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 investigate the effects of Shengmai Injection (, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2 (NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. METHODS: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI (0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of SMI on different cell growth states (cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% conflfluence, apoptosis was detected by flflow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. RESULTS: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/mL of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment (P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h (P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h (P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL (P<0.05). CONCLUSION The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI (2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Hepatic Stellate Cells ; drug effects ; physiology ; Humans ; Injections ; Liver Cirrhosis ; drug therapy ; Tumor Suppressor Proteins ; genetics

Activated nuclear factor-κB (NF-κB) plays an important role in the development of cardiovascular disease (CVD) through its regulated genes and microRNAs (miRNAs). However, the gene regulation profile remains unclear. In this study, primary mouse vascular endothelial cells (pMVECs) were employed to detect CVD-related NF-κB-regulated genes and miRNAs. Genechip assay identified 77 NF-κB-regulated genes, including 45 upregulated and 32 downregulated genes, in tumor necrosis factor α (TNFα)-treated pMVECs. Ten of these genes were also found to be regulated by NF-κB in TNFα-treated HeLa cells. Quantitative real-time PCR (qRT-PCR) assay confirmed the up-regulation of Egr1, Tnf, and Btg2 by NF-κB in the TNFα-treated pMVECs. The functional annotation revealed that many NF-κB-regulated genes identified in pMVECs were clustered into classical NF-κB-involved biological processes. Genechip assay also identified 26 NF-κB-regulated miRNAs, of which 21 were upregulated and 5 downregulated, in the TNFα-treated pMVECs. Further analysis showed that nine of the identified genes are regulated by seven of these miRNAs. Finally, among the identified NF-κB-regulated genes and miRNAs, 5 genes and 12 miRNAs were associated with CVD by miRWalk and genetic association database analysis. Taken together, these findings show an intricate gene regulation network raised by NF-κB in TNFα-treated pMVECs. The network provides new insights for understanding the molecular mechanism underlying the progression of CVD.
Animals ; Cardiovascular Diseases/genetics* ; Cells, Cultured ; Endothelial Cells/drug effects* ; Gene Regulatory Networks ; Mice ; MicroRNAs/physiology* ; NF-kappa B/physiology* ; Tumor Necrosis Factor-alpha/pharmacology*

OBJECTIVE: To investigate the expression of miR-33b in hepatocellular carcinoma (HCC) and to explore regulatory mechanism of miR-33b for cell proliferation of HCC.
 METHODS: HCC tissues and adjacent non-tumor tissues were collected for this study (n=32 for each). Real-time PCR and Western blot were conducted to examine the mRNA and protein expression, respectively. MTT assay was used to detect the cell proliferation. Luciferase reporter gene assay was performed to verify the target relationship between miR-33b and Sal-like 4 (SALL4).
 RESULTS: MiR-33b was significantly downregulated in HCC tissues compared with adjacent non-tumor tissues. Overexpression of miR-33b decreased the proliferation of HCC LH86 cells. SALL4 was identified as a target gene of miR-33b, and its protein expression was negatively regulated by miR-33b. Overexpression of SALL4 reversed the suppressive effect of miR-33b on LH86 cell proliferation. SALL4 was significantly upregulated in HCC tissues compared with adjacent non-tumor tissues.
 CONCLUSION The miR-33b suppresses HCC cell proliferation through down-regulation of SALL4.
Carcinoma, Hepatocellular ; chemistry ; genetics ; physiopathology ; Cell Proliferation ; genetics ; physiology ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; genetics ; physiology ; Humans ; Liver Neoplasms ; MicroRNAs ; analysis ; genetics ; physiology ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Transcription Factors ; genetics ; physiology ; Tumor Cells, Cultured ; Up-Regulation

To explore the role of the special AT rich sequence binding protein-1 (SATB1) and ribonucleotide reductase M2 (RRM2) in enhancing malignant progression of non-Hodgkin lymphoma (NHL). 
 Methods: A total of 42 NHL and 42 chronic lymphadenitis patients were recruited. The protein expressions of SATB1 and RRM2 in cervical lymph nodes were determined by Western blot. After overexpression of SATB1, siSATB1 or siRRM2, the mRNA levels of SATB1 and RRM2 in cells were analyzed via RT-PCR, the cell proliferation was evaluated via MTT and EdU assays, while the migration and invasion of cells were assessed by transwell assays.
 Results: Compared with chronic lymphadenitis, the expressions of SATB1 and RRM2 in NHL patients were up-regulated. There was positive correlation between SATB1 and RRM2 in NHL patients. RRM2 mRNA level was up-regulated after transfection of SATB1 and down-regulated after transfection of siSATB1. Overexpression of SATB1 increased tumor cell proliferation, migration and invasion, while knockdown of RRM2 reversed those phenomena.
 Conclusion: SATB1 functions as an oncogene and promotes tumor cell proliferation, migration and invasion by up-regulation of RRM2 in NHL.
Cell Cycle Proteins ; genetics ; physiology ; Cell Movement ; genetics ; Cell Proliferation ; genetics ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; genetics ; Humans ; Lymph Nodes ; chemistry ; Lymphoma, Non-Hodgkin ; Matrix Attachment Region Binding Proteins ; genetics ; physiology ; Neoplasm Invasiveness ; genetics ; Oncogenes ; genetics ; physiology ; RNA, Messenger ; RNA, Small Interfering ; Ribonucleoside Diphosphate Reductase ; Ribonucleotide Reductases ; genetics ; physiology ; Signal Transduction ; Transcription Factors ; Transcriptional Activation ; Transfection ; Tumor Cells, Cultured ; Up-Regulation

Wnt3a, one of Wnt family members, plays key roles in regulating pleiotropic cellular functions, including self-renewal, proliferation, differentiation, and motility. Accumulating evidence has suggested that Wnt3a promotes or suppresses tumor progression via the canonical Wnt signaling pathway depending on cancer type. In addition, the roles of Wnt3a signaling can be inhibited by multiple proteins or chemicals. Herein, we summarize the latest findings on Wnt3a as an important therapeutic target in cancer.
Cell Division ; physiology ; Gene Expression Regulation, Neoplastic ; physiology ; Humans ; Neoplasm Proteins ; metabolism ; physiology ; Neoplasms ; genetics ; metabolism ; pathology ; Tumor Cells, Cultured ; Wnt Signaling Pathway ; physiology ; Wnt3A Protein ; metabolism ; physiology

OBJECTIVETo investigate the effects of prostate stromal cells from different zones of normal prostate tissue on the growth of prostate cancer cells and their action mechanisms.

METHODSWe extracted stromal cells in the fresh normal prostatic tissue derived from the peripheral zone (PZ) or transitional zone (TZ), amplified them in vitro, and used the supernatants of the cells as conditioned media to culture hormone-resistant prostate cancer DU145 cells. We measured the growth curve of the tumor cells using the CCK8 method, determined the number and viability of the cells by trypan blue staining, evaluated their invasiveness by scratch test, and detected the effects of the stromal cells on the key enzymes in the glycolysis of the tumor cells by Western blot.

RESULTSThe conditioned medium with the PZ-derived stromal cells promoted, while that with the TZ-derived stromal cells inhibited the growth of the tumor cells. The former significantly increased, while the latter markedly decreased the expressions of the key enzymes hexokinase 2 (HK-2), pyruvate kinase 2 (PKM-2), lactate dehydrogenase (LDHA), and pyruvate dehydrogenase (PDH) in the glycolysis of the tumor cells.

CONCLUSIONProstate stromal cells from different zones exert different influences on the growth of tumor cells, which may be associated with their different effects on the glycolysis of tumor cells.

Blotting, Western ; Cell Culture Techniques ; Cell Proliferation ; Culture Media, Conditioned ; Glycolysis ; Humans ; Male ; Prostate ; cytology ; Prostatic Neoplasms ; pathology ; Stromal Cells ; physiology ; Tumor Cells, Cultured

Chronic neuroinflammation is an integral pathological feature of major neurodegenerative diseases. The recruitment of microglia to affected brain regions and the activation of these cells are the major events leading to disease-associated neuroinflammation. In a previous study, we showed that neuron-released alpha-synuclein can activate microglia through activating the Toll-like receptor 2 (TLR2) pathway, resulting in proinflammatory responses. However, it is not clear whether other signaling pathways are involved in the migration and activation of microglia in response to neuron-released alpha-synuclein. In the current study, we demonstrated that TLR2 activation is not sufficient for all of the changes manifested by microglia in response to neuron-released alpha-synuclein. Specifically, the migration of and morphological changes in microglia, triggered by neuron-released alpha-synuclein, did not require the activation of TLR2, whereas increased proliferation and production of cytokines were strictly under the control of TLR2. Construction of a hypothetical signaling network using computational tools and experimental validation with various peptide inhibitors showed that beta1-integrin was necessary for both the morphological changes and the migration. However, neither proliferation nor cytokine production by microglia was dependent on the activation of beta1-integrin. These results suggest that beta1-integrin signaling is specifically responsible for the recruitment of microglia to the disease-affected brain regions, where neurons most likely release relatively high levels of alpha-synuclein.
Animals ; Antigens, CD29/genetics/*metabolism ; Cell Line, Tumor ; *Cell Movement ; Cells, Cultured ; Culture Media, Conditioned/*pharmacology ; Gene Regulatory Networks ; Humans ; Mice ; Mice, Inbred C57BL ; Microglia/drug effects/metabolism/*physiology ; Neurons/*metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 2/metabolism ; alpha-Synuclein/*pharmacology

BACKGROUNDMitochondrial dysfunction has been reported to be one of the contributing factors of sepsis-associated encephalopathy (SAE). Mitochondrial biogenesis controls mitochondrial homeostasis and responds to changes in cellular energy demand. In addition, it is enhanced or decreased due to mitochondrial dysfunction during SAE. The aim of this study was to explore the changes of mitochondrial biogenesis of astrocytes under septic conditions.

METHODSLipopolysaccharide (LPS; 50 ng/ml) and interferon-γ (IFN-γ; 200 U/ml) were incubated with astrocytes to model the effects of a septic insult on astrocytes in vitro. The mitochondrial ultrastructure and volume density were evaluated by transmission electron microscopy. Intracellular adenosine triphosphate (ATP) levels were detected by the firefly luciferase system. The expression of protein markers of mitochondrial biogenesis and the binding ability of mitochondrial transcription factor A (TFAM) were determined by western blot and electrophoretic mobility shift assays, respectively. The mitochondrial DNA (mtDNA) content was detected by real-time polymerase chain reaction.

RESULTSThe number of mildly damaged mitochondria was found to be significantly greater after treatment for 6 hours, as compared with at 0 hour (P < 0.05). The mitochondrial volume density was significantly elevated at 24 hours, as compared with at 0 hour (P < 0.05). The ATP levels at 6 hours, 12 hours, and 24 hours were significantly greater than those at 0 hour (P < 0.05). The protein markers of mitochondrial biogenesis were significantly increased at 6 hours and 12 hours, as compared with at 0 hour (P < 0.05). The TFAM binding activity was not significantly changed among the four time points analyzed. The mtDNA contents were significantly increased at 12 hours and 24 hours, as compared with at 0 hour (P < 0.05).

CONCLUSIONSUnder septic conditions, mitochondrial biogenesis of astrocytes increased to meet the high-energy demand and to promote mitochondrial recovery. Furthermore, the TFAM-DNA binding ability was not sensitive to sepsis-induced injury.

Animals ; Astrocytes ; drug effects ; metabolism ; Blotting, Western ; Cells, Cultured ; DNA, Mitochondrial ; genetics ; Electrophoretic Mobility Shift Assay ; Interferon-gamma ; pharmacology ; Lipopolysaccharides ; pharmacology ; Microscopy, Electron, Transmission ; Mitochondrial Turnover ; drug effects ; physiology ; Nitric Oxide ; metabolism ; Rats ; Reactive Nitrogen Species ; metabolism ; Sepsis ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo study the effect of uncoupling protein 2 (UCP2)-siRNA on the inflammatory response of rat cardiomyocytes (H9C2) induced by septic serum and to investigate the possible role of UCP2 in the development of septic cardiomyopathy.

METHODSSerum samples were separately collected from normal rats and septic rats. Cultured rat cardiac cells (H9C2) were randomly divided into blank control, normal serum, 10% septic serum, UCP2-siRNA+10% septic serum and negative siRNA+10% septic serum groups. Stimulation with 10% septic serum was performed for 12 hours in relevant groups. The mRNA expression of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β) was measured by RT-PCR. The expression of phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) and nuclear factor-kappa B (NF-κB) was measured by Western blot.

RESULTSThe expression levels of p-p38 and NF-κB in the UCP2-siRNA+10% septic serum group were significantly higher than in the 10% septic serum group (P<0.05). The UCP2-siRNA+10% septic serum group had a significantly higher TNF-α mRNA expression than the 10% septic serum group (P<0.01), but IL-1β mRNA expression showed no significant difference between the two groups.

CONCLUSIONSUCP2 plays a regulatory role in the activation of p38 MAPK and NF-κB and the expression of downstream inflammatory mediators in H9C2 cells stimulated with septic serum.

Animals ; Cardiomyopathies ; etiology ; Cells, Cultured ; Inflammation ; etiology ; Interleukin-1beta ; genetics ; Ion Channels ; genetics ; physiology ; Male ; Mitochondrial Proteins ; genetics ; physiology ; Myocytes, Cardiac ; metabolism ; NF-kappa B ; metabolism ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley ; Sepsis ; blood ; complications ; Tumor Necrosis Factor-alpha ; genetics ; Uncoupling Protein 2 ; p38 Mitogen-Activated Protein Kinases ; metabolism