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: To explore the protective effect of NANOG against hydrogen peroxide (H2O2) -induced cell damage in the human hair follicle mesenchymal stem cells (hHF-MSCs). METHODS: NANOG was expressed from a lentiviral vector, pLVX-IRES-ZsGreen. NANOG hHF-MSCs and vector hHF-MSCs were treated with 400 μmol/L hydrogen peroxide (H2O2) for 2 h, the cell survival rate, cell morphology, ROS production, apoptosis and expression of AKT, ERK, and p21 were determined and compared. RESULTS: Our results showed that NANOG could activate AKT and upregulate the expression of p-AKT, but not p-ERK. When treated with 400 μmol/L H2O2, NANOG hHF-MSCs showed higher cell survival rate, lower ROS production and apoptosis, higher expression of p-AKT, higher ratio of p-AKT/AKT. CONCLUSION Our results suggest that NANOG could protect hHF-MSCs against cell damage caused by H2O2 through activating AKT signaling pathway.
Cell Survival ; Drug Evaluation, Preclinical ; Hair Follicle ; cytology ; Humans ; Hydrogen Peroxide ; Lentivirus ; Mesenchymal Stem Cells ; drug effects ; metabolism ; Nanog Homeobox Protein ; metabolism ; pharmacology ; Oxidative Stress ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction

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

OBJECTIVESTo investigate the hair growth-promoting effect of Miscanthus sinensis var. purpurascens (MSP) flower extracton on in vitro and in vivo models.

METHODSMSP flower extract was extracted in 99.9% methanol and applied to examine the proliferation of human dermal papilla cells (hDPCs) in vitro at the dose of 3.92-62.50 μg/mL and hair growth of C57BL/6 mice in vivo at the dose of 1000 μg/mL. The expression of transforming growth factor β1 (TGF-β1), hepatocyte growth factor (HGF), β-catenin, substance P was measured by relative quantitative realtime polymerase chain reaction. Histopathological and immunohistochemical analysis were performed.

RESULTSMSP (7.81 μg/mL) down-regulated TGF-β1 and up-regulated HGF and β-catenin in hDPCs (P<0.01). MSP (1000 μg/mL)-treated mice showed the earlier transition of hair follicles from the telogen to the anagen phase. The number of mast cells was lower in the MSP-treated mice than in other groups (P<0.05 vs. NCS group). Substance P and TGF-β1 were expressed in hair follicles and skin of the MSP group lower than that in negative control. Stem cell factor in hair follicles was up-regulated in the MSP-treated mice (P<0.01).

CONCLUSIONSThe MSP flower extract may have hair growth-promotion activities.

Animals ; Antioxidants ; pharmacology ; Cell Count ; Cell Proliferation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Flowers ; chemistry ; Hair Follicle ; cytology ; drug effects ; growth & development ; Hepatocyte Growth Factor ; metabolism ; Humans ; Mast Cells ; cytology ; Mice, Inbred C57BL ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; Poaceae ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Skin ; metabolism ; Stem Cell Factor ; metabolism ; Stress, Psychological ; pathology ; Substance P ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; beta Catenin ; metabolism

OBJECTIVES: To investigate the role of tetramethylpyrazine(TMP) nitrone in proliferation and differentiation of neural stem cells (NSCs). METHODS: We separated and cultivated the original generation of NSCs from cerebral cortex of 14 days rat embryo, and the phenotype characteristics of the third-generation NSCs was tested by immunofluorescence. The experiment was divided into control group, β-mercaptoethanol positive control group, tetramethylpyrazine nitrone group and tetramethylpyrazine nitrone + ethylene glycol tetraacetic acid(EGTA) group (=4). The third-generation cultivation of NSCs was used in the experiment. The effect of tetramethylpyrazine nitrone on the number of NSCs proliferation was determined by BrdU and MTT, and the differentiation of NSCs was determined by Western blot. RESULTS: The primary NSCs was isolated successfully, neurospheres with typical NSCs morphology and expressing nestin was formed at 3-5 days. As BrdU and MTT assay results shown, compared with the control group andβ-mercaptoethanol positive control group, the NSCs proliferation numbers of tetramethylpyrazine nitrone group increased significantly(<0.05). The results of Western blot showed that the neuronal differentiation rate of NSCs was increased significantly in both the tetramethylpyrazine nitrone group and tetramethylpyrazine nitrone + EGTA group, and the differentiation rate of NSCs in tetramethylpyrazine nitrone + EGTA group increased more significantly(<0.05). CONCLUSIONS Tetramethylpyrazine nitrone can significantly enhance the proliferation and neuronal differentiation rate of NSCs. Decrease in extracellular Ca can promote the differentiation of NSCs into neurons induced by tetramethylpyrazine nitrone. Ca signaling plays an important role in the differentiation of NSCs into neurons.
Animals ; Calcium Signaling ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Neural Stem Cells ; cytology ; drug effects ; Nitrogen Oxides ; pharmacology ; Pyrazines ; pharmacology ; Rats

Bone formation is important for the reconstruction of bone-related structures in areas that have been damaged by inflammation. Inflammatory conditions such as those that occur in patients with rheumatoid arthritis, cystic fibrosis, and periodontitis have been shown to inhibit osteoblastic differentiation. This study focussed on dental follicle stem cells (DFSCs), which are found in developing tooth germ and participate in the reconstruction of alveolar bone and periodontal tissue in periodontal disease. After bacterial infection of inflamed dental tissue, the destruction of bone was observed. Currently, little is known about the relationship between the inflammatory environment and bone formation. Osteogenic differentiation of inflamed DFSCs resulted in decreased alkaline phosphatase (ALP) activity and alizarin red S staining compared to normal DFSCs. Additionally, in vivo transplantation of inflamed and normal DFSCs demonstrated severe impairment of osteogenesis by inflamed DFSCs. Protein profile analysis via liquid chromatography coupled with tandem mass spectrometry was performed to analyse the differences in protein expression in inflamed and normal tissue. Comparison of inflamed and normal DFSCs showed significant changes in the level of expression of transforming growth factor (TGF)-β2. Porphyromonas gingivalis (P.g.)-derived lipopolysaccharide (LPS) was used to create in vitro inflammatory conditions similar to periodontitis. The osteogenic differentiation of LPS-treated DFSCs was suppressed, and the cells displayed low levels of TGF-β1 and high levels of TGF-β2. DFSCs treated with TGF-β2 inhibitors showed significant increases in alizarin red S staining and ALP activity. TGF-β1 expression was also increased after inhibition of TGF-β2. By examining inflamed DFSCs and LPS-triggered DFSCs, these studies showed both clinically and experimentally that the increase in TGF-β2 levels that occurs under inflammatory conditions inhibits bone formation.
Adolescent ; Alkaline Phosphatase ; metabolism ; Animals ; Cell Differentiation ; Cell Proliferation ; Cell Survival ; Cells, Cultured ; Dental Sac ; cytology ; metabolism ; Down-Regulation ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunohistochemistry ; Male ; Mass Spectrometry ; Mice ; Nitric Oxide ; metabolism ; Osteogenesis ; drug effects ; Polymerase Chain Reaction ; Staining and Labeling ; Stem Cells ; cytology ; metabolism ; Transforming Growth Factor beta2 ; pharmacology ; Young Adult

OBJECTIVEThis study aimed to investigate the expression pattern and function of Nuclear receptor subfamily 2 group E member 1 (Nr2e1) in retinoic acid (RA)-induced brain abnormality.

METHODSThe mouse model of brain abnormality was established by administering 28 mg/kg RA, and neural stem cells (NSCs) were isolated from the mouse embryo and cultured in vitro. Nr2e1 expression was detected by whole mount in situ hybridization, RT-PCR, and Western blotting. Nr2e1 function was determined by transducing Nr2e1 shRNA into NSCs, and the effect on the sonic hedgehog (Shh) signaling pathway was assessed in the cells. In addition, the regulation of Nr2e1 expression by RA was also determined in vitro.

RESULTSNr2e1 expression was significantly downregulated in the brain and NSCs of RA-treated mouse embryos, and knockdown of Nr2e1 affected the proliferation of NSCs in vitro. In addition, a similar expression pattern of Nr2e1 and RA receptor (RAR) α was observed after treatment of NSCs with different concentrations of RA.

CONCLUSIONOur study demonstrated that Nr2e1 could be regulated by RA, which would aid a better understanding of the mechanism underlying RA-induced brain abnormality.

Animals ; Brain ; cytology ; embryology ; Cell Proliferation ; Down-Regulation ; Gene Expression Regulation ; Gene Expression Regulation, Developmental ; drug effects ; Mice ; Mice, Inbred C57BL ; Neural Stem Cells ; drug effects ; physiology ; Receptors, Cytoplasmic and Nuclear ; genetics ; metabolism ; Tretinoin ; pharmacology

Objective: To study the effects of muskolibanum combination on the proliferation and differentiation of prostate stem cells. METHODS: We cultured prostate epithelial cells and urogenital sinus mesenchymal (UGSM) cells from 7－10 d old C57BL/6 mice and 16－18 d old pregnant C57BL/6 mice, transplanted the mixed suspension of the two types of cells under the kidney envelope of SCIDCB.17 male mice, and harvested the transplants 30 days later. We randomly divided the SCIDCB.17 mice into four groups to be treated intragastrically with musk (n = 8), olibanum (n = 8), musk+olibanum (n = 7), and normal saline (blank control, n = 8)) respectively, all for 14 days. Then we collected the kidney tissue for observation of the morphology of the glandular tubes and differentiation of different subsets of stem cells by HE staining and determination of the expressions and distribution of P63, CD133, CD117 and Sca1 by immunohistochemistry and Western blot. RESULTS: A system was successfully established for the isolation and mixed culture of Sca1 Lin+ CD49f+ (LSC) cells of prostate stem cells and UGSM cells of the mouse embryonic prostate. Immunohistochemistry showed positive expressions of P63, CD133, Sca1, and CD117 in the prostatic acinar epithelia and proved the presence of prostatic acinar epithelial structure in the transplants. Compared with the blank control group, the expressions of CD133, Sca1 and CD117 were significantly increased in the musk, olibanum, and musk+olibanum groups (P< 0.05), higher in the musk+olibanum than in the musk or olibanum group (P< 0.05), and their protein expressions were even more elevated in the musk+olibanum group (P< 0.01), with statistically significant difference from the olibanum group (P< 0.05). CONCLUSIONS The combination of musk and olibanum can improve the proliferation and differentiation of prostate stem cells.
Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Drug Therapy, Combination ; Epithelial Cells ; cytology ; drug effects ; Fatty Acids, Monounsaturated ; pharmacology ; Female ; Frankincense ; pharmacology ; Male ; Mesenchymal Stem Cells ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Mice, SCID ; Pregnancy ; Prostate ; cytology ; Random Allocation ; Receptor Protein-Tyrosine Kinases ; Receptors, Cholinergic ; Stem Cells ; cytology ; drug effects

Tissue damage induces cells into reprogramming-like cellular state, which contributes to tissue regeneration. However, whether factors promoting the cell reprogramming favor tissue regeneration remains elusive. Here we identified combination of small chemical compounds including drug cocktails robustly promoting in vitro cell reprogramming. We then administrated the drug cocktails to mice with acute liver injuries induced by partial hepatectomy or toxic treatment. Our results demonstrated that the drug cocktails which promoted cell reprogramming in vitro improved liver regeneration and hepatic function in vivo after acute injuries. The underlying mechanism could be that expression of pluripotent genes activated after injury is further upregulated by drug cocktails. Thus our study offers proof-of-concept evidence that cocktail of clinical compounds improving cell reprogramming favors tissue recovery after acute damages, which is an attractive strategy for regenerative purpose.
Animals ; Cellular Reprogramming ; drug effects ; Cellular Reprogramming Techniques ; methods ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; Mice

Objective To study the effect of endometrial stem cells (EnSCs) derived cytokine cocktail (EdCC) on myocardial ischemic reperfusion injury (I/R) in a mouse model. Methods EdCC was concentrated from the culture medium of EnSCs with Millipore ultra-filtration technology and was administrated to a myocardial I/R mouse models through tail vein injection. The infarct area was determined by TTC/Evans Blue staining. The apoptotic cells were counted by TUNEL assay and the protein level of cleaved caspase 3 was evaluated by Western blotting. Results The EdCC extraction efficiency was (222.4±29.3) Μg/10(6) cells in every 24 h,but the protein gradually degraded under-80 ℃ storage. As compared with I/R group,100 Μg fresh EdCC decreased infarct area (P=0.001),reduced apoptotic nuclei in the infarct border (P=0.019),and inhibited cleaved caspase 3 expression (P=0.002). Increasing EdCC dosage did not further reduce the infarct area. The myocardial protective effect of EdCC diminished after 90 days' storage under-80 ℃. Conclusion EdCC reduces myocardial I/R injury through protecting cardiomyocytes from apoptosis within 30 days storage under-80 ℃.
Animals ; Apoptosis ; Caspase 3 ; metabolism ; Cells, Cultured ; Cytokines ; therapeutic use ; Disease Models, Animal ; Endometrium ; cytology ; Female ; Mice ; Myocardial Reperfusion Injury ; therapy ; Myocytes, Cardiac ; drug effects ; Stem Cells ; cytology