent. When ROS was blocked with N-acetylcysteine (NAC), HVJ-E-induced LC3-II conversion and apoptosis were reversed. Interestingly, HVJ-E-induced apoptosis was significantly increased by an inducer of autophagy, rapamycin pretreatment, both in vitro and in vivo.

CONCLUSIONHVJ-E exerts anticancer effects via autophagic cell death in prostate cancer cells.

Apoptosis ; physiology ; Autophagy ; physiology ; Cell Line, Tumor ; Cell Survival ; Humans ; Male ; Oncolytic Virotherapy ; Prostatic Neoplasms ; metabolism ; Reactive Oxygen Species ; metabolism ; Sendai virus ; immunology ; physiology ; Virus Inactivation

OBJECTIVE: To explore the role of mitochondrial permeability transition pore (mPTP) in mediating the protective effect of gastrodin against oxidative stress damage in H9c2 cardiac myocytes. METHODS: H9c2 cardiac myocytes were treated with HO, gastrodin, gastrodin+HO, cyclosporin A (CsA), or CsA+gas+HO group. MTT assay was used to detect the survival ratio of H9c2 cells, and flow cytometry with Annexin V-FITC/PI double staining was used to analyze the early apoptosis rate after the treatments. The concentration of ATP and level of reactive oxygen species (ROS) in the cells were detected using commercial kits. The mitochondrial membrane potential of the cells was detected with laser confocal microscopy. The expression of cytochrome C was detected with Western blotting, and the activity of caspase-3 was also assessed in the cells. RESULTS: Gastrodin pretreatment could prevent oxidative stress-induced reduction of mitochondrial membrane potential, and this effect was inhibited by the application of CsA. Gastrodin significantly lowered the levels of ROS and apoptosis-related factors in HO-exposed cells, and such effects were reversed by CsA. CsA significantly antagonized the protective effect of gastrodin against apoptosis in HO-exposed cells. CONCLUSIONS Gastrodin prevents oxidative stress-induced injury in H9c2 cells by inhibiting mPTP opening to reduce the cell apoptosis.
Adenosine Triphosphate ; analysis ; Apoptosis ; drug effects ; Benzyl Alcohols ; antagonists & inhibitors ; pharmacology ; Caspase 3 ; analysis ; Cell Line ; Cell Survival ; drug effects ; Cyclosporine ; pharmacology ; Cytochromes c ; analysis ; Glucosides ; antagonists & inhibitors ; pharmacology ; Humans ; Hydrogen Peroxide ; antagonists & inhibitors ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondrial Membrane Transport Proteins ; physiology ; Myocytes, Cardiac ; drug effects ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; analysis