Male meiosis is a complex process whereby spermatocytes undergo cell division to form haploid cells. This review focuses on the role of retinoic acid (RA) in meiosis, as well as several processes regulated by RA before cell entry into meiosis that are critical for proper meiotic entry and completion. Here, we discuss RA metabolism in the testis as well as the roles of stimulated by retinoic acid gene 8 (STRA8) and MEIOSIN, which are responsive to RA and are critical for meiosis. We assert that transcriptional regulation in the spermatogonia is critical for successful meiosis.
Animals ; Cell Differentiation/genetics* ; Humans ; Meiosis/drug effects* ; Spermatogenesis/physiology* ; Tretinoin/metabolism*

As a member of the AFF (AF4/FMR2) family, AFF4 is a transcription elongation factor that is a component of the super elongation complex. AFF4 serves as a scaffolding protein that connects transcription factors and promotes gene transcription through elongation and chromatin remodelling. Here, we investigated the effect of AFF4 on human dental follicle cells (DFCs) in osteogenic differentiation. In this study, we found that small interfering RNA-mediated depletion of AFF4 resulted in decreased alkaline phosphatase (ALP) activity and impaired mineralization. In addition, the expression of osteogenic-related genes (DLX5, SP7, RUNX2 and BGLAP) was significantly downregulated. In contrast, lentivirus-mediated overexpression of AFF4 significantly enhanced the osteogenic potential of human DFCs. Mechanistically, we found that both the mRNA and protein levels of ALKBH1, a critical regulator of epigenetics, changed in accordance with AFF4 expression levels. Overexpression of ALKBH1 in AFF4-depleted DFCs partially rescued the impairment of osteogenic differentiation. Our data indicated that AFF4 promoted the osteogenic differentiation of DFCs by upregulating the transcription of ALKBH1.
Biomarkers ; metabolism ; Cell Differentiation ; Cells, Cultured ; Dental Sac ; drug effects ; metabolism ; Gene Expression Regulation ; Humans ; Osteogenesis ; genetics ; Repressor Proteins ; Transcription Factors ; genetics ; metabolism ; Transcriptional Elongation Factors ; metabolism

Once pulp necrosis or apical periodontitis occurs on immature teeth, the weak root and open root apex are challenging to clinicians. Berberine (BBR) is a potential medicine for bone disorders, therefore, we proposed to apply BBR in root canals to enhance root repair in immature teeth. An in vivo model of immature teeth with apical periodontitis was established in rats, and root canals were filled with BBR, calcium hydroxide or sterilized saline for 3 weeks. The shape of the roots was analyzed by micro-computed tomography and histological staining. In vitro, BBR was introduced into stem cells from apical papilla (SCAPs). Osteogenic differentiation of stem cells from apical papilla was investigated by alkaline phosphatase activity, mineralization ability, and gene expression of osteogenic makers. The signaling pathway, which regulated the osteogenesis of SCAPs was evaluated by quantitative real time PCR, Western blot analysis, and immunofluorescence. In rats treated with BBR, more tissue was formed, with longer roots, thicker root walls, and smaller apex diameters. In addition, we found that BBR promoted SCAPs osteogenesis in a time-dependent and concentration-dependent manner. BBR induced the expression of β-catenin and enhanced β-catenin entering into the nucleus, to up-regulate more runt-related nuclear factor 2 downstream. BBR enhanced root repair in immature teeth with apical periodontitis by activating the canonical Wnt/β-catenin pathway in SCAPs.
Animals ; Berberine ; pharmacology ; Cell Differentiation ; drug effects ; Dental Papilla ; Male ; Osteogenesis ; drug effects ; Periapical Periodontitis ; therapy ; Rats ; Stem Cells ; cytology ; drug effects ; metabolism ; Wnt Signaling Pathway ; drug effects ; Wnt3A Protein ; genetics ; metabolism ; X-Ray Microtomography

OBJECTIVE: We aimed to explore how fermented barley extracts with Lactobacillus plantarum dy-1 (LFBE) affected the browning in adipocytes and obese rats. METHODS: In vitro, 3T3-L1 cells were induced by LFBE, raw barley extraction (RBE) and polyphenol compounds (PC) from LFBE to evaluate the adipocyte differentiation. In vivo, obese SD rats induced by high fat diet (HFD) were randomly divided into three groups treated with oral gavage: (a) normal control diet with distilled water, (b) HFD with distilled water, (c) HFD with 800 mg LFBE/kg body weight (bw). RESULTS: In vitro, LFBE and the PC in the extraction significantly inhibited adipogenesis and potentiated browning of 3T3-L1 preadipocytes, rather than RBE. In vivo, we observed remarkable decreases in the body weight, serum lipid levels, white adipose tissue (WAT) weights and cell sizes of brown adipose tissues (BAT) in the LFBE group after 10 weeks. LFBE group could gain more mass of interscapular BAT (IBAT) and promote the dehydrogenase activity in the mitochondria. And LFBE may potentiate process of the IBAT thermogenesis and epididymis adipose tissue (EAT) browning via activating the uncoupling protein 1 (UCP1)-dependent mechanism to suppress the obesity. CONCLUSION These results demonstrated that LFBE decreased obesity partly by increasing the BAT mass and the energy expenditure by activating BAT thermogenesis and WAT browning in a UCP1-dependent mechanism.
3T3 Cells ; Adipocytes ; drug effects ; physiology ; Adipose Tissue, Brown ; drug effects ; physiology ; Adipose Tissue, White ; drug effects ; physiology ; Animal Feed ; analysis ; Animals ; Anti-Obesity Agents ; administration & dosage ; metabolism ; Cell Differentiation ; drug effects ; Diet ; Fermentation ; Hordeum ; chemistry ; Lactobacillus plantarum ; chemistry ; Male ; Mice ; Obesity ; drug therapy ; genetics ; Plant Extracts ; chemistry ; Probiotics ; administration & dosage ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Uncoupling Protein 1 ; genetics ; metabolism

To explore the effect of down-regulation of growth arrest and DNA damage inducible protein 45β (GADD45β) on the PC9 lung adenocarcinoma cells.
 Methods: GADD45β gene siRNA sequence was designed and synthesized, which was transfected into PC9 lung adenocarcinoma cells through lentivirus transfection. Quantitative real-time PCR (qRT-PCR) and Western blot are used to examine the mRNA and protein levels of GADD45β in PC9 cells before and after the transfection. Annexin V-allophycocyanin (APC) double-staining flow cytometry was used to detect the apoptosis level after the transfection. The intracellular DNA content after transfection was detected by flow cytometry. The percentage of the cells at each period of cell cycle was calculated, and the effect of RNA interference on the cell growth were analyzed. The effects of RNA interference on the tumor-formation ability of cells were tested by counting the number of clones. MTT assay was used to test the half maximal inhibitory concentration (IC50) of PC9 cells for gefitinib. 
 Results: The 5'-AAATCCACTTCACGCTCAT-3' sequence was identified as the effective sequence for GADD45β gene RNA interference. The mRNA and protein expression levels of GADD45β were markedly decreased (both P<0.05) at 48 h after transfection of GADD45β-siRNA, which resulted in the increased apoptosis rate (P<0.05), decreased tumor clone number (P<0.05) and increased percentage of PC9 cell at the S stage and G2/M stage (P<0.05). The IC50 for gefitinib was decreased obviously (P<0.05).
 Conclusion: Down-regulation of GADD45β can reduce the colony-forming ability of PC9 cells, promote the cell apoptosis, and enhance the sensitivity of PC9 cells to gefitinib.
Adenocarcinoma of Lung ; Antigens, Differentiation ; genetics ; metabolism ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Gefitinib ; pharmacology ; Humans ; RNA, Small Interfering

Oligodendrocytes (OLs) are myelinating glial cells that form myelin sheaths around axons to ensure rapid and focal conduction of action potentials. Here, we found that an axonal outgrowth regulatory molecule, AATYK (apoptosis-associated tyrosine kinase), was up-regulated with OL differentiation and remyelination. We therefore studied its role in OL differentiation. The results showed that AATYK knockdown inhibited OL differentiation and the expression of myelin genes in vitro. Moreover, AATYK-deficiency maintained the proliferation status of OLs but did not affect their survival. Thus, AATYK is essential for the differentiation of OLs.
Animals ; Animals, Newborn ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Cell Differentiation ; drug effects ; physiology ; Cell Proliferation ; drug effects ; genetics ; Cells, Cultured ; Cuprizone ; toxicity ; Demyelinating Diseases ; chemically induced ; metabolism ; pathology ; Embryo, Mammalian ; Gene Expression Regulation, Developmental ; genetics ; Ki-67 Antigen ; metabolism ; Mice ; Mice, Inbred C57BL ; Myelin Basic Protein ; metabolism ; Myelin Proteolipid Protein ; metabolism ; Myelin Sheath ; drug effects ; metabolism ; Oligodendroglia ; drug effects ; metabolism ; Protein-Tyrosine Kinases ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley

Bromodomain-containing 4 (BRD4) has been considered as an important requirement for disease maintenance and an attractive therapeutic target for cancer therapy. This protein can be targeted by JQ1, a selective small-molecule inhibitor. However, few studies have investigated whether BRD4 influenced acute promyelocytic leukemia (APL), and whether BRD4 had interaction with promyelocytic leukemia-retinoic acid receptor α (PML/RARα) fusion protein to some extent. Results from cell viability assay, cell cycle analysis, and Annexin-V/PI analysis indicated that JQ1 inhibited the growth of NB4 cells, an APL-derived cell line, and induced NB4 cell cycle arrest at G1 and apoptosis. Then, we used co-immunoprecipitation (co-IP) assay and immunoblot to demonstrate the endogenous interaction of BRD4 and PML/RARα in NB4 cells. Moreover, downregulation of PML/RARα at the mRNA and protein levels was observed upon JQ1 treatment. Furthermore, results from the RT-qPCR, ChIP-qPCR, and re-ChIP-qPCR assays showed that BRD4 and PML/RARα co-existed on the same regulatory regions of their target genes. Hence, we showed a new discovery of the interaction of BRD4 and PML/RARα, as well as the decline of PML/RARα expression, under JQ1 treatment.
Apoptosis ; drug effects ; Azepines ; pharmacology ; Cell Differentiation ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Leukemia, Promyelocytic, Acute ; drug therapy ; genetics ; Nuclear Proteins ; genetics ; Promyelocytic Leukemia Protein ; genetics ; RNA, Messenger ; genetics ; Retinoic Acid Receptor alpha ; genetics ; Transcription Factors ; genetics ; Triazoles ; pharmacology ; Tumor Cells, Cultured

OBJECTIVE: To construct a novel non-viral vector loaded with growth and differentiation factor-5 (GDF-5) plasmid using chitosan, hyaluronic acid, and chondroitin sulfate for osteoarthritis (OA) gene therapy. METHODS: Nano-microspheres (NMPs) were prepared by mixing chitosan, hyaluronic acid, and chondroitin sulfate. GDF-5 plasmid was encapsulated in the NMPs through electrostatic adsorption. The basic characteristics of the NMPs were observed, and then they were co-cultured with chondrocytes to observe their effects on extracellular matrix (ECM) protein expression. Finally, NMPs loaded with GDF-5 were injected into the articular cavities of rabbits to observe their therapeutic effects on OA in vivo. RESULTS: NMPs exhibited good physicochemical properties and low cytotoxicity. Their average diameter was (0.61±0.20) μm, and encapsulation efficiency was (38.19±0.36)%. According to Cell Counting Kit-8 (CCK-8) assay, relative cell viability was 75%-99% when the total weight of NMPs was less than 560 μg. Transfection efficiency was (62.0±2.1)% in a liposome group, and (60.0±1.8)% in the NMP group. There was no significant difference between the two groups (P>0.05). Immunohistochemical staining results suggested that NMPs can successfully transfect chondrocytes and stimulate ECM protein expression in vitro. Compared with the control groups, the NMP group significantly promoted the expression of chondrocyte ECM in vivo (P<0.05), as shown by analysis of the biochemical composition of chondrocyte ECM. When NMPs were injected into OA model rabbits, the expression of ECM proteins in chondrocytes was significantly promoted and the progression of OA was slowed down. CONCLUSIONS Based on these data, we think that these NMPs with excellent physicochemical and biological properties could be promising non-viral vectors for OA gene therapy.
Animals ; Cell Differentiation ; Cell Survival/drug effects* ; Chitosan/chemistry* ; Chondrocytes/cytology* ; Chondroitin Sulfates/chemistry* ; Drug Carriers ; Extracellular Matrix/metabolism* ; Genetic Therapy/methods* ; Growth Differentiation Factor 5/genetics* ; Hyaluronic Acid/chemistry* ; Microspheres ; Nanomedicine ; Osteoarthritis/therapy* ; Plasmids/metabolism* ; Rabbits

OBJECTIVETo further investigate the {ptin vitro} effects of an osteoprotective herbal formula "ELP" (Herba Epimedii, Fructus Ligustri Lucidi and Fructus Psoraleae) using seropharmacological approach.

METHODSRats were fed with ELP or its individual component herbs for 2 days. The serum containing the postabsorbed ingredients of the herbal items were collected for cell culture using UMR106 cell, RAW264.7 cell and mesenchymal stem cell (MSC) isolated from the bone marrow of the rats. The effects of the herbal-containing serum on cell toxicity were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay; bromodeoxyuridine assay was conducted to measure the cell proliferation of UMR106 cell and MSC; cell activity was measured using colorimetric method, and mRNA expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteopontin (OPN) of UMR106 and MSC as well as matrix metalloproteinase 9 (MMP-9), tartrate-resistant acid phosphatase (TRAP) and cathepsin K of RAW264.7 were analyzed using real-time reverse-transcription polymerase chain reaction.

RESULTSELP and its component serum exhibited no cytotoxic effects on the cells. The ELP-containing serum increased the proliferation of UMR106 cell and MSC by 25.7% and 14.4 %, respectively and the alkaline phosphatase activity of MSC was increased by 42.6%. On the contrary, it inhibited the RAW264.7 cell differentiation by 29.2 %. ELP serum upregulated the Runx2 expression of UMR and MSC by 1.18 fold and 1.27 fold, respectively. It also upregulated ALP and OPN expression in MSC by 1.69- and 2.12-fold, respectively. On the other hand, ELP serum down-regulated MMP-9 and cathepsin K expression of RAW264.7 cell by 0.46- and 0.36-fold, respectively.

CONCLUSIONSThe serum of the animals fed with ELP contains active ingredients which are effective in promoting osteogenesis and inhibiting osteoclastogenesis.

Absorption, Physiological ; drug effects ; Animals ; Bone and Bones ; drug effects ; pathology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Male ; Mice ; Osteoclasts ; drug effects ; metabolism ; pathology ; Osteogenesis ; drug effects ; Protective Agents ; pharmacology ; RAW 264.7 Cells ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Serum ; metabolism

Cancer stem cells (CSCs) have tumor initiation, self-renewal, metastasis and chemo-resistance properties in various tumors including colorectal cancer. Targeting of CSCs may be essential to prevent relapse of tumors after chemotherapy. Phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signals are central regulators of cell growth, proliferation, differentiation, and apoptosis. These pathways are related to colorectal tumorigenesis. This study focused on PI3K and mTOR pathways by inhibition which initiate differentiation of SW620 derived CSCs and investigated its effect on tumor progression. By using rapamycin, LY294002, and NVP-BEZ235, respectively, PI3K and mTOR signals were blocked independently or dually in colorectal CSCs. Colorectal CSCs gained their differentiation property and lost their stemness properties most significantly in dual-blocked CSCs. After treated with anti-cancer drug (paclitaxel) on the differentiated CSCs cell viability, self-renewal ability and differentiation status were analyzed. As a result dual-blocking group has most enhanced sensitivity for anti-cancer drug. Xenograft tumorigenesis assay by using immunodeficiency mice also shows that dual-inhibited group more effectively increased drug sensitivity and suppressed tumor growth compared to single-inhibited groups. Therefore it could have potent anti-cancer effects that dual-blocking of PI3K and mTOR induces differentiation and improves chemotherapeutic effects on SW620 human colorectal CSCs.
AC133 Antigen/genetics/metabolism ; Animals ; Antineoplastic Agents/pharmacology/therapeutic use ; Cell Differentiation/*drug effects ; Cell Line, Tumor ; Cell Survival/drug effects ; Chromones/pharmacology/therapeutic use ; Colorectal Neoplasms/drug therapy/metabolism/pathology ; Humans ; Imidazoles/pharmacology/therapeutic use ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Morpholines/pharmacology/therapeutic use ; Neoplastic Stem Cells/cytology/drug effects/metabolism ; Paclitaxel/pharmacology/therapeutic use ; Phosphatidylinositol 3-Kinases/*antagonists & inhibitors/metabolism ; Quinolines/pharmacology/therapeutic use ; SOXB1 Transcription Factors/genetics/metabolism ; Signal Transduction/*drug effects ; Sirolimus/pharmacology/therapeutic use ; TOR Serine-Threonine Kinases/*antagonists & inhibitors/metabolism ; Xenograft Model Antitumor Assays